The Candida albicans cell wall is the immediate point of contact with the host and is implicated in the host-fungal interaction and virulence. To date, a number of cell wall proteins have been identified and associated with virulence. Analysis of the C. albicans genome has identified the IFF gene family as encoding the largest family of cell wall-related proteins. This family is also conserved in a range of other Candida species. Iff11 differs from other family members in lacking a GPI anchor, and we have demonstrated it to be 0 glycosylated and secreted in C. albicans. A null mutant lacking IFF11 was hypersensitive to cell wall-damaging agents, suggesting a role in cell wall organization. In a murine model of systemic infection the null mutant was highly attenuated in virulence, and survival-standardized infections suggest it is required to establish an infection. This work provides the first evidence of the importance of this gene family in the host-fungal interaction and virulence.

The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc,Man,GlcNAc, N-glycan is processed by alpha-glucosidases I and II and alpha 1,2-mannosidase to generate Man,GlcNAc. This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CHW41, ROT2, and MNSI encode for alpha-glucosidase I, alpha-glucosidase II catalytic subunit, and alpha 1,2-mannosidase, respectively. We disrupted the C. albicans CHW41, ROT2, and MNS1 homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41 Delta, Carot2 Delta, and Camns1 Delta null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated beta-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.

Beta (1,3)-glucans represent 40% of the cell wall of the yeast Candida albicans. The dectin-1 lectin-like receptor has shown to recognize fungal beta (1,3)-glucans and induce innate immune responses. The importance of beta-glucan-dectin-1 pathways for the recognition of C. albicans by human primary blood cells has not been firmly established. In this study we demonstrate that cytokine production by both human peripheral blood mononuclear cells and murine macrophages is dependent on the recognition of beta-glucans by dectin-1. Heat killing of C. albicans resulted in exposure of beta-glucans on the surface of the cell wall and subsequent recognition by dectin-1, whereas live yeasts stimulated monocytes mainly via recognition of cell-surface mannans. Dectin-1 induced cytokine production through the following 2 pathways: Syk-dependent production of the T-helper (Th) 2-type anti-inflammatory cytokine interleukin-10 and Toll-like receptor-Myd88-dependent stimulation of monocyte-derived proinflammatory cytokines, such as tumor necrosis factor-alpha. In contrast, stimulation of Th1-type cytokines, such as interferon-gamma , by C. albicans was independent of the recognition of beta-glucans by dectin-1. In conclusion, C. albicans induces production of monocyte-derived and T cell-derived cytokines through distinct pathways dependent on or independent of dectin-1.

The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of beta-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of beta-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant alpha-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Delta and mnt1Delta single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Deltamnt2Delta mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections.

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated mannoproteins that are the immediate point of contact with the host and strongly influence the host-fungal interaction. N-Glycans are the major form of mannoprotein modification and consist of a core structure, common to all eukaryotes, that is further elaborated in the Golgi to form the highly branched outer chain that is characteristic of fungi. In yeasts, outer chain branching is initiated by the action of the alpha1,6-mannosyltransferase Och1p; therefore, we disrupted the C. albicans OCH1 homolog to determine the importance of outer chain N-glycans on the host-fungal interaction. Loss of CaOCH1 resulted in a temperature-sensitive growth defect and cellular aggregation. Outer chain elongation of N-glycans was absent in the null mutant, demonstrated by the lack of the alpha1,6-linked polymannose backbone and the underglycosylation of N-acetylglucosaminidase. A null mutant lacking OCH1 was hypersensitive to a range of cell wall perturbing agents and had a constitutively activated cell wall integrity pathway. These mutants had near normal growth rates in vitro but were attenuated in virulence in a murine model of systemic infection. However, tissue burdens for the Caoch1delta null mutant were similar to control strains with normal N-glycosylation, suggesting the host-fungal interaction was altered such that high burdens were tolerated. This demonstrates the importance of N-glycan outer chain epitopes to the host-fungal interaction and virulence.

Calcium (Ca2+) is an important second messenger for activating stress response signaling and cell adaptation in eukaryotic cells yet intracellular Ca2+-dynamics in fungi are poorly understood due to lack of effective real-time Ca2+ reporters. We engineered the GCaMP6f construct for use in the fungal pathogen, Candida albicans, and used live-cell imaging to observe both dynamic Ca2+ spiking and slower changes in non-spiking Ca2+-GCaMP signals elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca2+-GCaMP spiking was inhibited by 1 M NaCl but not by 0.666 M CaCl2. Treatment with sodium dodecylsulfate (SDS) caused a spike-burst, raised the non-spiking Ca2+-GCaMP signals, and caused significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H2O2 abolished spiking and caused transient non-GCaMP-related autofluorescence, but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H2O2 was dependent on Cap1, extracellular Ca2+, and calcineurin but not on its downstream target, Crz1. Ca2+-dynamics were not affected by H2O2 in the hog1Δ or yvc1Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca2+-GCaMP responses in individual cells has, therefore, revealed the dynamics of Ca2+-influx, signaling and homeostasis, and their role in the temporal stress response signatures of C. albicans. IMPORTANCE Intracellular calcium signaling plays an important role in the resistance and adaptation to stresses encountered by fungal pathogens within the host. This study reports the optimization of the GCaMP fluorescent calcium reporter for live-cell imaging of dynamic calcium responses in single cells of the pathogen, Candida albicans, for the first time. Exposure to membrane, osmotic or oxidative stress generated both specific changes in single cell intracellular calcium spiking and longer calcium transients across the population. Repeated treatments showed that calcium dynamics become unaffected by some stresses but not others, consistent with known cell adaptation mechanisms. By expressing GCaMP in mutant strains and tracking the viability of individual cells over time, the relative contributions of key signaling pathways to calcium flux, stress adaptation, and cell death were demonstrated. This reporter, therefore, permits the study of calcium dynamics, homeostasis, and signaling in C. albicans at a previously unattainable level of detail.

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Most microbes have developed responses that protect them against stresses relevant to their niches. Some that inhabit reasonably predictable environments have evolved anticipatory responses that protect against impending stresses that are likely to be encountered in their niches-termed "adaptive prediction". Unlike yeasts such as Saccharomyces cerevisiae, Kluyveromyces lactis and Yarrowia lipolytica and other pathogenic Candida species we examined, the major fungal pathogen of humans, Candida albicans, activates an oxidative stress response following exposure to physiological glucose levels before an oxidative stress is even encountered. Why? Using competition assays with isogenic barcoded strains, we show that "glucose-enhanced oxidative stress resistance" phenotype enhances the fitness of C. albicans during neutrophil attack and during systemic infection in mice. This anticipatory response is dependent on glucose signalling rather than glucose metabolism. Our analysis of C. albicans signalling mutants reveals that the phenotype is not dependent on the sugar receptor repressor pathway, but is modulated by the glucose repression pathway and down-regulated by the cyclic AMP-protein kinase a pathway. Changes in catalase or glutathione levels do not correlate with the phenotype, but resistance to hydrogen peroxide is dependent on glucose-enhanced trehalose accumulation. The data suggest that the evolution of this anticipatory response has involved the recruitment of conserved signalling pathways and downstream cellular responses, and that this phenotype protects C. albicans from innate immune killing, thereby promoting the fitness of C. albicans in host niches.

In 2009 Candida auris was first isolated as fungal pathogen of human disease from ear canal of a patient in Japan. In less than a decade, this pathogen has rapidly spread around the world and has now become a major health challenge that is of particular concern because many strains are resistant to multiple class of antifungal drugs. The lack of available antifungals and rapid increase of this fungal pathogen provides an incentive for the development of new and more potent anticandidal drugs and drug combinatorial treatments. Here we have explored the growth inhibitory activity against C. auris of a synthetic dipeptide glutamine analogue, L-norvalyl-N3-(4-methoxyfumaroyl)-L-2,3- diaminopropanoic acid (Nva-FMDP), that acts as an inhibitor of glucosamine-6-phosphate (GlcN-6-P) synthase - a key enzyme in the synthesis of cell wall chitin. We observed that in contrast to FLC susceptible isolates of C. auris, FLC resistant isolates had elevated cell wall chitin and were susceptible to inhibition by Nva-FMDP. The growth kinetics of C. auris in RPMI-1640 medium revealed that the growth of FLC resistant isolates were 50–60% more inhibited by Nva-FMDP (8 μ g/ml) compared to a FLC susceptible isolate. Fluconazole resistant strains displayed increased transcription of CHS1, CHS2 and CHS3, and the chitin content of the fluconazole resistant strains was reduced following the Nva-FMDP treatment. Therefore, the higher chitin content in FLC resistant C. auris isolates may make the strain more susceptible to inhibition of the antifungal activity of the Nva-FMDP peptide conjugate.

The immunogenicity of Candida albicans cells is influenced by changes in the exposure of microbe-associated molecular patterns (MAMPs) on the fungal cell surface. Previously, the degree of exposure on the C. albicans cell surface of the immunoinflammatory MAMP β-(1,3)-glucan was shown to correlate inversely with colonisation levels in the gastrointestinal (GI) tract. This is important because life-threatening systemic candidiasis in critically ill patients often arises from translocation of C. albicans strains present in the patient's GI tract. Therefore, using a murine model, we have examined the impact of gut-related factors upon β-glucan exposure and colonisation levels in the GI tract. The degree of β-glucan exposure was examined by imaging flow cytometry of C. albicans cells taken directly from GI compartments, and compared with colonisation levels. Fungal β-glucan exposure was lower in the cecum than the small intestine, and fungal burdens were correspondingly higher in the cecum. This inverse correlation did not hold for the large intestine. The gut fermentation acid, lactate, triggers β-glucan masking in vitro, leading to attenuated anti-Candida immune responses. Additional fermentation acids are present in the GI tract, including acetate, propionate, and butyrate. We show that these acids also influence β-glucan exposure on C. albicans cells in vitro and, like lactate, they influence β-glucan exposure via Gpr1/Gpa2-mediated signalling. Significantly, C. albicans gpr1Δ gpa2Δ cells displayed elevated β-glucan exposure in the large intestine and a corresponding decrease in fungal burden, consistent with the idea that Gpr1/Gpa2-mediated β-glucan masking influences colonisation of this GI compartment. Finally, extracts from the murine gut and culture supernatants from the mannan grazing gut anaerobe Bacteroides thetaiotaomicron promote β-glucan exposure at the C. albicans cell surface. Therefore, the local microbiota influences β-glucan exposure levels directly (via mannan grazing) and indirectly (via fermentation acids), whilst β-glucan masking appears to promote C. albicans colonisation of the murine large intestine.

Candida albicans exists as a commensal of mucosal surfaces and the gastrointestinal tract without causing pathology. However, this fungus is also a common cause of mucosal and systemic infections when antifungal immune defenses become compromised. The activation of antifungal host defenses depends on the recognition of fungal pathogen-associated molecular patterns (PAMPs), such as β-1,3-glucan. In C. albicans, most β-1,3-glucan is present in the inner cell wall, concealed by the outer mannan layer, but some β-1,3-glucan becomes exposed at the cell surface. In response to host signals, such as lactate, C. albicans induces the Xog1 exoglucanase, which shaves exposed β-1,3-glucan from the cell surface, thereby reducing phagocytic recognition. We show here that β-1,3-glucan is exposed at bud scars and punctate foci on the lateral wall of yeast cells, that this exposed β-1,3-glucan is targeted during phagocytic attack, and that lactate-induced masking reduces β-1,3-glucan exposure at bud scars and at punctate foci. β-1,3-Glucan masking depends upon protein kinase a (PKA) signaling. We reveal that inactivating PKA, or its conserved downstream effectors, Sin3 and Mig1/Mig2, affects the amounts of the Xog1 and Eng1 glucanases in the C. albicans secretome and modulates β-1,3-glucan exposure. Furthermore, perturbing PKA, Sin3, or Mig1/Mig2 attenuates the virulence of lactate-exposed C. albicans cells in Galleria. Taken together, the data are consistent with the idea that β-1,3-glucan masking contributes to Candida pathogenicity. IMPORTANCE Microbes that coexist with humans have evolved ways of avoiding or evading our immunological defenses. These include the masking by these microbes of their "pathogen-associated molecular patterns" (PAMPs), which are recognized as "foreign" and used to activate protective immunity. The commensal fungus Candida albicans masks the proinflammatory PAMP β-1,3-glucan, which is an essential component of its cell wall. Most of this β-1,3-glucan is hidden beneath an outer layer of the cell wall on these microbes, but some can become exposed at the fungal cell surface. Using high-resolution confocal microscopy, we examine the nature of the exposed β-1,3-glucan at C. albicans bud scars and at punctate foci on the lateral cell wall, and we show that these features are targeted by innate immune cells. We also reveal that downstream effectors of protein kinase a (Mig1/Mig2, Sin3) regulate the secretion of major glucanases, modulate the levels of β-1,3-glucan exposure, and influence the virulence of C. albicans in an invertebrate model of systemic infection. Our data support the view that β-1,3-glucan masking contributes to immune evasion and the virulence of a major fungal pathogen of humans.

The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.

Prior to the SARS-CoV-2 pandemic, antibiotic resistance was listed as the major global health care priority. Some analyses, including the O'Neill report, have predicted that deaths due to drug-resistant bacterial infections may eclipse the total number of cancer deaths by 2050. Although fungal infections remain in the shadow of public awareness, total attributable annual deaths are similar to, or exceeds, global mortalities due to malaria, tuberculosis or HIV. The impact of fungal infections has been exacerbated by the steady rise of antifungal drug resistant strains and species which reflects the widespread use of antifungals for prophylaxis and therapy, and in the case of azole resistance in Aspergillus, has been linked to the widespread agricultural use of antifungals. This review, based on a workshop hosted by the Medical Research Council and the University of Exeter, illuminates the problem of antifungal resistance and suggests how this growing threat might be mitigated.

Phagocytosis is an essential component of our immune defence against fungal pathogens. Differences in the dynamics of phagocyte migration, recognition, uptake and phagolysosome maturation are dependent on the characteristics of the fungal cargo, and in particular to differences in cell wall composition and cellular morphology. However, studies that have focused on phagocyte interactions with individual fungal species have not enabled comparisons in the kinetics of these interactions to be made between these different species. We therefore used live cell video microscopy to examine the temporal dynamics of phagocytosis for a range of fungal cargoes by thioglycollate-elicited peritoneal macrophages from C57BL/6 mice. Uniform populations of macrophages were challenged at the same time with yeast cells of Candida albicans, Candida glabrata, Saccharomyces cerevisiae and Cryptococcus neoformans (wild-type and an acapsular mutant, cap59Δ), and spores of Aspergillus fumigatus and Mucor circinelloides to enable standardized comparative interactions to be quantified from different stages of phagocytosis. Differences in the rate of uptake of fungal cells varied by up to 26-fold, whilst differences in time to induce phagosome acidification varied by as much as 29-fold. Heat-killing or opsonizing the fungal targets markedly affected the kinetics of the interaction in a species-specific manner. Fungal and macrophage killing assays further revealed cargo-specific differences in phagocytosis and diversity in fungal evasion mechanisms. Therefore, simultaneous assessment of the interaction of macrophages with different fungal pathogens highlighted major differences in the kinetics and growth responses during fungus-phagocyte interactions that are likely to impact on pathogenesis and virulence.

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Background:Candida species have long been recognised as aetiological agents of opportunistic infections of the oral mucosa, and more recently, as players of polymicrobial interactions driving caries, periodontitis and oral carcinogenesis. Methods: We studied the clonal structure of Candida spp. at oral niche resolution in patients (n = 20) with a range of oral health profiles over 22 months. Colonies from oral micro-environments were examined with multilocus sequencing typing. Results:Candida spp. identified were C. albicans, C. glabrata and C. dubliniensis. Increased propensity for micro-variations giving rise to multiple diploid strain types (DST), as a result of loss of heterozygosity, was observed among C. albicans clade 1 isolates compared to other clades. Micro-variations among isolates were also observed in C. dubliniensis contra to expectations of stable population structures for this species. Multiple sequence types were retrieved from patients without clinical evidence of oral candidosis, while single sequence types were isolated from oral candidosis patients. Conclusion: This is the first study to describe the clonal population structure, persistence and stability of Candida spp. at oral niche level. Future research investigating links between Candida spp. clonality and oral disease should recognise the propensity to micro-variations amongst oral niches in C. albicans and C. dubliniensis identified here.

Echinocandins such as caspofungin are frontline antifungal drugs that compromise β-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by upregulation of chitin synthesis, thereby sustaining cell wall integrity (CWI). When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a subpopulation of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable supra-high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway. This article has an associated First Person interview with the first author of the paper.

More than 95% of invasive Candida infections are caused by four Candida spp. (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis). C-type lectin-like receptors (CLRs), such as Dectin-1, Dectin-2, and Mincle mediate immune responses to C. albicans. Dectin-1 promotes clearance of C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis, however, dependence on Dectin-1 for specific immune responses varies with the different Candida spp. Dectin-2 is important for host immunity to C. albicans and C. glabrata, and Mincle is important for the immune response to C. albicans. However, whether Dectin-2 drives host immunity to C. tropicalis or C. parapsilosis, and whether Mincle mediates host immunity to C. glabrata, C. tropicalis or C. parapsilosis is unknown. Therefore, we compared the roles of Dectin-2 and Mincle in response to these four Candida spp. We demonstrate that these four Candida spp. cell walls have differential mannan contents. Mincle and Dectin-2 play a key role in regulating cytokine production in response to these four Candida spp. and Dectin-2 is also important for clearance of all four Candida spp. during systemic infection. However, Mincle was only important for clearance of C. tropicalis during systemic infection. Our data indicate that multiple Candida spp. have different mannan contents, and dependence on the mannan-detecting CLRs, Mincle, and Dectin-2 varies between different Candida spp. during systemic infection.

Invasive fungal infections remain highly problematic for human health. Collectively, they account for more than 1 million deaths a year in addition to more than 100 million mucosal infections and 1 billion skin infections. To be able to make progress it is important to understand the pathobiology of fungal interactions with the immune system. Here, we highlight new advancements pointing out the pivotal role of fungal cell wall components (β-glucan, mannan, galactosaminogalactan and melanin) in modulating host immunity and discuss how these open new opportunities for the development of immunomodulatory strategies to combat deadly fungal infectious diseases.

Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal pathogens are not a foregone conclusion because some pathogens have evolved mechanisms to evade phagocytic recognition, engulfment, and killing. For example, Candida albicans can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of C. albicans-macrophage interactions, we discovered that macrophages can counteract this by folding fungal hyphae. The folding of fungal hyphae is promoted by Dectin-1, β2-integrin, VASP, actin-myosin polymerization, and cell motility. Folding facilitates the complete engulfment of long hyphae in some cases and it inhibits hyphal growth, presumably tipping the balance toward successful fungal clearance.

Understanding the molecular mechanisms governing antifungal resistance is crucial for identifying new cellular targets for developing new antifungal therapeutics. In this study, we performed a transposon-mediated genome-wide genetic screen in haploid Candida albicans to identify mutants resistant to caspofungin, the first member of the echinocandin class of antifungal drugs. A mutant exhibiting the highest resistance possessed a transposon insertion that inactivates GPI7, a gene encoding the mannose-ethanolamine phosphotransferase. Deleting GPI7 in diploid C. albicans caused similar caspofungin resistance. gpi7Δ/Δ cells showed significantly elevated cell wall chitin content and enhanced phosphorylation of Mkc1, a core component of the PKC-MAPK cell-wall integrity pathway. Deleting MKC1 suppressed the chitin elevation and caspofungin resistance of gpi7Δ/Δ cells, but overexpressing the dominant inactive form of RHO1, an upstream activator of PKC-MAPK signaling, did not. Transcriptome analysis uncovered 406 differentially expressed genes in gpi7Δ/Δ cells, many related to cell wall construction. Our results suggest that GPI7 deletion impairs cell wall integrity, which triggers the cell-wall salvage mechanism via the PKC-MAPK pathway independently of Rho1, resulting in the compensatory chitin synthesis to confer caspofungin resistance.

While there is growing evidence that perturbation of the gut microbiota can result in a variety of pathologies including gut tumorigenesis, the influence of commensal fungi remains less clear. In this issue, Zhu et al (2021) show that mycobiota dysbiosis stimulates energy metabolism changes in subepithelial macrophages promoting colon cancer via enhancing innate lymphoid cell activity. These findings provide insights into a role of the gut flora in intestinal carcinogenesis and suggest opportunities for adjunctive antifungal or immunotherapeutic strategies to prevent colorectal cancer.

Independent studies from our group and others have provided evidence that sphingolipids (SLs) influence the antimycotic susceptibility of Candida species. We analyzed the molecular SL signatures of drug-resistant clinical isolates of Candida auris, which have emerged as a global threat over the last decade. This included Indian hospital isolates of C. auris, which were either resistant to fluconazole (FLCR) or amphotericin B (AmBR) or both drugs. Relative to Candida glabrata and Candida albicans strains, these C. auris isolates were susceptible to SL pathway inhibitors such as myriocin and aureobasidin A, suggesting that SL content may influence azole and AmB susceptibilities. Our analysis of SLs confirmed the presence of 140 SL species within nine major SL classes, namely the sphingoid bases, Cer, αOH-Cer, dhCer, PCer, αOH-PCer, αOH-GlcCer, GlcCer, and IPC. Other than for αOH-GlcCer, most of the SLs were found at higher concentrations in FLCR isolates as compared to the AmBR isolates. SLs were at intermediate levels in FLCR + AmBR isolates. The observed diversity of molecular species of SL classes based on fatty acyl composition was further reflected in their distinct specific imprint, suggesting their influence in drug resistance. Together, the presented data improves our understanding of the dynamics of SL structures, their synthesis, and link to the drug resistance in C. auris.

The unfolded protein response (UPR), crucial for the maintenance of endoplasmic reticulum (ER) homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here, we show that Candida albicans relies on an ER-resident protein, inositol-requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1-mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence-related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin-induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1-mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

The discovery of liquid water at several locations in the solar system raises the possibility that microbial life may have evolved outside Earth and as such could be accidently introduced into the Earth’s ecosystem. Unusual sugars or amino acids, like non-proteinogenic isovaline and α-aminoisobutyric acid that are vanishingly rare or absent from life forms on Earth, have been found in high abundance on non-terrestrial carbonaceous meteorites. It is therefore conceivable that exo-microorganisms might contain proteins that include these rare amino acids. We therefore asked whether the mammalian immune system would be able to recognize and induce appropriate immune responses to putative proteinaceous antigens that include these rare amino acids. To address this, we synthesised peptide antigens based on a backbone of ovalbumin and introduced isovaline and α-aminoisobutyric acid residues and demonstrated that these peptides can promote naïve OT-I cell activation and proliferation, but did so less efficiently than the canonical peptides. This is relevant to the biosecurity of missions that may retrieve samples from exoplanets and moons that have conditions that may be permissive for life, suggesting that accidental contamination and exposure to exo-microorganisms with such distinct proteomes might pose an immunological challenge.

Pathogenic fungi represent an increasing infectious disease threat to humans, especially with an increasing challenge of antifungal drug resistance. Over the decades, numerous tools have been developed to expedite the study of pathogenicity, initiation of disease, drug resistance and host-pathogen interactions. In this review, we highlight advances that have been made in the use of molecular tools using CRISPR technologies, RNA interference and transposon targeted mutagenesis. We also discuss the use of animal models in modelling disease of human fungal pathogens, focusing on zebrafish, the silkworm, Galleria mellonella and the murine model.

Early detection is critical to the successful treatment of life-threatening infections caused by fungal pathogens, as late diagnosis of systemic infection almost always equates with a poor prognosis. The field of fungal diagnostics has some tests that are relatively simple, rapid to perform and are potentially suitable at the point of care. However, there are also more complex high-technology methodologies that offer new opportunities regarding the scale and precision of fungal diagnosis, but may be more limited in their portability and affordability. Future developments in this field are increasingly incorporating new technologies provided by the use of new format biosensors. This overview provides a critical review of current fungal diagnostics and the development of new biophysical technologies that are being applied for selective new sensitive fungal biosensors to augment traditional diagnostic methodologies.

Breakthrough invasive fungal infections (bIFI) cause significant morbidity and mortality. Their diagnosis can be challenging due to reduced sensitivity to conventional culture techniques, serologic tests, and PCR-based assays in patients undergoing antifungal therapy, and their diagnosis can be delayed contributing to poor patient outcomes. In this review, we provide consensus recommendations on behalf of the European Confederation for Medical Mycology (ECMM) for the diagnosis of bIFI caused by invasive yeasts, molds, and endemic mycoses, to guide diagnostic efforts in patients receiving antifungals and support the design of future clinical trials in the field of clinical mycology. The cornerstone of lab-based diagnosis of breakthrough infections for yeast and endemic mycoses remain conventional culture, to accurately identify the causative pathogen and allow for antifungal susceptibility testing. The impact of non-culture-based methods are not well-studied for the definite diagnosis of breakthrough invasive yeast infections. Non-culture-based methods have an important role for the diagnosis of breakthrough invasive mold infections, in particular invasive aspergillosis, and a combination of testing involving conventional culture, antigen-based assays, and PCR-based assays should be considered. Multiple diagnostic modalities, including histopathology, culture, antibody, and/or antigen tests and occasionally PCR-based assays may be required to diagnose breakthrough endemic mycoses. A need exists for diagnostic tests that are effective, simple, cheap, and rapid to enable the diagnosis of bIFI in patients taking antifungals.

The publisher regrets that the ‘Declaration of Competing Interest’ statement was not included in the published article. The statement is as follows: Declaration of Competing Interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The publisher would like to apologise for any inconvenience caused.

Hypoxic adaptation pathways, essential for Candida albicans pathogenesis, are tied to its transition from a commensal to a pathogen. Herein, we identify a WW domain-containing protein, Ifu5, as a determinant of hypoxic adaptation that also impacts normoxic responses in this fungus. Ifu5 activity supports glycosylation homeostasis via the Cek1 mitogen-activated protein kinase-dependent up-regulation of PMT1, under normoxia. Transcriptome analysis of ifu5Δ/Δ under normoxia shows a significant up-regulation of the hypoxic regulator EFG1 and EFG1-dependent genes. We demonstrate physical interaction between Ifu5 by virtue of its WW domain and Efg1 that represses EFG1 expression under normoxia. This interaction is lost under hypoxic growth conditions, relieving EFG1 repression. Hypoxic adaptation processes such as filamentation and biofilm formation are affected in ifu5Δ/Δ cells revealing the role of Ifu5 in hypoxic signalling and modulating pathogenicity traits of C. albicans under varied oxygen conditions. Additionally, the WW domain of Ifu5 facilitates its role in hypoxic adaptation, revealing the importance of this domain in providing a platform to integrate various cellular processes. These data forge a relationship between Efg1 and Ifu5 that fosters the role of Ifu5 in hypoxic adaptation thus illuminating novel strategies to undermine the growth of C. albicans.

Human space travel is on the verge of visiting Mars and, in the future, even more distant places in the solar system. These journeys will be also made by terrestrial microorganisms (hitchhiking on the bodies of astronauts or on scientific instruments) that, upon arrival, will come into contact with new planetary environments, despite the best measures to prevent contamination. These microorganisms could potentially adapt and grow in the new environments and subsequently recolonize and infect astronauts. An even more challenging situation would be if truly alien microorganisms will be present on these solar system bodies: What will be their pathogenic potential, and how would our immune host defenses react? it will be crucial to anticipate these situations and investigate how the immune system of humans might cope with modified terrestrial or alien microbes. We propose several scenarios that may be encountered and how to respond to these challenges.

During the course of fungal infection, pathogen recognition by the innate immune system is critical to initiate efficient protective immune responses. The primary event that triggers immune responses is the binding of Pattern Recognition Receptors (PRRs), which are expressed at the surface of host immune cells, to Pathogen-Associated Molecular Patterns (PAMPs) located predominantly in the fungal cell wall. Most fungi have mannosylated PAMPs in their cell walls and these are recognized by a range of C-type lectin receptors (CTLs). However, the precise spatial distribution of the ligands that induce immune responses within the cell walls of fungi are not well defined. We used recombinant IgG Fc-CTLs fusions of three murine mannan detecting CTLs, including dectin-2, the mannose receptor (MR) carbohydrate recognition domains (CRDs) 4-7 (CRD4-7), and human DC-SIGN (hDC-SIGN) and of the β-1,3 glucan-binding lectin dectin-1 to map PRR ligands in the fungal cell wall of fungi grown in vitro in rich and minimal media. We show that epitopes of mannan-specific CTL receptors can be clustered or diffuse, superficial or buried in the inner cell wall. We demonstrate that PRR ligands do not correlate well with phylogenetic relationships between fungi, and that Fc-lectin binding discriminated between mannosides expressed on different cell morphologies of the same fungus. We also demonstrate CTL epitope differentiation during different phases of the growth cycle of Candida albicans and that MR and DC-SIGN labelled outer chain N-mannans whilst dectin-2 labelled core N-mannans displayed deeper in the cell wall. These immune receptor maps of fungal walls of in vitro grown cells therefore reveal remarkable spatial, temporal and chemical diversity, indicating that the triggering of immune recognition events originates from multiple physical origins at the fungal cell surface.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3-and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the in-tercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-D-glucan-and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-D-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans. IMPORTANCE Candida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans. Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

. Candida auris
. is a newly emerged fungal pathogen of humans. This species was first reported in 2009 when it was identified in an ear infection of a patient in Japan. However, despite intense interest in this organism as an often multidrug-resistant fungus, there is little knowledge about its cellular biology. During infection of human patients, fungi are able to change cell shape from ellipsoidal yeast cells to elongated filaments to adapt to various conditions within the host organism. There are different types of filaments, which are triggered by reactions to different cues.
. Candida auris
. fails to form filaments when exposed to triggers that stimulate yeast filament morphogenesis in other fungi. Here, we show that it does form filaments when its DNA is damaged. These conditions might arise when
. Candida auris
. cells interact with host immune cells or during growth in certain host tissues (kidney or bladder) or during treatment with antifungal drugs.
.

Despite the importance of fungal cell walls as the principle determinant of fungal morphology and the defining element determining fungal interactions with other cells, few scalar models have been developed that reconcile chemical and microscopic attributes of its structure. The cell wall of the fungal pathogen Candida albicans is comprised of an amorphous inner skeletal layer of β(1,3)- and β(1,6)-glucan and chitin and an outer fibrillar layer thought to be dominated by highly mannosylated cell wall proteins. The architecture of these two layers can be resolved at the electron microscopy level, but the visualised structure of the wall has not yet been defined precisely in chemical terms. We have therefore examined the precise structure, location and molecular sizes of the cell wall components using transmission electron microscopy and tomography and tested predictions of the cell wall models using mutants and agents that perturb the normal cell wall structure. We demonstrate that the fibrils are comprised of a frond of N-linked outer chain mannans linked to a basal layer of GPI-proteins concentrated in the mid-wall region and that the non-elastic chitin microfibrils are cantilevered with sufficient lengths of non-fibrillar chitin and/or β-glucan to enable the chitin-glucan cage to flex, e.g. during morphogenesis and osmotic swelling. We present the first three-dimensional nano-scalar model of the C. albicans cell wall which can be used to test hypotheses relating to the structure–function relationships that underpin the pathobiology of this fungal pathogen.

Candida auris is an emerging pathogenic yeast of significant clinical concern because of its frequent intrinsic resistance to fluconazole and often other antifungal drugs and the high mortality rates associated with systemic infections. Furthermore, C. auris has a propensity for persistence and transmission in health care environments. The reasons for this efficient transmission are not well understood, and therefore we tested whether enhanced resistance to environmental stresses might contribute to the ability of C. auris to spread in health care environments. We compared C. auris to other pathogenic Candida species with respect to their resistance to individual stresses and combinations of stresses. Stress resistance was examined using in vitro assays on laboratory media and also on hospital linen. In general, the 17 C. auris isolates examined displayed similar degrees of resistance to oxidative, nitrosative, cationic and cell wall stresses as clinical isolates of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. lusitaniae and C. kefyr. All of the C. auris isolates examined were more sensitive to low pH (pH 2, but not pH 4) compared to C. albicans, but were more resistant to high pH (pH 13). C. auris was also sensitive to low pH, when tested on contaminated hospital linen. Most C. auris isolates were relatively thermotolerant, displaying significant growth at 47°C. Furthermore, C. auris was relatively resistant to certain combinations of combinatorial stress (e.g. pH 13 plus 47°C). Significantly, C. auris was sensitive to the stress combinations imposed by hospital laundering protocol (pH > 12 plus heat shock at >80°C), suggesting that current laundering procedures are sufficient to limit the transmission of this fungal pathogen via hospital linen.

The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security.

We show that theC. albicansureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important forC. albicansgrowth and survival in microenvironments of the mammalian host.

ATP-binding cassette (ABC) superfamily members have a key role as nutrient importers and exporters in bacteria. However, their role as drug exporters in eukaryotes brought this superfamily member to even greater prominence. The capacity of ABC transporters to efflux a broad spectrum of xenobiotics represents one of the major mechanisms of clinical multidrug resistance in pathogenic fungi including Candida species. Candida auris, a newly emerged multidrug-resistant fungal pathogen of humans, has been responsible for multiple outbreaks of drug-resistant infections in hospitals around the globe. Our study has analyzed the entire complement of ABC superfamily transporters to assess whether these play a major role in drug resistance mechanisms of C. auris. Our bioinformatics analyses identified 28 putative ABC proteins encoded in the genome of the C. auris type-strain CBS 10913T; 20 of which contain transmembrane domains (TMDs). Quantitative real-time PCR confirmed the expression of all 20 TMD transporters, underlining their potential in contributing to the C. auris drug-resistant phenotype. Changes in transcript levels after short-term exposure of drugs and in drug-resistant C. auris isolates suggested their importance in the drug resistance phenotype of this pathogen. CAUR-02725 orthologous to CDR1, a major multidrug exporter in other yeasts, showed consistently higher expression in multidrug-resistant strains of C. auris. Homologs of other ABC transporter genes, such as CDR4, CDR6, and SNQ2, also displayed raised expression in a sub-set of clinical isolates. Together, our analysis supports the involvement of these transporters in multidrug resistance in C. auris.

The human fungal pathogen Candida albicans is known to require endocytosis to enable its adaptation to diverse niches and to maintain its highly polarized hyphal growth phase. While studies have identified changes in transcription leading to the synthesis and secretion of new proteins to facilitate hyphal growth, effective maintenance of hyphae also requires concomitant removal or relocalization of other cell surface molecules. The key molecules which must be removed from the cell surface, and the mechanisms behind this, have, however, remained elusive. In this study, we show that the AP-2 endocytic adaptor complex is required for the internalization of the major cell wall biosynthesis enzyme Chs3. We demonstrate that this interaction is mediated by the AP-2 mu subunit (Apm4) YXXΦ binding domain. We also show that in the absence of Chs3 recycling via AP-2, cells have abnormal cell wall composition, defective polarized cell wall deposition, and morphological defects. The study also highlights key distinctions between endocytic requirements of growth at yeast buds compared to that at hyphal tips and different requirements of AP-2 in maintaining the polarity of mannosylated proteins and ergosterol at hyphal tips. Together, our findings highlight the importance of correct cell wall deposition in cell shape maintenance and polarized growth and the key regulatory role of endocytic recycling via the AP-2 complex. IMPORTANCE Candida albicans is a human commensal yeast that can cause significant morbidity and mortality in immunocompromised individuals. Within humans, C. albicans can adopt different morphologies as yeast or filamentous hyphae and can occupy different niches with distinct temperatures, pHs, CO2 levels, and nutrient availability. Both morphological switching and growth in different environments require cell surface remodelling, which involves both the addition of newly synthesized proteins as well as the removal of other proteins. In our study, we demonstrate the importance of an adaptor complex AP-2 in internalizing and recycling a specific cell surface enzyme to maintain effective polarized hyphal growth. Defects in formation of the complex or in its ability to interact directly with cargo inhibit enzyme uptake and lead to defective cell walls and aberrant hyphal morphology. Our data indicate that the AP-2 adaptor plays a central role in regulating cell surface composition in Candida.

Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. Candida albicans is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein "High affinity glucose transporter 1" (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased in vitro phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of C. albicans.

Background: Recent outbreaks of Candida auris further exemplify that invasive Candida infections are a substantial threat to patients and healthcare systems. Even short treatment delays are associated with higher mortality rates. Epidemiological shifts towards more resistant Candida spp. require careful surveillance. Objectives: Triggered by the emergence of C auris and by increasing antifungal resistance rates the European Confederation of Medical Mycology developed an international Candida Registry (FungiScope™ CandiReg) to allow contemporary multinational surveillance. Methods: CandiReg serves as platform for international cooperation to enhance research regarding invasive Candida infections. CandiReg uses the General Data Protection Regulation compliant data platform ClinicalSurveys.net that holds the electronic case report forms (eCRF). Data entry is supported via an interactive macro created by the software that can be accessed via any Internet browser. Results: CandiReg provides an eCRF for invasive Candida infections that can be used for a variety of studies from cohort studies on attributable mortality to evaluations of guideline adherence, offering to the investigators of the 28 ECMM member countries the opportunity to document their cases of invasive Candida infection. CandiReg allows the monitoring of epidemiology of invasive Candida infections, including monitoring of multinational outbreaks. Here, we describe the structure and management of the CandiReg platform. Conclusion: CandiReg supports the collection of clinical information and isolates to improve the knowledge on epidemiology and eventually to improve management of invasive Candida infections. CandiReg promotes international collaboration, improving the availability and quality of evidence on invasive Candida infection and contributes to improved patient management.

Despite the high prevalence of women in graduate degree programs and equal or more women earning PhDs, MDs, and MD/PhDs, and despite efforts at individual and institutional levels to promote women in STEM fields, there remains a disparity in pay and academic advancement of women. Likewise, there is a paucity of women in top scientific and academic leadership positions. The causes of this gender disparity are complex and multi-factorial and to date no "magic bullet" approach has been successful in changing the landscape for women in academic and scientific fields. In this report we detail our experiences with a novel mechanism for promoting discussion and raising awareness of the challenges of gender disparity in the sciences. The Gordon Research Conferences (GRC) launched the Power Hour at its meetings in 2016: a dedicated, scheduled session held during the scientific meeting to facilitate discussion of challenges specific to women in science. Here we share our experience with hosting the second Power Hour at the 2019 GRC Immunology of Fungal Infections (IFI) meeting held in Galveston, TX. We will discuss the overall structure, key discussion points, and feedback from participants with the aim of supporting future efforts to empower women and underrepresented minority groups in science.

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By analogy with Pavlov's dogs, certain pathogens have evolved anticipatory behaviours that exploit specific signals in the human host to prepare themselves against imminent host challenges. This adaptive prediction, a type of history-dependent microbial behaviour, represents a primitive form of microbial memory. For fungal pathogens, adaptive prediction helps them circumvent nutritional immunity, protects them against phagocytic killing, and activates immune evasion strategies. We describe how these anticipatory responses, and the contrasting lifestyles and evolutionary trajectories of fungal pathogens, have influenced the evolution of such adaptive behaviours, and how these behaviours affect host colonisation and infection.

To colonise their host, pathogens must counter local environmental and immunological challenges. Here, we reveal that the fungal pathogen Candida albicans exploits diverse host-associated signals to promote immune evasion by masking of a major pathogen-associated molecular pattern (PAMP), β-glucan. Certain nutrients, stresses and antifungal drugs trigger β-glucan masking, whereas other inputs, such as nitrogen sources and quorum sensing molecules, exert limited effects on this PAMP. In particular, iron limitation triggers substantial changes in the cell wall that reduce β-glucan exposure. This correlates with reduced phagocytosis by macrophages and attenuated cytokine responses by peripheral blood mononuclear cells. Iron limitation-induced β-glucan masking depends on parallel signalling via the iron transceptor Ftr1 and the iron-responsive transcription factor Sef1, and the protein kinase a pathway. Our data reveal that C. albicans exploits a diverse range of specific host signals to trigger protective anticipatory responses against impending phagocytic attack and promote host colonisation.

Candida auris is a newly emerging pathogenic microbe, having been identified as a medically relevant fungus as recently as 2009. It is the most drug-resistant yeast species known to date and its emergence and population structure are unusual. Because of its recent emergence we are largely ignorant about fundamental aspects of its general biology, life cycle, and population dynamics. Here we report the karyotype variability of 26 C. auris strains representing the four main clades. We demonstrate that all strains are haploid and have a highly plastic karyotype containing five to seven chromosomes, which can undergo marked alterations within a short time-frame when the fungus is put under genotoxic, heat, or osmotic stress. No simple correlation was found between karyotype pattern, drug resistance, and clade affiliation indicating that karyotype heterogeneity is rapidly evolving. As with other Candida species, these marked karyotype differences between isolates are likely to have an important impact on pathogenic traits of C. auris.

Phagocytosis is a receptor-mediated process critical to innate immune clearance of pathogens. It proceeds in a regulated sequence of stages: (a) migration of phagocytes towards pathogens, (b) recognition of PAMPs and binding through PRRs, (c) engulfment and internalisation into phagosomes, (d) phagosome maturation, and (e) killing of pathogen or host cells. However, little is known about the role that individual receptors play in these discrete stages in the recognition of fungal cells. In a previous study, we found that dectin-2 deficiency impacted some but not all stages of macrophage-mediated phagocytosis of Candida glabrata. Because the C-type lectin receptor dectin-2 critically requires coupling to the FcRγ chain for signalling, we hypothesised that this coupling may be important for regulating phagocytosis of fungal cargo. We therefore examined how deficiency in FcRγ itself or two receptors to which it couples (dectin-2 and mincle) impacts phagocytosis of six fungal organisms representing three different fungal taxa. Our data show that deficiency in these proteins impairs murine bone marrow-derived macrophage migration, engulfment, and phagosome maturation, but not macrophage survival. Therefore, FcRγ engagement with selective C-type lectin receptors (CLRs) critically affects the spatio-temporal dynamics of fungal phagocytosis.

Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the Sporothrix genus. A new emerging species, Sporothrix brasiliensis, is related to cat-transmitted sporotrichosis and has severe clinical manifestations. The cell wall of pathogenic fungi is a unique structure and impacts directly on the host immune response. We reveal and compare the cell wall structures of Sporothrix schenckii and S. brasiliensis using high-pressure freezing electron microscopy to study the cell wall organization of both species. To analyze the components of the cell wall, we also used infrared and 13C and 1H NMR spectroscopy and the sugar composition was determined by quantitative high-performance anion-exchange chromatography. Our ultrastructural data revealed a bi-layered cell wall structure for both species, including an external microfibrillar layer and an inner electron-dense layer. The inner and outer layers of the S. brasiliensis cell wall were thicker than those of S. schenckii s. str. correlating with an increase in the chitin and rhamnose contents. Moreover, the outer microfibrillar layer of the S. brasiliensis cell wall had longer microfibrils interconnecting yeast cells. Distinct from those of other dimorphic fungi, the cell wall of Sporothrix spp. lacked α-glucan component. Interestingly, glycogen α-particles were identified in the cytoplasm close to the cell wall and the plasma membrane. The cell wall structure as well as the presence of glycogen α-particles varied over time during cell culture. The structural differences observed in the cell wall of these Sporothrix species seemed to impact its uptake by monocyte-derived human macrophages. The data presented here show a unique cell wall structure of S. brasiliensis and S. schenckii during the yeast parasitic phase. A new cell wall model for Sporothrix spp. is therefore proposed that suggests that these fungi molt sheets of intact cell wall layers. This observation may have significant effects on localized and disseminated immunopathology.

Microbes rarely exist in isolation, rather, they form intricate multi-species communities that colonize our bodies and inserted medical devices. However, the efficacy of antimicrobials is measured in clinical laboratories exclusively using microbial monocultures. Here, to determine how multi-species interactions mediate selection for resistance during antibiotic treatment, particularly following drug withdrawal, we study a laboratory community consisting of two microbial pathogens. Single-species dose responses are a poor predictor of community dynamics during treatment so, to better understand those dynamics, we introduce the concept of a dose-response mosaic, a multi-dimensional map that indicates how species' abundance is affected by changes in abiotic conditions. We study the dose-response mosaic of a two-species community with a 'Gene × Gene × Environment × Environment' ecological interaction whereby Candida glabrata, which is resistant to the antifungal drug fluconazole, competes for survival with Candida albicans, which is susceptible to fluconazole. The mosaic comprises several zones that delineate abiotic conditions where each species dominates. Zones are separated by loci of bifurcations and tipping points that identify what environmental changes can trigger the loss of either species. Observations of the laboratory communities corroborated theory, showing that changes in both antibiotic concentration and nutrient availability can push populations beyond tipping points, thus creating irreversible shifts in community composition from drug-sensitive to drug-resistant species. This has an important consequence: resistant species can increase in frequency even if an antibiotic is withdrawn because, unwittingly, a tipping point was passed during treatment.

In the version of this Article originally published, the following sentence was missing from the Acknowledgements: “R.E.B. is an EPSRC Healthcare Technologies Impact Fellow EP/N033671/1; I.G. is funded by ERC Consolidator grant 647292 MathModExp; A.J.P.B. N.A.R.G. and A.T. were funded by BBSRC grant BB/F00513X/1; K.H. I.G. S.N. and E.C. were funded by BBSRC grant BB/F005210/2.” This text has now been added.

With the Birmingham Annual Conference behind us, I can look back over the three Microbiology Society meetings of my Presidency and see the evolution of a brand and mechanism for delivery of scientific meetings that I believe will serve us well in the future.

Knowing the full set of essential genes for a given organism provides important information about ways to promote, and to limit, its growth and survival. For many non-model organisms, the lack of a stable haploid state and low transformation efficiencies impede the use of conventional approaches to generate a genome-wide comprehensive set of mutant strains and the identification of the genes essential for growth. Here we report on the isolation and utilization of a highly stable haploid derivative of the human pathogenic fungus Candida albicans, together with a modified heterologous transposon and machine learning (ML) analysis method, to predict the degree to which all of the open reading frames are required for growth under standard laboratory conditions. We identified 1,610 C. albicans essential genes, including 1,195 with high “essentiality confidence” scores, thereby increasing the number of essential genes (currently 66 in the Candida Genome Database) by >20-fold and providing an unbiased approach to determine the degree of confidence in the determination of essentiality. Among the genes essential in C. albicans were 602 genes also essential in the model budding and fission yeasts analyzed by both deletion and transposon mutagenesis. We also identified essential genes conserved among the four major human pathogens C. albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Histoplasma capsulatum and highlight those that lack homologs in humans and that thus could serve as potential targets for the design of antifungal therapies. IMPORTANCE Comprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power of in vivo transposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogen Candida albicans and to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicans genes that are essential for growth under standard laboratory conditions. Among these C. albicans essential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

Candida auris has recently emerged as an important, multidrug-resistant fungal pathogen of humans. Comparative studies indicate that despite high levels of genetic divergence, C. auris is as virulent as the most pathogenic member of the genus, Candida albicans However, key virulence attributes of C. albicans, such as morphogenetic switching, are not utilized by C. auris, indicating that this emerging pathogen employs alternative strategies to infect and colonize the host. An important trait required for the pathogenicity of many fungal pathogens is the ability to adapt to host-imposed stresses encountered during infection. Here, we investigated the relative resistance of C. auris and other pathogenic Candida species to physiologically relevant stresses and explored the role of the evolutionarily conserved Hog1 stress-activated protein kinase (SAPK) in promoting stress resistance and virulence. In comparison to C. albicans, C. auris is relatively resistant to hydrogen peroxide, cationic stress, and cell-wall-damaging agents. However, in contrast to other Candida species examined, C. auris was unable to grow in an anaerobic environment and was acutely sensitive to organic oxidative-stress-inducing agents. An analysis of C. aurishog1Δ cells revealed multiple roles for this SAPK in stress resistance, cell morphology, aggregation, and virulence. These data demonstrate that C. auris has a unique stress resistance profile compared to those of other pathogenic Candida species and that the Hog1 SAPK has pleiotropic roles that promote the virulence of this emerging pathogen.IMPORTANCE the rapid global emergence and resistance of Candidaauris to current antifungal drugs highlight the importance of understanding the virulence traits exploited by this human fungal pathogen to cause disease. Here, we characterize the stress resistance profile of C. auris and the role of the Hog1 stress-activated protein kinase (SAPK) in stress resistance and virulence. Our findings that C. auris is acutely sensitive to certain stresses may facilitate control measures to prevent persistent colonization in hospital settings. Furthermore, our observation that the Hog1 SAPK promotes C. auris virulence akin to that reported for many other pathogenic fungi indicates that antifungals targeting Hog1 signaling would be broad acting and effective, even on emerging drug-resistant pathogens.

Organisms must adapt to changes in oxygen tension if they are to exploit the energetic benefits of reducing oxygen while minimizing the potentially damaging effects of oxidation. Consequently, organisms in all eukaryotic kingdoms display robust adaptation to hypoxia (low oxygen levels). This is particularly important for fungal pathogens that colonize hypoxic niches in the host. We show that adaptation to hypoxia in the major fungal pathogen of humans Candida albicans includes changes in cell wall structure and reduced exposure, at the cell surface, of β-glucan, a key pathogen-associated molecular pattern (PAMP). This leads to reduced phagocytosis by murine bone marrow-derived macrophages and decreased production of IL-10, RANTES, and TNF-α by peripheral blood mononuclear cells, suggesting that hypoxia-induced β-glucan masking has a significant effect upon C. albicans-host interactions. We show that hypoxia-induced β-glucan masking is dependent upon both mitochondrial and cAMP-protein kinase a (PKA) signaling. The decrease in β-glucan exposure is blocked by mutations that affect mitochondrial functionality (goa1Δ and upc2Δ) or that decrease production of hydrogen peroxide in the inner membrane space (sod1Δ). Furthermore, β-glucan masking is enhanced by mutations that elevate mitochondrial reactive oxygen species (aox1Δ). The β-glucan masking defects displayed by goa1Δ and upc2Δ cells are suppressed by exogenous dibutyryl-cAMP. Also, mutations that inactivate cAMP synthesis (cyr1Δ) or PKA (tpk1Δ tpk2Δ) block the masking phenotype. Our data suggest that C. albicans responds to hypoxic niches by inducing β-glucan masking via a mitochondrial cAMP-PKA signaling pathway, thereby modulating local immune responses and promoting fungal colonization.IMPORTANCE Animal, plant, and fungal cells occupy environments that impose changes in oxygen tension. Consequently, many species have evolved mechanisms that permit robust adaptation to these changes. The fungal pathogen Candida albicans can colonize hypoxic (low oxygen) niches in its human host, such as the lower gastrointestinal tract and inflamed tissues, but to colonize its host, the fungus must also evade local immune defenses. We reveal, for the first time, a defined link between hypoxic adaptation and immune evasion in C. albicans As this pathogen adapts to hypoxia, it undergoes changes in cell wall structure that include masking of β-glucan at its cell surface, and it becomes better able to evade phagocytosis by innate immune cells. We also define the signaling mechanisms that mediate hypoxia-induced β-glucan masking, showing that they are dependent on mitochondrial signaling and the cAMP-protein kinase pathway. Therefore, hypoxia appears to trigger immune evasion in this fungal pathogen.

Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis.

The Wellcome Trust Strategic Award in Medical Mycology and Fungal Immunology is a unique investment that aimed to bolster capacity, training and research activity throughout the UK. This article summarises the rationale for collective collaboration of multiple institutions to achieve synergies and address a common medical problem.

The fungal cell wall is a critically important structure that represents a permeability barrier and protective shield. We probed Candida albicans and Cryptococcus neoformans with liposomes containing amphotericin B (AmBisome), with or without 15-nm colloidal gold particles. The liposomes have a diameter of 60 to 80 nm, and yet their mode of action requires them to penetrate the fungal cell wall to deliver amphotericin B to the cell membrane, where it binds to ergosterol. Surprisingly, using cryofixation techniques with electron microscopy, we observed that the liposomes remained intact during transit through the cell wall of both yeast species, even though the predicted porosity of the cell wall (pore size, ~5.8 nm) is theoretically too small to allow these liposomes to pass through intact. C. albicans mutants with altered cell wall thickness and composition were similar in both their in vitro AmBisome susceptibility and the ability of liposomes to penetrate the cell wall. AmBisome exposed to ergosterol-deficient C. albicans failed to penetrate beyond the mannoprotein-rich outer cell wall layer. Melanization of C. neoformans and the absence of amphotericin B in the liposomes were also associated with a significant reduction in liposome penetration. Therefore, AmBisome can reach cell membranes intact, implying that fungal cell wall viscoelastic properties are permissive to vesicular structures. The fact that AmBisome can transit through chemically diverse cell wall matrices when these liposomes are larger than the theoretical cell wall porosity suggests that the wall is capable of rapid remodeling, which may also be the mechanism for release of extracellular vesicles.IMPORTANCE AmBisome is a broad-spectrum fungicidal antifungal agent in which the hydrophobic polyene antibiotic amphotericin B is packaged within a 60- to 80-nm liposome. The mode of action involves perturbation of the fungal cell membrane by selectively binding to ergosterol, thereby disrupting membrane function. We report that the AmBisome liposome transits through the cell walls of both Candida albicans and Cryptococcus neoformans intact, despite the fact that the liposome is larger than the theoretical cell wall porosity. This implies that the cell wall has deformable, viscoelastic properties that are permissive to transwall vesicular traffic. These observations help explain the low toxicity of AmBisome, which can deliver its payload directly to the cell membrane without unloading the polyene in the cell wall. In addition, these findings suggest that extracellular vesicles may also be able to pass through the cell wall to deliver soluble and membrane-bound effectors and other molecules to the extracellular space.

Candida auris has recently emerged as a multi-drug resistant fungal pathogen that poses a serious global health threat, especially for patients in hospital intensive care units (ICUs). C. auris can colonize human skin and can spread by physical contact or contaminated surfaces and equipment. Here, we show that the mycoparasitic yeast Saccharomycopsis schoenii efficiently kills both sensitive and multi-drug resistant isolates of C. auris belonging to the same clade, as well as clinical isolates of other pathogenic species of the Candida genus suggesting novel approaches for biocontrol.

© 2018, the Author(s). Interactions between bacterial and fungal cells shape many polymicrobial communities. Bacteria elaborate diverse strategies to interact and compete with other organisms, including the deployment of protein secretion systems. The type VI secretion system (T6SS) delivers toxic effector proteins into host eukaryotic cells and competitor bacterial cells, but, surprisingly, T6SS-delivered effectors targeting fungal cells have not been reported. Here we show that the ‘antibacterial’ T6SS of Serratia marcescens can act against fungal cells, including pathogenic Candida species, and identify the previously undescribed effector proteins responsible. These antifungal effectors, Tfe1 and Tfe2, have distinct impacts on the target cell, but both can ultimately cause fungal cell death. ‘In competition’ proteomics analysis revealed that T6SS-mediated delivery of Tfe2 disrupts nutrient uptake and amino acid metabolism in fungal cells, and leads to the induction of autophagy. Intoxication by Tfe1, in contrast, causes a loss of plasma membrane potential. Our findings extend the repertoire of the T6SS and suggest that antifungal T6SSs represent widespread and important determinants of the outcome of bacterial–fungal interactions.

Journal clubs are important mechanisms for teaching how to approach the scientific literature critically and for disseminating findings. Papers from high-impact journals often dominate journal club selections, a practice that reinforces the unscientific emphasis of placing high value on publishing venue rather than scientific content and critical analysis of the publications. We suggest improving journal clubs by including preprints rather than focusing completely on published papers. This change in practice might benefit the scientific enterprise in numerous ways, including by providing direct criticisms to preprint authors before publication, deemphasizing publishing venue, teaching students the art of reviewing papers, and making journal clubs more current by discussing unpublished data.

The opportunistic human fungal pathogen Candida albicans can cause a variety of diseases, ranging from superficial mucosal infections to life-threatening systemic infections. Phagocytic cells of the innate immune response, such as neutrophils and macrophages, are important first-line responders to an infection and generate reactive oxygen and nitrogen species as part of their protective antimicrobial response. During an infection, host cells generate nitric oxide through the enzyme inducible nitric oxide synthase (iNOS) to kill the invading pathogen. Inside the phagocyte, iNOS competes with the enzyme arginase-1 for a common substrate, the amino acidL-arginine. Several pathogenic species, including bacteria and parasitic protozoans, actively modulate the production of nitric oxide by inducing their own arginases or the host’s arginase activity to prevent the conversion ofL-arginine to nitric oxide. We report here that C. albicans blocks nitric oxide production in human-monocyte-derived macrophages by induction of host arginase activity. We further determined that purified chitin (a fungal cell wall polysaccharide) and increased chitin exposure at the fungal cell wall surface induces this host arginase activity. Blocking the C. albicans-induced arginase activity with the arginase-specific substrate inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) or the chitinase inhibitor bisdionin F restored nitric oxide production and increased the efficiency of fungal killing. Moreover, we determined that C. albicans influences macrophage polarization from a classically activated phenotype toward an alternatively activated phenotype, thereby reducing antimicrobial functions and mediating fungal survival. Therefore, C. albicans modulatesL-arginine metabolism in macrophages during an infection, potentiating its own survival. IMPORTANCE the availability and metabolism of amino acids are increasingly recognized as crucial regulators of immune functions. In acute infections, the conversion of the “conditionally essential” amino acid L-arginine by the inducible nitric oxide synthase to nitric oxide is a resistance factor that is produced by the host to fight pathogens. Manipulation of these host defense mechanisms by the pathogen can be key to successful host invasion. We show here that the human opportunistic fungal pathogen Candida albicans influences L-arginine availability for nitric oxide production by induction of the substrate-competing host enzyme arginase-1. This led to a reduced production of nitric oxide and, moreover, reduced eradication of the fungus by human macrophages. We demonstrate that blocking of host arginase-1 activity restored nitric oxide production and increased the killing potential of macrophages. These results highlight the therapeutic potential of L-arginine metabolism in fungal diseases.

Candida albicans is a human opportunist pathogen that can grow as yeast, pseudohyphae, or true hyphae in vitro and in vivo, depending on environmental conditions. Reversible cellular morphogenesis is an important virulence factor that facilitates invasion of host tissues, escape from phagocytes, and dissemination in the blood stream. The innate immune system is the first line of defense against C. albicans infections and is influenced by recognition of wall components that vary in composition in different morphological forms. However, the relationship between cellular morphogenesis and immune recognition of this fungus is not fully understood. We therefore studied various vegetative cell types of C. albicans, singly and in combination, to assess the consequences of cellular morphogenesis on selected immune cytokine outputs from human monocytes. Hyphae stimulated proportionally lower levels of certain cytokines from monocytes per unit of cell surface area than yeast cells, but did not suppress cytokine response when copresented with yeast cells. Pseudohyphal cells induced intermediate cytokine responses. Yeast monomorphic mutants had elevated cytokine responses under conditions that otherwise supported filamentous growth and mutants of yeast and hyphal cells that were defective in cell wall mannosylation or lacking certain hypha-specific cell wall proteins could variably unmask or deplete the surface of immunostimulatory ligands. These observations underline the critical importance of C. albicans morphology and morphology-associated changes in the cell wall composition that affect both immune recognition and pathogenesis.

Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.

Candida albicans is an opportunistic fungal pathogen that infects immunocompromised patients. Infection control requires phagocytosis by innate immune cells, including macrophages. Migration towards, and subsequent recognition of, C. albicans fungal cell wall components by macrophages is critical for phagocytosis. Using live-cell imaging of phagocytosis, the macrophage cell line J774.1 showed enhanced movement in response to C. albicans cell wall mutants, particularly during the first 30 min, irrespective of the infection ratio. However, phagocyte migration was reduced up to 2-fold within a C. albicans biofilm compared to planktonic fungal cells. Biofilms formed from C. albicans glycosylation mutant cells also inhibited macrophage migration to a similar extent as wildtype Candida biofilms, suggesting that the physical structure of the biofilm, rather than polysaccharide matrix composition, may hamper phagocyte migration. These data illustrate differential macrophage migratory capacities, dependent upon the form of C. albicans encountered. Impaired migration of macrophages within a C. albicans biofilm may contribute to the recalcitrant nature of clinical infections in which biofilm formation occurs.

Candida albicans is normally a harmless commensal of human beings, but it can cause superficial infections of the mucosa (oral/vaginal thrush) in healthy individuals and (rarely) infections of the skin or nails. It can also become invasive, causing life-threatening systemic and bloodstream infections in immunocompromised hosts, where the mortality rate can be as high as 50 %. It is the most common cause of serious fungal infection and is a common cause of nosocomial infections in hospitals. Some strains have been recognized that are resistant to azoles or echinocandins, which are the first-line antifungals for treatment of C. albicans infections.

Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to MNN4, C. albicans has a MNN4-like gene family composed of seven other homologous members that have no known function. Here, using the classical mini-Ura-blaster approach and the new gene knockout CRISPR-Cas9 system for gene disruption, we generated mutants lacking single and multiple genes of the MNN4 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, MNN42 was also required for full protein phosphomannosylation. The reintroduction of MNN41, MNN42, MNN46, or MNN47 in a genetic background lacking MNN4 partially restored the phenotype associated with the mnn4Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of MNN4 over other genes could be due to its relative abundance within the cell. We observed that additional copies of alleles number of any of the other family members, with the exception of MNN46, restored the phosphomannan content in cells lacking both MNT3 and MNT5. We, therefore, suggest that phosphomannosylation is achieved by three groups of proteins: [i] enzymes solely activated by Mnn4, [ii] enzymes activated by the dual action of Mnn4 and any of the products of other MNN4-like genes, with exception of MNN46, and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the MNN4-like genes have the potential to functionally redundant with Mnn4, they apparently do not play a major role in cell wall mannosylation under most in vitro growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose C. albicans cells.

The molecular composition of the cell wall is critical for the biology and ecology of each fungal species. Fungal walls are composed of matrix components that are embedded and linked to scaffolds of fibrous load-bearing polysaccharides. Most of the major cell wall components of fungal pathogens are not represented in humans, other mammals, or plants, and therefore the immune systems of animals and plants have evolved to recognize many of the conserved elements of fungal walls. For similar reasons the enzymes that assemble fungal cell wall components are excellent targets for antifungal chemotherapies and fungicides. However, for fungal pathogens, the cell wall is often disguised since key signature molecules for immune recognition are sometimes masked by immunologically inert molecules. Cell wall damage leads to the activation of sophisticated fail-safe mechanisms that shore up and repair walls to avoid catastrophic breaching of the integrity of the surface. The frontiers of research on fungal cell walls are moving from a descriptive phase defining the underlying genes and component parts of fungal walls to more dynamic analyses of how the various components are assembled, cross-linked, and modified in response to environmental signals. This review therefore discusses recent advances in research investigating the composition, synthesis, and regulation of cell walls and how the cell wall is targeted by immune recognition systems and the design of antifungal diagnostics and therapeutics.

Pathogenic microorganisms often face acute micronutrient limitation during infection due to the action of host-mediated nutritional immunity. The human fungal pathogen Candida albicans is polymorphic and its morphological plasticity is one of its most widely recognized pathogenicity attributes. Here we investigated the effect of zinc, iron, manganese, and copper limitation on C. albicans morphology. Restriction of zinc specifically resulted in the formation of enlarged, spherical yeasts, a phenotype which we term Goliath cells. This cellular response to zinc restriction was conserved in C. albicans, C. dubliniensis and C. tropicalis, but not in C. parapsilosis, C. lusitaniae or Debaryomyces hansenii, suggesting that it may have emerged in the last common ancestor of these related pathogenic species. Cell wall analysis revealed proportionally more chitin exposure on the Goliath cell surface. Importantly, these cells were hyper-adherent, suggesting a possible role in pathogenicity. Interestingly, the zincophore-encoding gene PRA1 was expressed by Goliath cells in zinc limited media and lack of Pra1 inhibited both cellular enlargement and adhesion. Goliath cells represent a further layer of Candida phenotypic plasticity.

Candida albicans is a commensal coloniser of most people and a pathogen of the immunocompromised or patients in which barriers that prevent dissemination have been disrupted. Both the commensal and pathogenic states involve regulation and adaptation to the host microenvironment. The pathogenic potential can be downregulated to sustain commensalism or upregulated to damage host tissue and avoid and subvert immune surveillance. In either case it seems as though the cell biology of this fungus has evolved to enable the establishment of different types of relationships with the human host. Here we summarise latest advances in the analysis of mechanisms that enable C. albicans to occupy different body sites whilst avoiding being eliminated by the sentinel activities of the human immune system.

Chronic mucocutaneous candidiasis (CMC) is a primary immunodeficiency disorder characterised by susceptibility to chronic Candida and fungal dermatophyte infections of the skin, nails and mucous membranes. Molecular epidemiology studies of CMC infection are limited in number and scope and it is not clear whether single or multiple strains inducing CMC persist stably or are exchanged and replaced. We subjected 42 C. albicans individual single colony isolates from 6 unrelated CMC patients to multilocus sequence typing (MLST). Multiple colonies were typed from swabs taken from multiple body sites across multiple time points over a 17-month period. Among isolates from each individual patient, our data show clonal and persistent diploid sequence types (DSTs) that were stable over time, identical between multiple infection sites and exhibit azole resistant phenotypes. No shared origin or common source of infection was identified among isolates from these patients. Additionally, we performed C. albicans MLST SNP genotype frequency analysis to identify signatures of past loss of heterozygosity (LOH) events among persistent and azole resistant isolates retrieved from patients with autoimmune disorders including CMC.

Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies.

As the summer months arrive and the academic terms have been wound up, more time may be available for us to complete manuscripts and grant applications. For Microbiology Society staff, Executive Officers and Committee members this is also a period of intense planning for the future. We are looking forward to seeing our plans from last year transform into action in the coming months. For example the Early Career Microbiologists' Forum will shortly be formed.

Pathogenic and non-pathogenic fungi synthesize glycosphingolipids, which have a crucial role in growth and viability. Glycosphingolipids also contribute to fungal-associated pathogenesis. The opportunistic yeast pathogen Candida albicans synthesizes phospholipomannan (PLM), which is a glycosphingolipid of the mannosylinositol phosphorylceramide family. Through its lipid and glycan moieties, PLM contributes to the initial recognition of the yeast, causing immune system disorder and persistent fungal disease through activation of host signaling pathways. The lipid moiety of PLM activates the deregulation signaling pathway involved in yeast phagocytosis whereas its glycan moiety, composed of β-1,2 mannosides (β-Mans), participates to inflammatory processes through a mechanism involving Galectin-3. Biosynthesis of PLM β-Mans involves two β-1,2 mannosyltransferases (Bmts) that initiate (Bmt5) and elongate (Bmt6) the glycan chains. After generation of double bmtsΔ mutants, we show that Bmt5 has redundant activity with Bmt2, which can replace Bmt5 in bmt5Δ mutant. We also report that PLM is located in the inner layer of the yeast cell wall. PLM seems to be not essential for systemic infection of the yeast. However, defect of PLM β-mannosylation increases resistance of C. albicans to inhibitors of β-glucans and chitin synthesis, highlighting a role of PLM in cell wall homeostasis.

The surveillance and elimination of fungal pathogens rely heavily on the sentinel behaviour of phagocytic cells of the innate immune system, especially macrophages and neutrophils. The efficiency by which these cells recognize, uptake and kill fungal pathogens depends on the size, shape and composition of the fungal cells and the success or failure of various fungal mechanisms of immune evasion. In this Review, we describe how fungi, particularly Candida albicans, interact with phagocytic cells and discuss the many factors that contribute to fungal immune evasion and prevent host elimination of these pathogenic microorganisms.

As they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of β-glucan, a key pathogen-associated molecular pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in the carbon source. Exposure to lactate induces β-glucan masking in C. albicans via a signalling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other well-characterized pathways. In response to lactate, these regulators control the expression of cell-wall-related genes that contribute to β-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system.

Fungi cause more than a billion skin infections, more than 100 million mucosal infections, 10 million serious allergies and more than a million deaths each year. Global mortality owing to fungal infections is greater than for malaria and breast cancer and is equivalent to that owing to tuberculosis (TB) and HIV. These statistics evidence fungal infections as a major threat to human health and a major burden to healthcare budgets worldwide. Those patients who are at greatest risk of life-threatening fungal infections include those who have weakened immunity or have suffered trauma or other predisposing infections such as HIV. To address these global threats to human health, more research is urgently needed to understand the immunopathology of fungal disease and human disease susceptibility in order to augment the advances being made in fungal diagnostics and drug development. Here, we highlight some recent advances in basic research in medical mycology and fungal immunology that are beginning to inform clinical decisions and options for personalized medicine, vaccine development and adjunct immunotherapies.

Emerging infections caused by fungi have become a widely recognized global phenomenon. Their notoriety stems from their causing plagues and famines, driving species extinctions, and the difficulty in treating human mycoses alongside the increase of their resistance to antifungal drugs. This special issue comprises a collection of articles resulting from a Royal Society discussion meeting examining why pathogenic fungi are causing more disease now than they did in the past, and how we can tackle this rapidly emerging threat to the health of plants and animals worldwide.
. This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’.

Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is “Crabtree positive”, displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for yeasts in environmental niches, the more flexible carbon assimilation strategies offered by Crabtree negativity enhance the ability of yeasts to colonize and infect the mammalian host.

Despite the fact that Candida albicans is an important human fungal pathogen and Dectin-2 is a major pattern recognition receptor for fungi, our knowledge regarding the role of Dectin-2 for the host defense against disseminated candidiasis is limited. Dectin-2 deficient (Dectin-2-/-) mice were more susceptible to systemic candidiasis, and the susceptibility was mirrored by an elevated fungal load in the kidneys that correlated with the presence of large inflammatory foci. Phagocytosis of Candida by the macrophages lacking the Dectin-2 receptor was moderately decreased, while production of most of the macrophage-derived cytokines from Dectin-2-/- mice with systemic candidiasis was decreased. No striking differences among several Candida mutants defective in mannans could be detected between naïve wild-type and Dectin-2-/- mice, apart from the β-mannan-deficient bmt1Δ/bmt2Δ/bmt5Δ triple mutant, suggesting that β-mannan may partially mask α-mannan detection, which is the major fungal structure recognized by Dectin-2. Deciphering the mechanisms responsible for host defense against the majority of C. albicans strains represents an important step in understanding the pathophysiology of systemic candidiasis, which might lead to the development of novel immunotherapeutic strategies.

Chitin represents one of the most important components of the fungal cell wall. The multiplicity of chitin synthase (Chs) enzymes found in filamentous fungi underlines the importance of chitin in these organisms. Among this group of fungal enzymes, two classes, V and VII, are armed with myosin motors, constituting the MMD-Chs (Myosin Motor Domain - Chitin synthases) that are found in filamentous fungi and are absent in most yeast species. These enzymes play a critical role in promoting the synthesis of chitin at the hyphal tip, thus influencing fungal growth and the architecture of fungal infection structures. Other processes in which these enzymes are important are in osmo- and H2O2-tolerance, the ability to grow at 37 °C and in conidiogenesis. This review is focussed on the classification, structure and function of these enzymes describing the fundamental role of these enzymes in the ability of filamentous fungi to infect plants and their possible involvement in infections of animals. Moreover, data obtained with deletant mutants of this family of proteins indicates that they have potential as targets for novel antifungals.

The ability of Candida albicans to cause disease is associated with its capacity to undergo morphological transition between yeast and filamentous forms, but the role of morphology in colonization and dissemination from the gastrointestinal (GI) tract remains poorly defined. To explore this, we made use of wild-type and morphological mutants of C.albicans in an established model of GI tract colonization, induced following antibiotic treatment of mice. Our data reveal that GI tract colonization favours the yeast form of C.albicans, that there is constitutive low level systemic dissemination in colonized mice that occurs irrespective of fungal morphology, and that colonization is not controlled by Th17 immunity in otherwise immunocompetent animals. These data provide new insights into the mechanisms of pathogenesis and commensalism of C.albicans, and have implications for our understanding of human disease. Candida albicans is a commensal of the human gastrointestinal (GI) tract but can also spread from this site to cause systemic disease following immune perturbation. Here, using morphologically-locked strains we show that although the yeast form is favoured in the GI tract, both the yeast and hyphal forms can disseminate from this site to distal tissues in healthy animals. Finally, we show that Th17 immunity has no role in fungal colonisation or dissemination from the GI tract.

Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca2+-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2δ. chs8δ mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses.

The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. IMPORTANCE the C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock.

The major fungal pathogen of humans, Candida albicans, is exposed to reactive nitrogen and oxygen species following phagocytosis by host immune cells. In response to these toxins, this fungus activates potent anti-stress responses that include scavenging of reactive nitrosative and oxidative species via the glutathione system. Here we examine the differential roles of two glutathione recycling enzymes in redox homeostasis, stress adaptation and virulence in C. albicans: glutathione reductase (Glr1) and the S-nitrosoglutathione reductase (GSNOR), Fdh3. We show that the NADPH-dependent Glr1 recycles GSSG to GSH, is induced in response to oxidative stress and is required for resistance to macrophage killing. GLR1 deletion increases the sensitivity of C. albicans cells to H2O2, but not to formaldehyde or NO. In contrast, Fdh3 detoxifies GSNO to GSSG and NH3, and FDH3 inactivation delays NO adaptation and increases NO sensitivity. C. albicans fdh3Δ cells are also sensitive to formaldehyde, suggesting that Fdh3 also contributes to formaldehyde detoxification. FDH3 is induced in response to nitrosative, oxidative and formaldehyde stress, and fdh3Δ cells are more sensitive to killing by macrophages. Both Glr1 and Fdh3 contribute to virulence in the Galleria mellonella and mouse models of systemic infection. We conclude that Glr1 and Fdh3 play differential roles during the adaptation of C. albicans cells to oxidative, nitrosative and formaldehyde stress, and hence during the colonisation of the host. Our findings emphasise the importance of the glutathione system and the maintenance of intracellular redox homeostasis in this major pathogen.

The major fungal pathogen of humans, Candida albicans, mounts robust responses to oxidative stress that are critical for its virulence. These responses counteract the reactive oxygen species (ROS) that are generated by host immune cells in an attempt to kill the invading fungus. Knowledge of the dynamical processes that instigate C. albicans oxidative stress responses is required for a proper understanding of fungus-host interactions. Therefore, we have adopted an interdisciplinary approach to explore the dynamical responses of C. albicans to hydrogen peroxide (H2O2). Our deterministic mathematical model integrates two major oxidative stress signalling pathways (Cap1 and Hog1 pathways) with the three major antioxidant systems (catalase, glutathione and thioredoxin systems) and the pentose phosphate pathway, which provides reducing equivalents required for oxidative stress adaptation. The model encapsulates existing knowledge of these systems with new genomic, proteomic, transcriptomic, molecular and cellular datasets. Our integrative approach predicts the existence of alternative states for the key regulators Cap1 and Hog1, thereby suggesting novel regulatory behaviours during oxidative stress. The model reproduces both existing and new experimental observations under a variety of scenarios. Time- and dose-dependent predictions of the oxidative stress responses for both wild type and mutant cells have highlighted the different temporal contributions of the various antioxidant systems during oxidative stress adaptation, indicating that catalase plays a critical role immediately following stress imposition. This is the first model to encapsulate the dynamics of the transcriptional response alongside the redox kinetics of the major antioxidant systems during H2O2 stress in C. albicans.

The theoretical physicist and Nobel laureate Richard Feynman outlined in his 1959 lecture, “There’s plenty of room at the bottom”, the enormous possibility of producing and visualising things at smaller scales. The advent of advanced scanning and transmission electron microscopy and high-resolution microscopy has begun to open the door to visualise host-pathogen interactions at smaller scales, and spinning disc confocal and two-photon microscopy has improved our ability to study these events in real time in three dimensions. The aim of this review is to illustrate some of the advances in understanding host-fungal interactions that have been made in recent years in particular those relating to the interactions of live fungal pathogens with phagocytes. Dynamic imaging of host-pathogen interactions has recently revealed novel detail and unsuspected mechanistic insights, facilitating the dissection of the phagocytic process into its component parts. Here, we will highlight advances in our knowledge of host-fungal pathogen interactions, including the specific effects of fungal cell viability, cell wall composition and morphogenesis on the phagocytic process and try to define the relative contributions of neutrophils and macrophages to the clearance of fungal pathogens in vitro and the infected host.

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogen Candida albicans undergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated by C. albicans hyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment of C. albicans cells. Phagosomes containing live C. albicans cells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing by C. albicans. Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

The yeast exocyst is a multiprotein complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) which orchestrates trafficking of exocytic vesicles to specific docking sites on the plasma membrane during polarized secretion. To study SEC6 function in Candida albicans, we generated a conditional mutant strain in which SEC6 was placed under the control of a tetracycline-regulated promoter. In the repressed state, the tetR-SEC6 mutant strain (denoted tSEC6) was viable for up to 27 h; thus, all phenotypic analyses were performed at 24 h or earlier. Strain tSEC6 under repressing conditions had readily apparent defects in cytokinesis and endocytosis and accumulated both post-Golgi apparatus secretory vesicles and structures suggestive of late endosomes. Strain tSEC6 was markedly defective in secretion of aspartyl proteases and lipases as well as filamentation under repressing conditions. Lack of SEC6 expression resulted in markedly reduced lateral hyphal branching, which requires the establishment of a new axis of polarized secretion. Aberrant localization of chitin at the septum and increased resistance to zymolyase activity were observed, suggesting that C. albicans Sec6 plays an important role in mediating trafficking and delivery of cell wall components. The tSEC6 mutant was also markedly defective in macrophage killing, indicating a role of SEC6 in C. albicans virulence. Taken together, these studies indicate that the late secretory protein Sec6 is required for polarized secretion, hyphal morphogenesis, and the pathogenesis of C. albicans.

The contribution of fungal infections to the morbidity and mortality of HIV-infected individuals is largely unrecognized. A recent meeting highlighted several priorities that need to be urgently addressed, including improved epidemiological surveillance, increased availability of existing diagnostics and drugs, more training in the field of medical mycology, and better funding for research and provision of treatment, particularly in developing countries.

Candida albicans is a major life-threatening human fungal pathogen in the immunocompromised host. Host defense against systemic Candida infection relies heavily on the capacity of professional phagocytes of the innate immune system to ingest and destroy fungal cells. A number of pathogens, including C. albicans, have evolved mechanisms that attenuate the efficiency of phagosome-mediated inactivation, promoting their survival and replication within the host. Here we visualize hostpathogen interactions using live-cell imaging and show that viable, but not heat- or UV-killed C. albicans cells profoundly delay phagosome maturation in macrophage cell lines and primary macrophages. The ability of C. albicans to delay phagosome maturation is dependent on cell wall composition and fungal morphology. Loss of cell wall O-mannan is associated with enhanced acquisition of phagosome maturation markers, distinct changes in Rab GTPase acquisition by the maturing phagosome, impaired hyphal growth within macrophage phagosomes, profound changes in macrophage actin dynamics, and ultimately a reduced ability of fungal cells to escape from macrophage phagosomes. The loss of cell wall O-mannan leads to exposure of β-glucan in the inner cell wall, facilitating recognition by Dectin-1, which is associated with enhanced phagosome maturation.

Polarized cells reorient their direction of growth in response toenvironmental cues. In the fungus Candida albicans, the Rho-familysmall GTPase, Cdc42, is essential for polarized hyphal growth andCa2+ influx is required for the tropic responses of hyphae to environmental cues, but the regulatory link between these systems isunclear. In this study, the interaction between Ca2+ influx and Cdc42polarity-complex dynamics was investigated using hyphal galvano-tropic and thigmotropic responses as reporter systems. During polarity establishment in an applied electric field, cathodal emergenceof hyphae was lost when either of the two Cdc42 apical recyclingpathways was disrupted by deletion of Rdi1, a guanine nucleotidedissociation inhibitor, or Bnr1, a formin, but was completely restored by extracellular Ca2+. Loss of the Cdc42 GTPase activatingproteins, Rga2 and Bem3, also abolished cathodal polarization, butthis was not rescued by Ca2+. Expression of GTP-locked Cdc42 reversed the polarity of hypha emergence from cathodal to anodal, aneffect augmented by Ca2+. The cathodal directional cue thereforerequires Cdc42 GTP hydrolysis. Ca2+ influx amplifies Cdc42-mediateddirectional growth signals, in part by augmenting Cdc42 apical trafficking. The Ca2+-binding EF-hand motif in Cdc24, the Cdc42 activator, was essential for growth in yeast cells but not in establishedhyphae. The Cdc24 EF-hand motif is therefore essential for polarityestablishment but not for polarity maintenance.

Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3+ regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3+ Treg-cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4+Foxp3+ population that correlated positively with fungal burden. Depletion from Foxp3hCD2 reporter mice in vivo confirmed that Foxp3+ cells exacerbated fungal burden and inflammatory renal disease. The CD4+Foxp3+ population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg-cell subset, together with conversion of Foxp3- cells to the induced Treg-cell form, and to a cell type sharing effector Th17-cell characteristics, expressing ROR-γt, and secreting IL-17A. The expanded Foxp3+ T cells inhibited Th1 and Th2 responses, but enhanced Th17-cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17-cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3+ T cells promotes Th17-cell responses that drive pathology. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chitin is an essential structural polysaccharide of fungal pathogens and parasites, but its role in human immune responses remains largely unknown. It is the second most abundant polysaccharide in nature after cellulose and its derivatives today are widely used for medical and industrial purposes. We analysed the immunological properties of purified chitin particles derived from the opportunistic human fungal pathogen Candida albicans, which led to the selective secretion of the anti-inflammatory cytokine IL-10. We identified NOD2, TLR9 and the mannose receptor as essential fungal chitin-recognition receptors for the induction of this response. Chitin reduced LPS-induced inflammation in vivo and may therefore contribute to the resolution of the immune response once the pathogen has been defeated. Fungal chitin also induced eosinophilia in vivo, underpinning its ability to induce asthma. Polymorphisms in the identified chitin receptors, NOD2 and TLR9, predispose individuals to inflammatory conditions and dysregulated expression of chitinases and chitinase-like binding proteins, whose activity is essential to generate IL-10-inducing fungal chitin particles in vitro, have also been linked to inflammatory conditions and asthma. Chitin recognition is therefore critical for immune homeostasis and is likely to have a significant role in infectious and allergic disease.Authors Summary:Chitin is the second most abundant polysaccharide in nature after cellulose and an essential component of the cell wall of all fungal pathogens. The discovery of human chitinases and chitinase-like binding proteins indicates that fungal chitin is recognised by cells of the human immune system, shaping the immune response towards the invading pathogen. We show that three immune cell receptors- the mannose receptor, NOD2 and TLR9 recognise chitin and act together to mediate an anti-inflammatory response via secretion of the cytokine IL-10. This mechanism may prevent inflammation-based damage during fungal infection and restore immune balance after an infection has been cleared. By increasing the chitin content in the cell wall pathogenic fungi may influence the immune system in their favour, by down-regulating protective inflammatory immune responses. Furthermore, gene mutations and dysregulated enzyme activity in the described chitin recognition pathway are implicated in inflammatory conditions such as Crohn's Disease and asthma, highlighting the importance of the discovered mechanism in human health. © 2014 Wagener et al.

Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity.

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host. © 2014 Scháfer et al.

Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro. We also show that the high fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as their pharmacological attenuation with apocynin or glibenclamide reduced phagocytic potency to a similar extent. The mechanistic basis for the extreme potency of combinatorial cationic plus oxidative stress--a phenomenon we term stress pathway interference--lies with the inhibition of hydrogen peroxide detoxification by the cations. In C. albicans this causes the intracellular accumulation of ROS, the inhibition of Cap1 (a transcriptional activator that normally drives the transcriptional response to oxidative stress), and altered readouts of the stress-activated protein kinase Hog1. This leads to a loss of oxidative and cationic stress transcriptional outputs, a precipitous collapse in stress adaptation, and cell death. This stress pathway interference can be suppressed by ectopic catalase (Cat1) expression, which inhibits the intracellular accumulation of ROS and the synergistic killing of C. albicans cells by combinatorial cationic plus oxidative stress. Stress pathway interference represents a powerful fungicidal mechanism employed by the host that suggests novel approaches to potentiate antifungal therapy. Importance: the immune system combats infection via phagocytic cells that recognize and kill pathogenic microbes. Human neutrophils combat Candida infections by killing this fungus with a potent mix of chemicals that includes reactive oxygen species (ROS) and cations. Yet, Candida albicans is relatively resistant to these stresses in vitro. We show that it is the combination of oxidative plus cationic stresses that kills yeasts so effectively, and we define the molecular mechanisms that underlie this potency. Cations inhibit catalase. This leads to the accumulation of intracellular ROS and inhibits the transcription factor Cap1, which is critical for the oxidative stress response in C. albicans. This triggers a dramatic collapse in fungal stress adaptation and cell death. Blocking either the oxidative burst or cationic fluxes in human neutrophils significantly reduces their ability to kill this fungal pathogen, indicating that combinatorial stress is pivotal to immune surveillance.

Metabolism is integral to the pathogenicity of Candida albicans, a major fungal pathogen of humans. As well as providing the platform for nutrient assimilation and growth in diverse host niches, metabolic adaptation affects the susceptibility of C. albicans to host-imposed stresses and antifungal drugs, the expression of key virulence factors, and fungal vulnerability to innate immune defences. These effects, which are driven by complex regulatory networks linking metabolism, morphogenesis, stress adaptation, and cell wall remodelling, influence commensalism and infection. Therefore, current concepts of Candida-host interactions must be extended to include the impact of metabolic adaptation upon pathogenicity and immunogenicity.

The present work reports the effects of caspofungin, a β-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred adVantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting β-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any adVantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals unDer study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the β-glucan synthase inhibitor against this fungus. Copyright © 2014, American Society for Microbiology.

The soil-borne plant pathogen Fusarium oxysporum causes life-threatening invasive fusariosis in immunocompromised individuals. The mechanism of infection in mammalian hosts is largely unknown. In the present study we show that the symptoms of disseminated fusariosis caused by F. oxysporum in immunosuppressed mice are remarkably similar to those reported in humans. Distinct fungal structures were observed inside the host, depending on the infected organ. Invasive hyphae developed in the heart and kidney, causing massive colonization of the organs. By contrast, chlamydospore-like survival structures were found in lung, spleen and liver. Systemically infected mice also developed skin and eye infections, as well as thrombosis and necrosis in the tail. We further show that F. oxysporum can disseminate and persist in the organs of immunocompetent animals, and that these latent infections can lead to lethal systemic fusariosis if the host is later subjected to immunosuppressive treatment. © 2014 Schäfer et al.

Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90-100% efficiency via site-specific recombination between loxP sites. Ironically, the utility of this system was hampered by the extreme efficiency of Cre, which prevented the construction in Escherichia coli of stable disruption cassettes carrying a methionine-regulatable CaMET3p-cre gene flanked by loxP sites. Therefore, we have significantly enhanced this system by engineering new Clox cassettes that carry a synthetic, intron-containing cre gene. The Clox kit facilitates efficient transformation and marker recycling, thereby simplifying and accelerating the process of gene disruption in C. albicans. Indeed, homozygous mutants can be generated and their markers resolved within two weeks. The Clox kit facilitates strategies involving single marker recycling or multi-marker gene disruption. Furthermore, it includes the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting the manipulation of clinical isolates as well as genetically marked strains of C. albicans. The accelerated gene disruption strategies afforded by this new Clox system are likely to have a profound impact on the speed with which C. albicans pathobiology can be dissected. © 2014 Shahana et al.

Thigmotropism is the ability of an organism to respond to a topographical stimulus by altering its axis of growth. The thigmotropic response of the model fungus Neurospora crassa was quantified using microfabricated glass slides with ridges of defined height. We show that the polarity machinery at the hyphal tip plays a role in the thigmotropic response of N. crassa. Deletion of N. crassa genes encoding the formin, BNI-1, and the Rho-GTPase, CDC-42, an activator of BNI-1 in yeast, CDC-24, its guanine nucleotide exchange factor (GEF), and BEM-1, a scaffold protein in the same pathway, were all shown to significantly decrease the thigmotropic response. In contrast, deletion of genes encoding the cell end-marker protein, TEA-1, and KIP-1, the kinesin responsible for the localisation of TEA-1, significantly increased the thigmotropic response. These results suggest a mechanism of thigmotropism involving vesicle delivery to the hyphal tip via the actin cytoskeleton and microtubules. Neurospora crassa thigmotropic response differed subtly from that of Candida albicans where the stretch-activated calcium channel, Mid1, has been linked with thigmotropic behaviour. The MID-1 deficient mutant of N. crassa (δ. mid-1) and the effects of calcium depletion were examined here but no changein the thigmotropic response was observed. However, SPRAY, a putative calcium channel protein, was shown to be required for N. crassa thigmotropism. We propose that the thigmotropic response is a result of changes in the polarity machinery at the hyphal tip which are thought to be downstream effects of calcium signalling pathways triggered by mechanical stress at the tip. © 2014 the British Mycological Society.

Although Candida glabrata is an important pathogenic Candida species, relatively little is known about its innate immune recognition. Here, we explore the potential role of Dectin-2 for host defense against C. glabrata. Dectin-2-deficient (Dectin-2-/-) mice were found to be more susceptible to C. glabrata infections, showing a defective fungal clearance in kidneys but not in the liver. The increased susceptibility to infection was accompanied by lower production of T helper 1 (Th1) and Th17-derived cytokines by splenocytes of Dectin-2-/- mice, while macrophage-derived cytokines were less affected. These defects were associated with a moderate yet significant decrease in phagocytosis of the fungus by the Dectin-2-/- macrophages and neutrophils. Neutrophils of Dectin-2-/- mice also displayed lower production of reactive oxygen species (ROS) upon challenge with opsonized C. glabrata or C. albicans. This study suggests that Dectin-2 is important in host defense against C. glabrata and provides new insights into the host defense mechanisms against this important fungal pathogen. © 2014, American Society for Microbiology.

Upon priming with Candida albicans or with the fungal cell wall component β-glucan, monocytes respond with an increased cytokine production upon restimulation, a phenomenon termed "trained immunity." in contrast, the prestimulation of monocytes with lipopolysaccharide has long been known to induce tolerance. Because the vast majority of commensal microorganisms belong to bacterial or viral phyla, we sought to systematically investigate the functional reprogramming of monocytes induced by the stimulation of pattern recognition receptors (PRRs) with various bacterial or viral ligands. Monocytes were functionally programmed for either enhanced (training) or decreased (tolerance) cytokine production, depending on the type and concentration of ligand they encountered. The functional reprogramming of monocytes was also associated with cell shape, granulocity, and cell surface marker modifications. The training effect required p38- and Jun N-terminal protein kinase (JNK)-mediated mitogen-activated protein kinase (MAPK) signaling, with specific signaling patterns directing the functional fate of the cell. The long-term effects on the function of monocytes were mediated by epigenetic events, with both histone methylation and acetylation inhibitors blocking the training effects. In conclusion, our experiments identify the ability of monocytes to acquire adaptive characteristics after prior activation with a wide variety of ligands. Trained immunity and tolerance are two distinct and opposing functional programs induced by the specific microbial ligands engaging the monocytes. Copyright © 2014 Ifrim et al.

Candida albicans is a frequent pathogen of immunologically compromised individuals, but it is an even more common commensal of healthy humans, where it resides in the gut in a benign state. A new study shows that a specific commensal form of the fungus is induced in the gut through a developmental program that downregulates virulence factors and induces metabolic functions, enabling it to thrive on the nutrients that are available in the large intestine without damaging its host. © 2013 Nature America, Inc. All rights reserved.

An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection. IMPORTANCE Extensive work investigating fungal cell phagocytosis by macrophages and PMNs of the innate immune system has been carried out. These studies have been informative but have examined this phenomenon only when one phagocyte subset is present. The current study employed live-cell video microscopy to break down C. albicans phagocytosis into its component parts and examine the effect of a single phagocyte subset, versus a mixed phagocyte population, on these individual stages. Through this approach, we identified that the rate of fungal cell engulfment and rate of phagocyte killing altered significantly when both macrophages and PMNs were incubated in coculture with C. albicans compared to the rate of either phagocyte subset incubated alone with the fungus. This research highlights the significance of studying pathogen-host cell interactions with a combination of phagocytes in order to gain a greater understanding of the interactions that occur between cells of the host immune system in response to fungal invasion. © 2013 Rudkin et al.

In addition to the bio-guided investigation of the antifungal activity of Plinia cauliflora leaves against different Candida species, the major aim of the present study was the search for targets on the fungal cell. The most active antifungal fraction was purified by chromatography and characterized by NMR and mass spectrometry. The antifungal activity was evaluated against five Candida strains according to referenced guidelines. Cytotoxicity against fibroblast cells was determined. The likely targets of Candida albicans cells were assessed through interactions with ergosterol and cell wall composition, porosity and architecture. The chemical major component within the most active antifungal fraction of P. cauliflora leaves identified was the hydrolysable tannin casuarinin. The cytotoxic concentration was higher than the antifungal one. The first indication of plant target on cellular integrity was suggested by the antifungal activity ameliorated when using an osmotic support. The most important target for the tannin fraction studied was suggested by ultrastructural analysis of yeast cell walls revealing a denser mannan outer layer and wall porosity reduced. It is possible to imply that P. cauliflora targeted the C. albicans cell wall inducing some changes in the architecture, notably the outer glycoprotein layer, affecting the cell wall porosity without alteration of the polysaccharide or protein level. © 2013 by the authors.

The immune system is essential to maintain homeostasis with resident microbial populations, ensuring that the symbiotic host-microbial relationship is maintained. In parallel, commensal microbes significantly shape mammalian immunity at the host mucosal surface, as well as systemically. Candida albicans is an opportunistic pathogen that lives as a commensal on skin and mucosa of healthy individuals. Little is known about its capacity to modulate responses toward other microorganisms, such as colonizing bacteria (e.g. intestinal microorganisms). The aim of this study was to assess the cytokine production of PBMCs induced by commensal bacteria when these cells were primed by C. albicans. We show that C. albicans and β-1,3-glucan induce priming of human primary mononuclear cells and this leads to enhanced cytokine production upon in vitro stimulation with TLR ligands and bacterial commensals. This priming requires the β-1,3-glucan receptor dectin-1 and the noncanonical Raf-1 pathway. In addition, although purified mannans cannot solely mediate the priming, the presence of mannosyl residues in the cell wall of C. albicans is nevertheless required. In conclusion, C. albicans is able to modify cytokine responses to TLR ligands and colonizing bacteria, which is likely to impact the inflammatory reaction during mucosal diseases. Copyright © 2013 by the American Association of Immunologists, Inc.

In fungi, as with all walled organisms, cytokinesis followed by septation marks the end of the cell cycle and is essential for cell division and viability. For yeasts, the septal cross-wall comprises a ring and primary septal plate composed of chitin, and a secondary septum thickened with β(1,3)-glucan. In the human pathogen Candida albicans, chitin synthase enzyme Chs1 builds the primary septum that is surrounded by a chitin ring made by Chs3. Here we show that the lethal phenotype induced by repression of CHS1 was abrogated by stress-induced synthesis of alternative and novel septal types synthesized by other chitin synthase enzymes that have never before been implicated in septation. Chs2 and Chs8 formed a functional salvage septum, even in the absence of both Chs1 and Chs3. A second type of salvage septum formed by Chs2 in combination with Chs3 or Chs8 was proximally offset in the mother-bud neck. Chs3 alone or in combination with Chs8 formed a greatly thickened third type of salvage septum. Therefore, cell wall stress induced alternative forms of septation that rescued cell division in the absence of Chs1, demonstrating that fungi have previously unsuspected redundant strategies to enable septation and cell division to be maintained, even under potentially lethal environmental conditions. © 2013. Published by the Company of Biologists Ltd.

The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α+/+ than cPLA2α-/- macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α+/+ than cPLA2α-/- macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α+/+ and cPLA2α-/- macrophages (3 h) was compared by microarray. cPLA2α+/+ macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α-/- macrophages (≥2-fold, p

The β-glucan receptor Dectin-1 is a member of the C-type lectin family and functions as an innate pattern recognition receptor in antifungal immunity. In both mouse and man, Dectin-1 has been found to play an essential role in controlling infections with Candida albicans, a normally commensal fungus in man which can cause superficial mucocutaneous infections as well as life-threatening invasive diseases. Here, using in vivo models of infection, we show that the requirement for Dectin-1 in the control of systemic Candida albicans infections is fungal strain-specific; a phenotype that only becomes apparent during infection and cannot be recapitulated in vitro. Transcript analysis revealed that this differential requirement for Dectin-1 is due to variable adaptation of C. albicans strains in vivo, and that this results in substantial differences in the composition and nature of their cell walls. In particular, we established that differences in the levels of cell-wall chitin influence the role of Dectin-1, and that these effects can be modulated by antifungal drug treatment. Our results therefore provide substantial new insights into the interaction between C. albicans and the immune system and have significant implications for our understanding of susceptibility and treatment of human infections with this pathogen. © 2013 Marakalala et al.

The fungus Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this, little is known about the attributes that allow this organism to cause disease or its interaction with the host immune system. However, in common with other fungi, the cell wall of C. glabrata is the initial point of contact between the host and pathogen, and as such, it is likely to play an important role in mediating interactions and hence virulence. Here, we show both through genetic complementation and polysaccharide structural analyses that C. glabrata ANP1, MNN2, and MNN11 encode functional orthologues of the respective Saccharomyces cerevisiae mannosyltransferases. Furthermore, we show that deletion of the C. glabrata Anp1, Mnn2, and Mnn11 mannosyltransferases directly affects the structure of the fungal N-linked mannan, in line with their predicted functions, and this has implications for cell wall integrity and consequently virulence. C. glabrata anp1 and mnn2 mutants showed increased virulence, compared with wild-type (and mnn11) cells. This is in contrast to Candida albicans where inactivation of genes involved in mannan biosynthesis has usually been linked to an attenuation of virulence. In the long term, a better understanding of the attributes that allow C. glabrata to cause disease will provide insights that can be adopted for the development of novel therapeutic and diagnostic approaches.

Purpose: There is increasing evidence that immunologic mechanisms play a role in the pathogenesis of endometriosis. A high incidence of infection with Candida albicans in women with endometriosis has been reported. There is evidence to suggest that C. albicans may contribute to the pathogenesis of endometriosis possibly by modulating cytokine production. Methods: Endometrial tissue was obtained from eight women attending Aberdeen Royal Infirmary for laparoscopic sterilization or investigation of infertility (n=7 patients without endometriosis, n= 1 patient with endometriosis). Culture of endometrial cells with inactivated Candida/Candida product was performed. The endometrial tissue was processed using a number of different assays including Ki-67 cell proliferation assay, DELFIA Ki-67 assay, MTT cell viability assay, and BrdU incorporation proliferation assay. Results: the results from seven women without endometriosis showed that Candida product significantly (P

The echinocandin antifungal drugs inhibit synthesis of the major fungal cell wall polysaccharide β(1,3)-glucan. Echinocandins have good efficacy against Candida albicans but reduced activity against other Candida species, in particular Candida parapsilosis and Candida guilliermondii. Treatment of Candida albicans with a sub-MIC level of caspofungin has been reported to cause a compensatory increase in chitin content and to select for sporadic echinocandin-resistant FKS1 point mutants that also have elevated cell wall chitin. Here we show that elevated chitin in response to caspofungin is a common response in various Candida species. Activation of chitin synthesis was observed in isolates of C. albicans, Candida tropicalis, C. parapsilosis, and C. guilliermondii and in some isolates of Candida krusei in response to caspofungin treatment. However, Candida glabrata isolates demonstrated no exposure-induced change in chitin content. Furthermore, isolates of C. albicans, C. krusei, C. parapsilosis, and C. guilliermondii which were stimulated to have higher chitin levels via activation of the calcineurin and protein kinase C (PKC) signaling pathways had reduced susceptibility to caspofungin. Isolates containing point mutations in the FKS1 gene generally had higher chitin levels and did not demonstrate a further compensatory increase in chitin content in response to caspofungin treatment. These results highlight the potential of increased chitin synthesis as a potential mechanism of tolerance to caspofungin for the major pathogenic Candida species. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

The cell cycle is a sequence of biochemical events that are controlled by complex but robust molecular machinery. This enables cells to achieve accurate self-reproduction under a broad range of different conditions. Environmental changes are transmitted by molecular signalling networks, which coordinate their action with the cell cycle. The cell cycle process and its responses to environmental stresses arise from intertwined nonlinear interactions among large numbers of simpler components. Yet, understanding of how these pieces fit together into a coherent whole requires a systems biology approach. Here, we present a novel mathematical model that describes the influence of osmotic stress on the entire cell cycle of S. cerevisiae for the first time. Our model incorporates all recently known and several proposed interactions between the osmotic stress response pathway and the cell cycle. This model unveils the mechanisms that emerge as a consequence of the interaction between the cell cycle and stress response networks. Furthermore, it characterises the role of individual components. Moreover, it predicts different phenotypical responses for cells depending on the phase of cells at the onset of the stress. The key predictions of the model are: (i) exposure of cells to osmotic stress during the late S and the early G2/M phase can induce DNA re-replication before cell division occurs, (ii) cells stressed at the late G2/M phase display accelerated exit from mitosis and arrest in the next cell cycle, (iii) osmotic stress delays the G1-to-S and G2-to-M transitions in a dose dependent manner, whereas it accelerates the M-to-G1 transition independently of the stress dose and (iv) the Hog MAPK network compensates the role of the MEN network during cell division of MEN mutant cells. These model predictions are supported by independent experiments in S. cerevisiae and, moreover, have recently been observed in other eukaryotes. © 2013 Radmaneshfar et al.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells; and how engulfment leads to alteration of the cell cycle and behavior of macrophages. In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

Summary: the fungal cell wall is a dynamic organelle required for cell shape, protection against the environment and, in pathogenic species, recognition by the innate immune system. The outer layer of the cell wall is comprised of glycosylated mannoproteins with the majority of these post-translational modifications being the addition of O- and N-linked mannosides. These polysaccharides are exposed on the outer surface of the fungal cell wall and are, therefore, the first point of contact between the fungus and the host immune system. This review focuses on O- and N-linked mannan biosynthesis in the fungal pathogen Candida albicans and highlights new insights gained from the characterization of mannosylation mutants into the role of these cell wall components in host-fungus interactions. In addition, we discuss the use of fungal mannan as a diagnostic marker of fungal disease. © 2013 the Authors.

A large proportion of Candida albicans cell surface proteins are decorated post-translationally by glycosylation. Indeed N-glycosylation is critical for cell wall biogenesis in this major fungal pathogen and for its interactions with host cells. A detailed understanding of N-glycosylation will yield deeper insights into host-pathogen interactions. However, the analysis of N-glycosylation is extremely challenging because of the complexity and heterogeneity of these structures. Therefore, in an attempt to reduce this complexity and facilitate the analysis of N-glycosylation, we have developed new synthetic C. albicans reporters that carry a single N-linked glycosylation site derived from Saccharomyces cerevisiae Suc2. These glycosylation reporters, which carry C.albicans Hex1 or Sap2 signal sequences plus carboxy-terminal FLAG3 and His6 tags, were expressed in C.albicans from the ACT1 promoter. The reporter proteins were successfully secreted and hyperglycosylated by C.albicans cells, and their outer chain glycosylation was dependent on Och1 and Pmr1, which are required for N-mannan synthesis, but not on Mnt1 and Mnt2 which are only required for O-mannosylation. These reporters are useful tools for the experimental dissection of N-glycosylation and other related processes in C.albicans, such as secretion. © 2013 the Authors.

BACKGROUND: the Candida albicans cell wall is the first point of contact with the host, and its outer surface is heavily enriched in mannoproteins modified through the addition of N- and O-mannan. Previous work, using mutants with gross defects in glycosylation, has clearly identified the importance of mannan in the host-pathogen interaction, immune recognition and virulence. Here we report the first analysis of the MNN1 gene family, which contains six members predicted to act as α-1,3 mannosyltransferases in the terminal stages of glycosylation. FINDINGS: We generated single null mutants in all members of the C. albicans MNN1 gene family, and disruption of MNN14 led to both in vitro and in vivo defects. Null mutants in other members of the family demonstrated no phenotypic defects, suggesting that these members may display functional redundancy. The mnn14Δ null mutant displayed hypersensitivity to agents associated with cell wall and glycosylation defects, suggesting an altered cell wall structure. However, no gross changes in cell wall composition or N-glycosylation were identified in this mutant, although an extension of phosphomannan chain length was apparent. Although the cell wall defects associated with the mnn14Δ mutant were subtle, this mutant displayed a severe attenuation of virulence in a murine infection model. CONCLUSION: Mnn14 plays a distinct role from other members of the MNN1 family, demonstrating that specific N-glycan outer chain epitopes are required in the host-pathogen interaction and virulence.

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.

BACKGROUND: Saccharomyces cerevisiae senses hyperosmotic conditions via the HOG signaling network that activates the stress-activated protein kinase, Hog1, and modulates metabolic fluxes and gene expression to generate appropriate adaptive responses. The integral control mechanism by which Hog1 modulates glycerol production remains uncharacterized. An additional Hog1-independent mechanism retains intracellular glycerol for adaptation. Candida albicans also adapts to hyperosmolarity via a HOG signaling network. However, it remains unknown whether Hog1 exerts integral or proportional control over glycerol production in C. albicans. RESULTS: We combined modeling and experimental approaches to study osmotic stress responses in S. cerevisiae and C. albicans. We propose a simple ordinary differential equation (ODE) model that highlights the integral control that Hog1 exerts over glycerol biosynthesis in these species. If integral control arises from a separation of time scales (i.e. rapid HOG activation of glycerol production capacity which decays slowly under hyperosmotic conditions), then the model predicts that glycerol production rates elevate upon adaptation to a first stress and this makes the cell adapts faster to a second hyperosmotic stress. It appears as if the cell is able to remember the stress history that is longer than the timescale of signal transduction. This is termed the long-term stress memory. Our experimental data verify this. Like S. cerevisiae, C. albicans mimimizes glycerol efflux during adaptation to hyperosmolarity. Also, transient activation of intermediate kinases in the HOG pathway results in a short-term memory in the signaling pathway. This determines the amplitude of Hog1 phosphorylation under a periodic sequence of stress and non-stressed intervals. Our model suggests that the long-term memory also affects the way a cell responds to periodic stress conditions. Hence, during osmohomeostasis, short-term memory is dependent upon long-term memory. This is relevant in the context of fungal responses to dynamic and changing environments. CONCLUSIONS: Our experiments and modeling have provided an example of identifying integral control that arises from time-scale separation in different processes, which is an important functional module in various contexts.

The cell surface of Candida albicans is enriched with highly glycosylated mannoproteins that are involved in the interaction with host tissues. N- and O-glycosylation are post-translational modifications that initiate in the endoplasmic reticulum, and finalize in the Golgi. The KRE2/MNT1 family encode a set of multifunctional mannosyltransferases that participate in O-, N- and phosphomannosylation. In order to gain insights into the substrate specificities of these enzymes, recombinant forms of Mnt1, Mnt2, and Mnt5 were expressed in Pichia pastoris and the enzyme activities characterized. Mnt1 and Mnt2 showed a high specificity for α-methylmannoside and α1,2-mannobiose as acceptor substrates. Notably, they also used Saccharomyces cerevisiae O-mannans as acceptors and generated products with more than three mannose residues, suggesting than Mnt1 and Mnt2 could be the mannosyltransferases adding the fourth and fifth mannose residue to the O-mannans in C. albicans. Mnt5 only recognized α-methylmannoside as acceptor, suggesting that participates in the addition of the second mannose residues to the N-glycan outer chain. © 2012 Elsevier Inc.

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to inhibit effective destruction by host phagocytes. Using live cell video microscopy, we show here for the first time that C. albicans inhibits cell division in macrophages undergoing mitosis. Inhibition of macrophage cell division is dependent on the ability of C. albicans to form hyphae, as it is rarely observed following phagocytosis of UV-killed or morphogenesis-defective mutant Candida. Interestingly, failed cell division following phagocytosis of hyphal C. albicans is surprisingly common, and leads to the formation of large multinuclear macrophages. This raises question as to whether inhibition of macrophage cell division is another virulence attribute of C. albicans or enables host macrophages to contain the pathogen. © 2012 Elsevier Inc.

Candida albicans is a common fungal pathogen of humans that colonizes the skin and mucosal surfaces of most healthy individuals. Until recently, little was known about the mechanisms by which mucosal antifungal defences tolerate colonizing C. albicans but react strongly when hyphae of the same microorganism attempt to invade tissue. In this Review, we describe the properties of yeast cells and hyphae that are relevant to their interaction with the host, and the immunological mechanisms that differentially recognize colonizing versus invading C. albicans. © 2012 Macmillan Publishers Limited. All rights reserved.

Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival in their hosts. The major fungal pathogens of humans, Candida albicans and Candida glabrata, are exposed to a range of environmental stresses in their hosts including osmotic, oxidative and nitrosative stresses. Significant efforts have been devoted to the characterization of the adaptive responses to each of these stresses. In the wild, cells are frequently exposed simultaneously to combinations of these stresses and yet the effects of such combinatorial stresses have not been explored. We have developed a common experimental platform to facilitate the comparison of combinatorial stress responses in C. glabrata and C. albicans. This platform is based on the growth of cells in buffered rich medium at 30°C, and was used to define relatively low, medium and high doses of osmotic (NaCl), oxidative (H(2)O(2)) and nitrosative stresses (e.g. dipropylenetriamine (DPTA)-NONOate). The effects of combinatorial stresses were compared with the corresponding individual stresses under these growth conditions. We show for the first time that certain combinations of combinatorial stress are especially potent in terms of their ability to kill C. albicans and C. glabrata and/or inhibit their growth. This was the case for combinations of osmotic plus oxidative stress and for oxidative plus nitrosative stress. We predict that combinatorial stresses may be highly significant in host defences against these pathogenic yeasts.

Candida albicans cells with increased cell wall chitin have reduced echinocandin susceptibility in vitro. The aim of this study was to investigate whether C. albicans cells with elevated chitin levels have reduced echinocandin susceptibility in vivo. BALB/c mice were infected with C. albicans cells with normal chitin levels and compared to mice infected with high-chitin cells. Caspofungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully treated with caspofungin, as indicated by reduced kidney fungal burdens, reduced weight loss, and decreased C. albicans density in kidney lesions. In contrast, mice infected with high-chitin C. albicans cells were less susceptible to caspofungin, as they had higher kidney fungal burdens and greater weight loss during early infection. Cells recovered from mouse kidneys at 24 h postinfection with highchitin cells had 1.6-fold higher chitin levels than cells from mice infected with chitin-normal cells and maintained a significantly reduced susceptibility to caspofungin when tested in vitro. At 48 h postinfection, caspofungin treatment induced a further increase in chitin content of C. albicans cells harvested from kidneys compared to saline treatment. Some of the recovered clones had acquired, at a low frequency, a point mutation in FKS1 resulting in a S645Y amino acid substitution, a mutation known to confer echinocandin resistance. This occurred even in cells that had not been exposed to caspofungin. Our results suggest that the efficacy of caspofungin against C. albicans was reduced in vivo due to either elevation of chitin levels in the cell wall or acquisition of FKS1 point mutations. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Although fungal infections contribute substantially to human morbidity and mortality, the impact of these diseases on human health is not widely appreciated. Moreover, despite the urgent need for efficient diagnostic tests and safe and effective new drugs and vaccines, research into the pathophysiology of human fungal infections lags behind that of diseases caused by other pathogens. In this Review, we highlight the importance of fungi as human pathogens and discuss the challenges we face in combating the devastating invasive infections caused by these microorganisms, in particular in immunocompromised individuals.

The survival of all microbes depends upon their ability to respond to environmental challenges. To establish infection, pathogens such as Candida albicans must mount effective stress responses to counter host defences while adapting to dynamic changes in nutrient status within host niches. Studies of C.albicans stress adaptation have generally been performed on glucose-grown cells, leaving the effects of alternative carbon sources upon stress resistance largely unexplored. We have shown that growth on alternative carbon sources, such as lactate, strongly influence the resistance of C.albicans to antifungal drugs, osmotic and cell wall stresses. Similar trends were observed in clinical isolates and other pathogenic Candida species. The increased stress resistance of C.albicans was not dependent on key stress (Hog1) and cell integrity (Mkc1) signalling pathways. Instead, increased stress resistance was promoted by major changes in the architecture and biophysical properties of the cell wall. Glucose- and lactate-grown cells displayed significant differences in cell wall mass, ultrastructure, elasticity and adhesion. Changes in carbon source also altered the virulence of C.albicans in models of systemic candidiasis and vaginitis, confirming the importance of alternative carbon sources within host niches during C.albicans infections. The major fungal pathogen of humans, Candida albicans, often exploits alternative carbon sources to grow and colonise glucose-poor niches in its host. Most investigations of stress adaptation have examined glucose-grown cells, but we demonstrate that growth on physiologically relevant carbon sources such as lactate alters C. albicans cell wall architecture and that this affects the resistance of cells to stress and antifungal drugs. Furthermore, growth on alternative carbon sources alters the pathogenicity of C. albicans during systemic and mucosal infections. © 2012 Blackwell Publishing Ltd.

Fungal vacuoles are involved in a diverse range of cellular functions, participating in cellular homeostasis, degradation of intracellular components, and storage of ions and molecules. In recent years there has been a significant increase in the number of studies linking these organelles with the regulation of growth and control of cellular morphology, particularly in those fungal species able to undergo yeast-hypha morphogenetic transitions. This has contributed to the refinement of previously published protocols and the development of new techniques, particularly in the area of live-cell imaging of membrane trafficking events and vacuolar dynamics. The current review outlines recent advances in the imaging of fungal vacuoles and assays for characterization of trafficking pathways, and other physiological activities of this important cell organelle. © 2012 Elsevier B.V.

An imbalance of the normal microbial flora, breakage of epithelial barriers or dysfunction of the immune system favour the transition of the human pathogenic yeast Candida albicans from a commensal to a pathogen. C. albicans has evolved to be adapted as a commensal on mucosal surfaces. As a commensal it has also acquired attributes, which are necessary to avoid or overcome the host defence mechanisms. The human host has also co-evolved to recognize and eliminate potential fungal invaders. Many of the fungal genes that have been the focus of this co-evolutionary process encode cell wall components. In this review, we will discuss the transition from commensalism to pathogenesis, the key players of the fungal cell surface that are important for this transition, the role of the morphology and the mechanisms of host recognition and response. © 2012 Elsevier Ltd.

The fungal cell wall is the armour that protects the cell from changes in the external environment. The wall of Candida albicans, an opportunistic human pathogen, is also the immediate point of contact with the host immune system and contains most of the pathogen-associated molecular patterns recognised by innate immune cells. Along with the use of mutants altered in cell wall composition, the isolation and purification of cell wall components has proven useful in the identification of receptors involved in the sensing of these molecules, and assessment of the relative relevance of ligand-receptor interactions during the sensing of C. albicans by the immune system. Here, we describe protocols for the isolation of cell wall chitin, N-linked and O-linked mannans from C. albicans, and how they can subsequently be used to assess immunological activities such as phagocytosis and cytokine production by myeloid cells.

Dendritic cells (DCs) detect and respond to microbes or their components by producing cytokines and other molecules that can activate the proliferation and differentiation pathways of T cells. Investigation of DC responses to pathogens would thus provide important insights into how T-cell responses most appropriate for the pathogen are induced. Here, we describe methods for the use of mixed leukocyte reactions, to determine the proliferative and cytokine responses of murine splenic T cells in response to co-culture with bone marrow-derived DCs stimulated with Candida albicans.

Candida albicans is an opportunistic pathogen and is recognised and phagocytosed by macrophages. Using live-cell imaging, non-lytic expulsion/exocytosis of C. albicans from macrophages is demonstrated for the first time. Following complete expulsion, both the phagocyte and pathogen remain intact and viable. Partial engulfment of hyphal C. albicans without macrophage lysis is also demonstrated. These observations underpin the complexity of interactions between C. albicans and innate immune cells. © 2012 Elsevier Inc.

Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 μm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages. © 2012 Lewis et al.

Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose triggers the degradation of the ICL1 and PCK1 transcripts in C. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the assimilation of carboxylic and fatty acids are not degraded in response to glucose. However, when expressed in C. albicans, S. cerevisiae Icl1 (ScIcl1) is subjected to glucose-accelerated degradation, indicating that like S. cerevisiae, this pathogen has the molecular apparatus required to execute ubiquitin-dependent catabolite inactivation. C. albicans Icl1 (CaIcl1) lacks analogous ubiquitination sites and is stable under these conditions, but the addition of a ubiquitination site programs glucose-accelerated degradation of CaIcl1. Also, catabolite inactivation is slowed in C. albicans ubi4 cells. Ubiquitination sites are present in gluconeogenic and glyoxylate cycle enzymes from S. cerevisiae but absent from their C. albicans homologues. We conclude that evolutionary rewiring of ubiquitination targets has meant that following glucose exposure, C. albicans retains key metabolic functions, allowing it to continue to assimilate alternative carbon sources. This metabolic flexibility may be critical during infection, facilitating the rapid colonization of dynamic host niches containing complex arrays of nutrients. IMPORTANCE Pathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeast Candida albicans are strongly influenced by the Saccharomyces cerevisiae paradigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case in C. albicans because there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available.

The fungal cell wall polymer β-(1,3)-D-glucan is synthesized by the enzyme β-(1,3)- D-glucan synthase that is a complex composed of at least two proteins, Rho1p and Fks1p. Here, we report the nucleotide sequence of a single FKS gene and of the regulatory unit, RHO1 from the dematiaceous pathogenic fungus Alternaria infectoria. The predicted AiFks and AiRho share, respectively, 93% and 100% identity with that of Drechslera tritici-repentis. We also report that the sensitivity to caspofungin of eight different A. infectoria clinical strains is similar, with a MIC >32 g/ml and a MEC of 1 g/ml, except for one strain which had a MEC of 1.4 g/ml. This same strain exhibited one substitution at the hot spot 2, S1405A, compatible with less susceptible phenotypes, with the other seven strains having no mutations in either hot spot 1 or 2. The relative quantification of the expression of AiFKS and of AiRHO demonstrated a decrease in response to an exposure to caspofungin at 0.5 g/ml. © 2012 ISHAM.

Oral epithelial cells discriminate between the yeast and hyphal forms of Candida albicans via the mitogenactivated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether β-glucan and N- or O-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of β-glucan and N- and O-mannan mutants, we found that N-mannosylation (via Δoch1 and Δpmr1 mutants) and O-mannosylation (via Δpmt1 and Δmnt1 Δmnt2 mutants), but not phosphomannan (via a Δmnn4 mutant) or β-1,2 mannosylation (via Δbmt1 to Δbmt6 mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N- and O-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the α-1,6-mannose backbone of the N-linked outer chain (corresponding to a Δmnn9 mutant) may be required for epithelial adhesion, while the α-1,2-mannose component of phospholipomannan (corresponding to a Δmit1 mutant) may contribute to epithelial cell damage. β-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N- and O-mannosylation defects affect the ability of C. albicans to induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/ MKP1/c-Fos hypha-mediated immune response. © 2011, American Society for Microbiology.

Cell surface polysaccharides are key determinants of host responses to fungal infection. We determined the effects of alterations in Candida albicans cell surface polysaccharide composition and gross changes in the host immune response in groups of mice challenged intravenously with five C. albicans strains at doses adjusted to give equal disease progression 3 days later. The five strains used were the parental strain NGY152, two mutants with defective cell wall mannosylation, pmr1Δ mutant and mnt1/2Δ mutant, and the same two strains with a copy of PMR1 and MNT1 reintegrated, respectively. Renal and spleen levels of chemokines and cytokines previously shown to be key components of early host response to C. albicans were determined at intervals up to 3 days after challenge. By 12 h after C. albicans challenge, the levels of granulocyte colony-stimulating factor (G-CSF), keratinocyte-derived chemokine (KC), interleukin 6 (IL-6), monocyte chemotactic peptide 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), MIP-1, and MIP-2 were higher in the kidneys of mice challenged with the pmr1Δ mutant than in animals challenged with the other strains and were lower by day 3, suggesting an earlier host response to the pmr1Δ mutant. The production of these chemokines also diminished earlier than controls in mice infected with the mnt1/2Δ strain. Although these differences were statistically significant, their magnitude was seldom great, and no unambiguous evidence was obtained for individual responses specific to any cell surface glycosylation change. We conclude that complex, multifactorial local responses offset and obscure any differences resulting from differences in surface mannosylation of C. albicans strains when infection results from pathology-standardized challenges. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Few mating-regulated genes have been characterized in Candida albicans. C. albicans FIG1 (CaFIG1) is a fungus-specific and mating-induced gene encoding a putative 4-transmembrane domain protein that shares sequence similarities with members of the claudin superfamily. In Saccharomyces cerevisiae, Fig1 is required for shmoo fusion and is upregulated in response to mating pheromones. Expression of CaFIG1 was also strongly activated in the presence of cells of the opposite mating type. CaFig1-green fluorescent protein (GFP) was visible only during the mating response, when it localized predominantly to the plasma membrane and perinuclear zone in mating projections and daughter cells. At the plasma membrane, CaFig1-GFP was visualized as discontinuous zones, but the distribution of perinuclear CaFig1-GFP was homogeneous. Exposure to pheromone induced a 5-fold increase in Ca2+ uptake in mating-competent opaque cells. Uptake was reduced substantially in the fig1Δ null mutant. CaFig1 is therefore involved in Ca2+ influx and localizes to membranes that are destined to undergo fusion during mating. © 2011, American Society for Microbiology. All Rights Reserved.

The cell wall of the opportunistic human fungal pathogen, Candida albicans is a complex, layered network of rigid structural polysaccharides composed of β-glucans and chitin that is covered with a fibrillar matrix of highly glycosylated mannoproteins. Polymorphonuclear cells (PMNs, neutrophils) are the most prevalent circulating phagocytic leukocyte in peripheral blood and they are pivotal in the clearance of invading fungal cells from tissues. The importance of cell-wall mannans for the recognition and uptake of C. albicans by human PMNs was therefore investigated. N- and O-glycosylation-deficient mutants were attenuated in binding and phagocytosis by PMNs and this was associated with reduced killing of C. albicans yeast cells. No differences were found in the production of the respiratory burst enzyme myeloperoxidase (MPO) and the neutrophil chemokine IL-8 in PMNs exposed to control and glycosylation-deficient C. albicans strains. Thus, the significant decrease in killing of glycan-deficient C. albicans strains by PMNs is a consequence of a marked reduction in phagocytosis rather than changes in the release of inflammatory mediators by PMNs. © 2011 ISHAM.

Chitin is a skeletal cell wall polysaccharide of the inner cell wall of fungal pathogens. As yet, little about its role during fungus-host immune cell interactions is known. We show here that ultrapurified chitin from Candida albicans cell walls did not stimulate cytokine production directly but blocked the recognition of C. albicans by human peripheral blood mononuclear cells (PBMCs) and murine macrophages, leading to significant reductions in cytokine production. Chitin did not affect the induction of cytokines stimulated by bacterial cells or lipopolysaccharide (LPS), indicating that blocking was not due to steric masking of specific receptors. Toll-like receptor 2 (TLR2), TLR4, and Mincle (the macrophage-inducible C-type lectin) were not required for interactions with chitin. Dectin-1 was required for immune blocking but did not bind chitin directly. Cytokine stimulation was significantly reduced upon stimulation of PBMCs with heat-killed chitin-deficient C. albicans cells but not with live cells. Therefore, chitin is normally not exposed to cells of the innate immune system but is capable of influencing immune recognition by blocking dectin-1-mediated engagement with fungal cell walls. © 2011, American Society for Microbiology.

In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL-1β in macrophages. Inflammasome activation in macrophages results from differences in cell-wall architecture between yeasts and hyphae and is partly mediated by the dectin-1/Syk pathway. These results define the dectin-1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans. © Society for Leukocyte Biology.

Candida spp. are opportunistic pathogens in humans, and their systemic infections display upwards of 30% mortality in immunocompromised patients. Current mammalian model systems have certain disadvantages in that obtaining results is time consuming owing to the relatively long life spans and these results have low statistical resolution because sample sizes are usually small. We have therefore evaluated the potential of Drosophila melanogaster as an additional model system with which to dissect the host-pathogen interactions that occur during Candida albicans systemic infection. To do this, we monitored the survival of wild-type flies infected with various C. albicans clinical isolates that were previously ranked for murine virulence. From our lifetime data we computed two metrics of virulence for each isolate. These correlated significantly with murine survival, and were also used to group the isolates, and this grouping made relevant predictions regarding their murine virulence. Notably, differences in virulence were not predictably resolvable using immune-deficient spz-/- flies, suggesting that Toll signalling might actually be required to predictably differentiate virulence. Our analysis reveals wild-type D. melanogaster as a sensitive and relevant model system; one that offers immense genetic tractability (having an extensive RNA interference library that enables tissue-specific gene silencing), and that is easy to manipulate and culture. Undoubtedly, it will prove to be a valuable addition to the model systems currently used to study C. albicans infection. © 2011. Published by the Company of Biologists Ltd.

The cell wall proteins of fungi are modified by N- and O-linked mannosylation and phosphomannosylation, resulting in changes to the physical and immunological properties of the cell. Glycosylation of cell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnTDelta null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of beta-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.

When colonising host-niches or non-animated medical devices, individual cells of the fungal pathogen Candida albicans expand into significant biomasses. Here we show that within such biomasses, fungal metabolically generated CO2 acts as a communication molecule promoting the switch from yeast to filamentous growth essential for C. albicans pathology. We find that CO2-mediated intra-colony signalling involves the adenylyl cyclase protein (Cyr1p), a multi-sensor recently found to coordinate fungal responses to serum and bacterial peptidoglycan. We further identify Lys 1373 as essential for CO2/bicarbonate regulation of Cyr1p. Disruption of the CO2/bicarbonate receptor-site interferes selectively with C. albicans filamentation within fungal biomasses. Comparisons between the Drosophila melanogaster infection model and the mouse model of disseminated candidiasis, suggest that metabolic CO2 sensing may be important for initial colonisation and epithelial invasion. Our results reveal the existence of a gaseous Candida signalling pathway and its molecular mechanism and provide insights into an evolutionary conserved CO2-signalling system. © 2010 Hall et al.

Chitin is an essential part of the carbohydrate skeleton of the fungal cell wall and is a molecule that is not represented in humans and other vertebrates. Complex regulatory mechanisms enable chitin to be positioned at specific sites throughout the cell cycle to maintain the overall strength of the wall and enable rapid, life-saving modifications to be made under cell wall stress conditions. Chitin has also recently emerged as a significant player in the activation and attenuation of immune responses to fungi and other chitin-containing parasites. This review summarises latest advances in the analysis of chitin synthesis regulation in the context of fungal pathogenesis. © 2010 Elsevier Ltd.

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects of C. albicans innate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion of C. albicans by macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4Δ, pmr1Δ, and mnt3 mnt5Δ), whereas O- and N-linked mannan defects (mnt1Δ mnt2Δ and mns1Δ) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1Δ, hwp1Δ, and als3Δ) and yeast-locked mutants (clb2Δ, hgc1Δ, cph1Δ, efg1Δ, and efg1Δ cph1Δ), was similar to that observed for wild-type C. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and process C. albicans. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Pattern recognition receptors (PRRs) recognize and respond to evolutionarily conserved microbe-specific molecular patterns and therefore play a critical role in host defense towards pathogenic organisms. The current dogma is that the expression and activation of PRRs in innate immune cells serve as an essential link between the innate and adaptive arms of immunity. This has been demonstrated in studies on PRRs, in particular the toll-like receptors (TLRs) and C-type lectins, which revealed that innate recognition of pathogens results not only in the expression of innate response genes, to control or eliminate infectious agents, but also in the expression of chemokines and cytokines that promote and shape the acquired immune reaction. Recent reports of TLR and other PRR expression and the presence of functional TLR pathways in B and T cell subpopulations indicate that PRR signaling could also induce or modulate adaptive immune responses directly, suggesting that cytokine production and survival/proliferation of activated B and T cells could be finely tuned in a pathogen-specific manner. This review analyzes the available evidence on the expression and possible functions of TLR and other PRRs in adaptive immune cells. © 2010 Bentham Science Publishers Ltd.

The echinocandins are the newest class of antifungal agents in the clinical armory. These secondary metabolites are non-competitive inhibitors of the synthesis of β-(1,3)-glucan, a major structural component of the fungal cell wall. Recent work has shown that spontaneous mutations can arise in two hot spot regions of Fks1 the target protein of echinocandins that reduce the enzyme's sensitivity to the drug. However, other strains have been isolated in which the sequence of FKS1 is unaltered yet the fungus has decreased sensitivity to echinocandins. In addition it has been shown that echinocandin-treatment can induce cell wall salvage mechanisms that result in the compensatory upregulation of chitin synthesis in the cell wall. This salvage mechanism strengthens cell walls damaged by exposure to echinocandins. Therefore, fungal resistance to echinocandins can arise due to the selection of either stable mutational or reversible physiological alterations that decrease susceptibility to these antifungal agents. © 2009 Elsevier Inc. All rights reserved.

The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in L-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization. © 2010, American Society for Microbiology.

The ability to undergo polarised cell growth is fundamental to the development of almost all walled organisms. Fungi are characterised by yeasts and moulds, and both cellular forms have been studied extensively as tractable models of cell polarity. Chitin is a hallmark component of fungal cell walls. Chitin synthesis is essential for growth, viability and rescue from many conditions that impair cell-wall integrity. In the polymorphic human pathogen Candida albicans, chitin synthase 3 (Chs3) synthesises the majority of chitin in the cell wall and is localised at the tips of growing buds and hyphae, and at the septum. An analysis of the C. albicans phosphoproteome revealed that Chs3 can be phosphorylated at Ser139. Mutation of this site showed that both phosphorylation and dephosphorylation are required for the correct localisation and function of Chs3. The kinase Pkc1 was not required to target Chs3 to sites of polarised growth. This is the first report demonstrating an essential role for chitin synthase phosphorylation in the polarised biosynthesis of fungal cell walls and suggests a new mechanism for the regulation of this class of glycosyl-transferase enzyme. © 2010. Published by the Company of Biologists Ltd.

Signal-mediated interactions between the human opportunistic pathogens Pseudomonas aeruginosa and Candida albicans affect virulence traits in both organisms. Phenotypic studies revealed that bacterial supernatant from four P. aeruginosa strains strongly reduced the ability of C. albicans to form biofilms on silicone. This was largely a consequence of inhibition of biofilm maturation, a phenomenon also observed with supernatant prepared from non-clinical bacterial species. The effects of supernatant on biofilm formation were not mediated via interference with the yeast-hyphal morphological switch and occurred regardless of the level of homoserine lactone (HSL) produced, indicating that the effect is HSL-independent. A transcriptome analysis to dissect the effects of the P. aeruginosa supernatants on gene expression in the early stages of C. albicans biofilm formation identified 238 genes that exhibited reproducible changes in expression in response to all four supernatants. In particular, there was a strong increase in the expression of genes related to drug or toxin efflux and a decrease in expression of genes associated with adhesion and biofilm formation. Furthermore, expression of YWP1, which encodes a protein known to inhibit biofilm formation, was significantly increased. Biofilm formation is a key aspect of C. albicans infections, therefore the capacity of P. aeruginosa to antagonize this has clear biomedical implications. © 2010 SGM.

Yeast-like and filamentous fungi contain vacuoles which are similar to mammalian lysosomes and plant vacuoles, and have a variety of important functions in solute storage, protein turnover, ion homeostasis and apoptosis. Fungal vacuoles are unusual in their wide variety of architectures and roles in different species and in different cell types - their morphology and dynamics reflecting their ecological specialisation. Filamentous fungi have a network of interconnected spherical and tubular vacuole structures which may form the basis of a solute transport system that acts as nutrient transport pipelines. Some filamentous fungi fill entire cellular compartments with vacuole, which reduces the metabolic demands for cytoplasm biosynthesis and markedly affects cell cycle timing. Vacuoles are also highly dynamic, undergoing a continuous balance of fusion and fission reactions to allow changes in size, shape and number during cell division and in response to osmotic stress. This article summarises recent developments in our understanding of the dynamics, regulation and functions of fungal vacuoles. © 2010 the British Mycological Society.

The role of TLR4 in the recognition of Candida albicans has been brought into question. In order to assess whether discrepancies in the literature are due to differences in the recognition of various C. albicans strains, we selected 14 different isolates of C. albicans to evaluate their recognition by TLR4 and lectin receptors. We demonstrate that recognition of cell wall structures by lectin receptors is a consistent characteristic independent of the C. albicans strain selected, while recognition by TLR4 is a more variable feature. These data were corroborated by the increased susceptibility of TLR4-/-mice to a C. albicans strain recognized by TLR4, but not to a strain in which recognition has been shown to be independent of this receptor. This suggests a heavier reliance of in vivo antifungal host defense on lectin receptors than on TLRs, a notion compatible with the clinical picture in individuals deficient in MyD88/TLRs or dectin-1/CARD9. © 2010 ISHAM.

Background. Interleukin (IL)-1β has an important role in antifungal defense mechanisms. The inflammasome is thought to be required for caspase-1 activation and processing of the inactive precursor pro-IL-1β. The aim of the present study was to investigate the pathways of IL-1β production induced by Candida albicans in human monocytes. Methods. Human mononuclear cells were stimulated with C. albicans or mutant strains defective in mannosylation or chitin. Receptors were blocked with specific antagonists, and the IL-1β concentration was measured. Results. Human primary monocytes produce bioactive IL-1β when stimulated with C. albicans. The transcription of IL-1β was induced through mannose receptor (MR), Toll-like receptor (TLR) 2, and dectin-1 but not through TLR4 and TLR9. N-mannan-linked residues, chitin, and β-glucan from C. albicans are important for IL-1β stimulation. Surprisingly, processing and secretion of IL-1β in monocytes did not require pathogen-mediated inflammasome activation, because of the constitutive activation of caspase-1 and the capability of monocytes to release endogenous adenosine-5'-triphosphate. Conclusions. This study is the first dissection of the molecular mechanisms of IL-1β production by a fungal pathogen. Transcription through mannan/chitin/MR and β-glucan/dectin-1/TLR2 induces production of IL-1β by C. albicans in human monocytes, whereas processing of IL-1β is mediated by constitutively active caspase-1. © 2009 by the Infectious Diseases Society of America.

Hyphae of the dimorphic fungus, Candida albicans, exhibit directional tip responses when grown in contact with surfaces. On hard surfaces or in liquid media, the trajectory of hyphal growth is typically linear, with tip re-orientation events limited to encounters with topographical features (thigmotropism). In contrast, when grown on semisolid surfaces, the tips of C. albicans hyphae grow in an oscillatory manner to form regular two-dimensional sinusoidal curves and three-dimensional helices. We show that, like thigmotropism, initiation of directional tip oscillation in C. albicans hyphae is severely attenuated when Ca2+ homeostasis is perturbed. Chelation of extracellular Ca2+ or deletion of the Ca2+ transporters that modulate cytosolic [Ca2+] (Mid1, Cch1 or Pmr1) did not affect hyphal length but curve formation was severely reduced in mid1Δ and cch1Δ and abolished in pmr1Δ. Sinusoidal hypha morphology was altered in the mid1Δ, chs3Δ and heterozygous pmr1Δ/PMR1 strains. Treatments that affect cell wall integrity, changes in surface mannosylation or the provision of additional carbon sources had significant but less pronounced effects on oscillatory growth. The induction of two- and three-dimensional sinusoidal growth in wild-type C. albicans hyphae is therefore the consequence of mechanisms that involve Ca2+ influx and signalling rather than gross changes in the cell wall architecture. © 2009 Blackwell Publishing Ltd.

The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis. © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

Candida dubliniensis is the closest known relative of Candida albicans, the most pathogenic yeast species in humans. However, despite both species sharing many phenotypic characteristics, including the ability to form true hyphae, C. dubliniensis is a significantly less virulent and less versatile pathogen. Therefore, to identify C. albicans-specific genes that may be responsible for an increased capacity to cause disease, we have sequenced the C. dubliniensis genome and compared it with the known C. albicans genome sequence. Although the two genome sequences are highly similar and synteny is conserved throughout, 168 species-specific genes are identified, including some encoding known hyphal-specific virulence factors, such as the aspartyl proteinases Sap4 and Sap5 and the proposed invasin Als3. Among the 115 pseudogenes confirmed in C. dubliniensis are orthologs of several filamentous growth regulator (FGR) genes that also have suspected roles in pathogenesis. However, the principal differences in genomic repertoire concern expansion of the TLO gene family of putative transcription factors and the IFA family of putative transmembrane proteins in C. albicans, which represent novel candidate virulence-associated factors. The results suggest that the recent evolutionary histories of C. albicans and C. dubliniensis are quite different. While gene families instrumental in pathogenesis have been elaborated in C. albicans, C. dubliniensis has lost genomic capacity and key pathogenic functions. This could explain why C. albicans is a more potent pathogen in humans than C. dubliniensis. © 2009 by Cold Spring Harbor Laboratory Press.

Maintenance of the integrity of the cell wall in fungi is essential. One mechanism that cells use to maintain cell wall integrity in response to cell wall damage is to up-regulate chitin synthesis. In Candida albicans, the PKC cell wall integrity, Ca2+/calcineurin and high osmolarity glycerol (HOG) signalling pathways co-ordinately regulate chitin synthesis in response to cell wall stress. The transcription factors downstream of these pathways and their DNA binding sites within the promoters of target genes are well characterised in Saccharomyces cerevisiae, but not in C. albicans. The promoters of the C. albicans class I CHS genes (CaCHS2 and CaCHS8) were functionally dissected with the aim of identifying and characterising the transcription factors and promoter elements that mediate the transcriptional up-regulation of CaCHS2 and CaCHS8 in response to cell wall stress. This analysis provided evidence that the PKC cell wall integrity pathway may operate through RLM1-elements in the CaCHS2 and CaCHS8 promoters, but that promoter sequences that respond to the Ca2+/calcineurin and HOG signalling pathways in S. cerevisiae did not directly regulate chitin synthase 2 and 8 gene transcription in C. albicans.

Background: the mouse intravenous challenge model of Candida albicans infection is widely used to determine aspects of host-fungus interaction. We investigated the production of cytokines in the kidneys and spleen of animals up to 48 h after challenge with virulent and attenuated isolates and related these responses to semi-quantitative estimations of histopathological changes in the kidney. Methodology/Principal Findings: Progression of Candida albicans infection of the kidney in response to highly virulent fungal strains was characterized by higher levels of host cellular infiltrate, higher lesion densities and greater quantities of fungal elements at 24 and 48 h, and by higher kidney concentrations of IL-1β, MCP-1, KC, IL-6, G-CSF, TNF, MIP-2 and MIP1β, among the immune effectors measured. Levels of the chemokine KC as early as 12 h after challenge correlated significantly with all later measurements of lesion severity. Early renal IL-6 and MIP-1β concentrations also correlated with subsequent damage levels, but less significantly than for KC. All chemokines tested appeared in kidney homogenates, while most of the cytokines were undetectable in kidney and spleen homogenates. GM-CSF and IL-10 showed inverse correlations with measures of lesion severity, suggesting these alone may have exerted a defensive role. Spleen levels of KC at all times showed significant associations with kidney lesion measurements. Conclusions/Significance: Elevated chemokine levels, including KC, represent the earliest responses to C. albicans infection in the mouse kidney. Fungal strains of low mouse virulence stimulate a lower innate response and less host infiltrate than more virulent strains. These findings are consistent with immunopathological damage to kidneys in the mouse C. albicans infection model and with growing evidence implicating some TLR pathways as the main point of interaction between fungal surface polysaccharides and leukocytes. © 2009 MacCallum et al.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

The ability to reversibly convert between budding yeast-like and filamentous hyphal forms is important for the virulence of Candida albicans. A new study now provides insight into the relationship between environmental temperature and the signalling mechanisms that regulate morphogenesis in this fungal pathogen. © 2009 Elsevier Ltd. All rights reserved.

Global analysis of the molecular responses of microbial pathogens to their mammalian hosts represents a major challenge. To date few microarray studies have been performed on Candida albicans cells derived from infected tissues. In this study we examined the C. albicans SC5314 transcriptome from renal infections in the rabbit. Genes involved in adhesion, stress adaptation and the assimilation of alternative carbon sources were up-regulated in these cells compared with control cells grown in RPMI 1640, whereas genes involved in morphogenesis, fermentation and translation were down-regulated. When we compared the congenic virulent C. albicans strains NGY152 and SC5314, there was minimal overlap between their transcriptomes during kidney infections. This suggests that much of the gene regulation observed during infections is not essential for virulence. Indeed, we observed a poor correlation between the transcriptome and phenome for those genes that were regulated during kidney infection and that have been virulence tested. © 2008 Elsevier Inc. All rights reserved.

Metabolic adaptation, and in particular the modulation of carbon assimilatory pathways during disease progression, is thought to contribute to the pathogenicity of Candida albicans. Therefore, we have examined the global impact of glucose upon the C. albicans transcriptome, testing the sensitivity of this pathogen to wide-ranging glucose levels (0.01, 0.1, and 1.0%). We show that, like Saccharomyces cerevisiae, C. albicans is exquisitely sensitive to glucose, regulating central metabolic genes even in response to 0.01% glucose. This indicates that glucose concentrations in the bloodstream (approximate range 0.05-0.1%) have a significant impact upon C. albicans gene regulation. However, in contrast to S. cerevisiae where glucose down-regulates stress responses, some stress genes were induced by glucose in C. albicans. This was reflected in elevated resistance to oxidative and cationic stresses and resistance to an azole antifungal agent. Cap1 and Hog1 probably mediate glucose-enhanced resistance to oxidative stress, but neither is essential for this effect. However, Hog1 is phosphorylated in response to glucose and is essential for glucose-enhanced resistance to cationic stress. The data suggest that, upon entering the bloodstream, C. albicans cells respond to glucose increasing their resistance to the oxidative and cationic stresses central to the armory of immunoprotective phagocytic cells. © 2009 by the American Society for Cell Biology.

The outermost layer of the Candida albicans cell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants of C. albicans, which are defective to varying degrees in cell wall protein mannosylation, to investigate the role of the outermost layer of the yeast cell wall in mediating the fungicidal action of the cationic, α-helical antimicrobial peptide dermaseptin S3(1-16) [DsS3(1-16)]. The degree of phosphomannan loss, and concomitant reduction in surface negative charge, from the series of glycosylation mutants correlated with reduced levels of peptide binding to the cells. In turn, the reduced peptide binding correlated with enhanced resistance to DsS3(1-16). To ascertain whether DsS3(1-16) binds to negatively charged phosphate, we studied the effect of exogenous glucosamine 6-phosphate, and glucosamine hydrochloride as a negative control, on the antifungal efficacy of DsS3(1-16). Glucosamine 6-phosphate retarded the efficacy of DsS3(1-16), and this was attributed to the presence of phosphate, because addition of identical concentrations of glucosamine hydrochloride had little detrimental effect on peptide efficacy. Fluorescence microscopy with DsS3(1-16) tagged with fluorescein revealed that the peptide binds to the outer surface of the yeast cell, supporting our previous conclusion that the presence of exterior phosphomannan is a major determinant of the antifungal potency of DsS3(1-16). The binding of the peptide to the cell surface was a transient event that was followed by apparent localization of DsS3(1-16) in the vacuole or dissemination throughout the entire cytosol. The presence of glucosamine 6-phosphate clearly reduced the proportion of cells in the population that showed complete cytosolic staining, implying that the binding and entry of the peptide into the cytosol is significantly reduced due to the exogenous phosphate sequestering the peptide and reducing the amount of peptide able to bind to the surface phosphomannan. In conclusion, we present evidence that an antimicrobial peptide, similar to those employed by cells of the human immune system, has evolved to recognize molecular patterns on the surface of pathogens in order to maximize efficacy. © 2009 SGM.

Hypha orientation is an essential aspect of polarised growth and the morphogenesis, spatial ecology and pathogenesis of fungi. The ability to re-orient tip growth in response to environmental cues is critical for colony ramification, the penetration of diverse host tissues and the formation of mating structures. Recent studies have begun to describe the molecular machinery regulating hypha orientation. Calcium signalling, the polarisome Bud1-GTPase module and the Tea cell-end marker proteins of the microtubule cytoskeleton, along with specific kinesins and sterol-rich apical microdomains, are involved in hypha orientation. Mutations that affect these processes generate normal-shaped, growing hyphae that have either abnormal meandering trajectories or attenuated tropic responses. Hyphal tip orientation and tip extension are, therefore, distinct regulatory mechanisms that operate in parallel during filamentous growth, thereby allowing fungi to orchestrate their reproduction in relation to gradients of effectors in their environments. © 2009 Elsevier Ltd. All rights reserved.

Aim: to find sustainable alternatives to the application of synthetic chemicals for oomycete pathogen suppression. Methods and Results: Here, we present experiments on an Arabidopsis thaliana model system in which we studied the antagonistic properties of rhizobacterium Paenibacillus polymyxa strains towards the oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum. We carried out studies on agar plates, in liquid media and in soil. Our results indicate that P. polymyxa strains significantly reduced P. aphanidermatum and P. palmivora colonization in liquid assays. Most plants that had been treated with P. polymyxa survived the P. aphanidermatum inoculations in soil assays. Conclusions: the antagonistic abilities of both systems correlated well with mycoidal substance production and not with the production of antagonistic substances from the biocontrol bacteria. Significance and Impact of the Study: Our experiments highlight the need to take biofilm formation and niche exclusion mechanisms into consideration for biocontrol assays performed under natural conditions. © 2009 the Society for Applied Microbiology.

The β-glucan receptor Dectin-1 is an archetypical non-toll-like pattern recognition receptor expressed predominantly by myeloid cells, which can induce its own intracellular signalling and can mediate a variety of cellular responses, such as cytokine production. Recent identification of the components of these signalling pathways, such as Syk kinase, CARD9 and Raf-1, has provided novel insights into the molecular mechanisms underlying Dectin-1 function. Furthermore, a broader appreciation of the cellular responses mediated by this receptor and the effects of interactions with other receptors, including the TLRs, have greatly furthered our understanding of innate immunity and how this drives the development of adaptive immunity, particularly Th17 responses. Recent studies have highlighted the importance of Dectin-1 in anti-fungal immunity, in both mice and humans, and have suggested a possible involvement of this receptor in the control of mycobacterial infections. © 2009 Elsevier Ltd. All rights reserved.

A selection of 43 Candida albicans isolates, chosen to represent the four major strain clades of the species and also intraclade diversity, was screened for their virulence in the murine intravenous challenge model of C. albicans infection, for a range of properties measurable in vitro that might relate to virulence, and for the numbers of midrepeat sequences in genes of the ALS and HYR families. Heterozygosity at the mating type locus and low whole-cell acid phosphatase activity and growth rate at 40°C were found to be significantly positively associated with the most virulent isolates. Acid phosphatase activity and growth in 2 M NaCl were statistically significant variables between clades by univariate analysis. Isolates in different clades also differed significantly in midrepeat sequence alleles of ALS2, ALS4, ALS6, ALS7, ALS9, HYR1, and HYR2. There was no association between the midrepeat alleles of any ALS or HYR gene and the virulence of isolates to mice. Genome-wide transcript profiles of 20 isolates (5 per clade) grown under two conditions showed considerable variation between individual isolates, but only a small number of genes showed statistically significant differential gene expression between clades. Analysis of the expression profiles by overall strain virulence revealed 18 open reading frames differing significantly between isolates of high, intermediate, and low virulence. Four of these genes encoded functions related to phosphate uptake and metabolism. This finding and the significant association between whole-cell acid phosphatase activity and virulence led us to disrupt PHO100, which encodes a predicted periplasmic acid phosphatase. The pho100Δ mutant was mildly but significantly attenuated in terms of survival curves in the mouse model. The study has extended the range of properties known to differ between C. albicans clades and suggests a possible but minor role of phosphate metabolism in the virulence of the species. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Candidiasis is a significant cause of invasive human mycosis with associated mortality rates that are equivalent to, or worse than, those cited for most cases of bacterial septicemia. As a result, considerable efforts are being made to understand how the fungus invades host cells and to identify new targets for fungal chemotherapy. This has led to an increasing interest in Candida glycobiology, with an emphasis on the identification of enzymes essential for glycoprotein and adhesion metabolism, and the role of N-and O-linked glycans in host recognition and virulence. Here, we refer to studies dealing with the identification and characterization of enzymes such as dolichol phosphate mannose synthase, dolichol phosphate glucose synthase and processing glycosidases and synthesis, structure and recognition of mannans and discuss recent findings in the context of Candida albicans pathogenesis. © 2009.

Population genetics of the amphibian pathogen Batrachochytrium dendrobatidis (Bd) show that isolates are highly related and globally homogenous, data that are consistent with the recent epidemic spread of a previously endemic organism. Highly related isolates are predicted to be functionally similar due to low levels of heritable genetic diversity. To test this hypothesis, we took a global panel of Bd isolates and measured (i) the genetic relatedness among isolates, (ii) proteomic profiles of isolates, (iii) the susceptibility of isolates to the antifungal drug caspofungin, (iv) the variation among isolates in growth and phenotypic characteristics, and (v) the virulence of isolates against the European common toad Bufo bufo. Our results show (i) genotypic differentiation among isolates, (ii) proteomic differentiation among isolates, (iii) no significant differences in susceptibility to caspofungin, (iv) differentiation in growth and phenotypic/morphological characters, and (v) differential virulence in B. bufo. Specifically, our data show that Bd isolates can be profiled by their genotypic and proteomic characteristics, as well as by the size of their sporangia. Bd genotypic and phenotypic distance matrices are significantly correlated, showing that less-related isolates are more biologically unique. Mass spectrometry has identified a set of candidate genes associated with inter-isolate variation. Our data show that, despite its rapid global emergence, Bd isolates are not identical and differ in several important characters that are linked to virulence. We argue that future studies need to clarify the mechanism(s) and rate at which Bd is evolving, and the impact that such variation has on the host-pathogen dynamic.

Pseudohyphal growth of Candida albicans has been recognized as a morphological growth form that exhibits characteristics that are distinct from those of the budding yeast phase and true hyphal form of this pathogenic fungus. In Saccharomyces cerevisiae, pseudohypha growth involves synchronous unipolar cell divisions that are a modification of the bipolar budding pattern of diploid cells. While pseudohyphae of C. albicans also exhibit unipolar cell divisions, live cell imaging demonstrated departures from the normal unipolar pattern. Buds occasionally followed a bipolar or axial budding event in which buds could be formed from the proximal or distal ends of a parent pseudohypha. This extends the known morphological repertoire of cell division patterns in C. albicans pseudohyphal cells.

Background. Pentraxin-3 (PTX3) may be a useful biomarker in sepsis, but its regulatory mechanisms are still unclear. Oxidative stress is well defined in patients with sepsis and has a role in regulation of inflammatory pathways which may include PTX3. We undertook an in vitro study of the effect of antioxidants on regulation of PTX3 in endothelial cells combined with a prospective observational pilot study of PTX3 in relation to markers of antioxidant capacity and oxidative stress in patients with sepsis.Methods. Human endothelial cells were cultured with lipopolysaccharide 2 μg ml-1, peptidoglycan G 20 μg ml-1, tumour necrosis factor (TNF) α 10 ng ml -1, interleukin-1 (IL-1) β 20 ng ml-1, or killed Candida albicans yeast cells plus either N-acetylcysteine (NAC) 25 mM, trolox 100 mM, or idebenone 1 μM. Plasma samples were obtained from 15 patients with sepsis and 11 healthy volunteers. Results. PTX3 levels in plasma were higher in patients with sepsis than in healthy people [26 (1-202) ng ml-1 compared with 6 (1-12) ng ml-1, P=0.01]. Antioxidant capacity was lower in patients with sepsis than healthy controls [0.99 (0.1-1.7) mM compared with 2.2 (1.3-3.3) mM, P=0.01]. In patients with sepsis, lipid hydroperoxide levels were 3.32 (0.3-10.6) nM and undetectable in controls. We found no relationship between PTX3 and antioxidant capacity or lipid hydroperoxides. Cell expression of PTX3 increased with all inflammatory stimulants but was highest in cells treated with TNF α plus IL-1β. PTX3 concentrations were lower in cells co-treated with antioxidants (all P

The innate immune system of humans recognizes the human pathogenic fungus Candida albicans via sugar polymers present in the cell wall, such as mannan and β-glucan. Here, we examined whether nucleic acids from C. albicans activate dendritic cells. C. albicans DNA induced interleukin-12p40 (IL-12p40) production and CD40 expression by murine bone marrow-derived myeloid dendritic cells (BM-DCs) in a dose-dependent manner. BM-DCs that lacked Toll-like receptor 4 (TLR4), TLR2, and dectin-1, which are pattern recognition receptors for fungal cell wall components, produced IL-12p40 at levels comparable to the levels produced by BM-DCs from wild-type mice, and DNA from a C. albicans pmr1Δ null mutant, which has a gross defect in mannosylation, retained the ability to activate BM-DCs. This stimulatory effect disappeared completely after DNase treatment. In contrast, RNase treatment increased production of the cytokine. A similar reduction in cytokine production was observed when BM-DCs from TLR9-/- and MyD88-/- mice were used. In a luciferase reporter assay, NF-κB activation was detected in TLR9-expressing HEK293T cells stimulated with C. albicans DNA. Confocal microscopic analysis showed similar localization of C. albicans DNA and CpG-oligodeoxynucleotide (CpG-ODN) in BM-DCs. Treatment of C. albicans DNA with methylase did not affect its ability to induce IL-12p40 synthesis, whereas the same treatment completely eliminated the ability of CpG-ODN to induce IL-12p40 synthesis. Finally, impaired clearance of this fungal pathogen was not found in the kidneys of TLR9-/- mice. These results suggested that C. albicans DNA activated BM-DCs through a TLR9-mediated signaling pathway using a mechanism independent of the unmethylated CpG motif. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Hyphal growth of Candida albicans is characterized by asymmetric cell divisions in which the subapical mother cell inherits most of the vacuolar space and becomes cell cycle arrested in G1, while the apical daughter cell acquires most of the cell cytoplasm and progresses through G1 into the next mitotic cell cycle. Consequently, branch formation in hyphal compartments is delayed until sufficient cytoplasm is synthesized to execute the G1 'START' function. To test the hypothesis that this mode of vacuole inheritance determines cell cycle progression and therefore the branching of hyphae, eight tetracycline-regulated conditional mutants were constructed that were affected at different stages of the vacuole inheritance pathway. Under repressing conditions, vac7, vac8 and fab1 mutants generated mycelial compartments with more symmetrically distributed vacuoles and increased branching frequencies. Repression of VAC1, VAM2 and VAM3 resulted in sparsely branched hyphae, with large vacuoles and enlarged hyphal compartments. Therefore, during hyphal growth of C. albicans the cell cycle, growth and branch formation can be uncoupled, resulting in the investment of cytoplasm to support hyphal extension at the expense of hyphal branching. © 2008 the Authors.

The innate immune response was once considered to be a limited set of responses that aimed to contain an infection by primitive 'ingest and kill' mechanisms, giving the host time to mount a specific humoral and cellular immune response. In the mid-1990s, however, the discovery of Toll-like receptors heralded a revolution in our understanding of how microorganisms are recognized by the innate immune system, and how this system is activated. Several major classes of pathogen-recognition receptors have now been described, each with specific abilities to recognize conserved bacterial structures. The challenge ahead is to understand the level of complexity that underlies the response that is triggered by pathogen recognition. In this Review, we use the fungal pathogen Candida albicans as a model for the complex interaction that exists between the host pattern-recognition systems and invading microbial pathogens.

Directional growth is a function of polarized cells such as neurites, pollen tubes, and fungal hyphae. Correct orientation of the extending cell tip depends on signaling pathways and effectors that mediate asymmetric responses to specific environmental cues. In the hyphal form of the eukaryotic fungal pathogen Candida albicans, these responses include thigmotropism and galvanotropism (hyphal turning in response to changes in substrate topography and imposed electrical fields, respectively) and penetration into semisolid substrates. During vegetative growth in C. albicans, as in the model yeast Saccharomyces cerevisiae, the Ras-like GTPase Rsr1 mediates internal cellular cues to position new buds in a prespecified pattern on the mother cell cortex. Here, we demonstrate that Rsr1 is also important for hyphal tip orientation in response to the external environmental cues that induce thigmotropic and galvanotropic growth. In addition, Rsr1 is involved in hyphal interactions with epithelial cells in vitro and its deletion diminishes the hyphal invasion of kidney tissue during systemic infection. Thus, Rsr1, an internal polarity landmark in yeast, is also involved in polarized growth responses to asymmetric environmental signals, a paradigm that is different from that described for the homologous protein in S. cerevisiae. Rsr1 may thereby contribute to the pathogenesis of C. albicans infections by influencing hyphal tip responses triggered by interaction with host tissues. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Objectives: Expression of fungal virulence factors can be influenced by exposure to antifungal agents. To test this hypothesis, we determined the effects of subinhibitory concentrations of three antifungal agents on expression of three secreted proteinase genes associated with virulence in filamentous forms of Candida albicans. Methods: GFP-SAP promoter constructs and fluorescence measurement, transcript profiling and RT-PCR in vitro and in an animal model of disseminated Candida infection. Results: Exposure of C. albicans to subinhibitory concentrations of fluconazole in RPMI 1640 in the absence of serum led to up-regulation of the virulence-associated genes SAP4, SAP5 and SAP6 in hyphae and long pseudohyphae. Measurements with green fluorescent protein (GFP)-tagged promoters showed that the fluorescence of SAP4 and SAP6 under these conditions was strongest in the apical tip compartments of these filamentous cells and declined in compartments more proximal to the parent yeast cell. By contrast, SAP5- GFP fluorescence was expressed at similar levels in all cell compartments. Exposure to fluconazole led to significant increases in GFP- SAP4 and - SAP6 fluorescence in the filaments; itraconazole exposure also significantly increased GFP- SAP4 fluorescence, whereas flucytosine had no effect on any of the constructs. In experimentally infected animals, fluorescence of the GFP- SAP promoter fungal cells in kidney tissues was greater than that was seen in vitro for all four SAP constructs: treatment of animals with fluconazole did not significantly increase SAP promoter expression as measured by GFP fluorescence. Conclusions: Azole antifungal agents stimulated up-regulation of SAP4 and SAP6 genes in filamentous C. albicans cells in vitro and may therefore influence virulence as well as growth of the fungus. However, such effects appear to be transient in vivo. © the Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

The fungus, Candida albicans, and the bacterium, Pseudomonas aeruginosa, are opportunistic human pathogens that have been coisolated from diverse body sites. Pseudomonas aeruginosa suppresses C. albicans proliferation in vitro and potentially in vivo but it is the C. albicans hyphae that are killed while yeast cells are not. We show that hyphal killing involves both contact-mediated and soluble factors. Bacterial culture filtrates contained heat-labile soluble factors that killed C. albicans hyphae. In cocultures, localized points of hyphal lysis were observed, suggesting that adhesion and subsequent bacteria-mediated cell wall lysis is involved in the killing of C. albicans hyphae. The glycosylation status of the C. albicans cell wall affected the rate of contact-dependent killing because mutants with severely truncated O-linked, but not N-linked, glycans were hypersensitive to Pseudomonas-mediated killing. Deletion of HWP1, ALS3 or HYR1, which encode major hypha-associated cell wall proteins, had no effect on fungal susceptibility. © 2008 the Authors.

Multi-locus sequence typing data for 217 Candida albicans isolates cultured since 1990 from blood and vaginal samples in Japan, England/Wales and the USA were analysed for geographically related variations. While no significant differences were found between distributions of diploid sequence types (DSTs) in blood vs. vaginal isolates, there were highly significant differences in the clade distributions of isolates from the three geographical sources. Clade 2 strains were predominantly isolates from England/Wales, while clade 3 strains came mainly from the USA. The isolates from Japan were highly prevalent among strains in clades 5-17, and provided the first example seen so far in C. albicans of an amino acid encoded by three separate codons. Within clade 1, the most commonly encountered clade for isolates from all three regions, 15 Japanese isolates and 1 English isolate formed a separate clonal cluster in eBURST analysis. A similarly well demarcated clonal cluster rich in isolates from Japan was also found among the clade 4 strains. The data suggest C. albicans undergoes localized evolution, but human movements and person-to-person spread considerably blur the boundaries of such evolution.

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, because a detailed characterization at the structural level is lacking. Antigen-presenting dendritic cells (DCs), strategically located at mucosal surfaces and in the skin, may play an important role in anti-Candida protective immunity. However, the contribution of the various Candida-associated molecular patterns and their counter-receptors to DC function remains unknown. Here, we demonstrate that two C-type lectins, DC-SIGN and the macrophage mannose receptor, specifically mediate C. albicans binding and internalization by human DCs. Moreover, by combining a range of C. albicans glycosylation mutants with receptor-specific blocking and cytokine production assays, we determined that N-linked mannan but not O-linked or phosphomannan is the fungal carbohydrate structure specifically recognized by both C-type lectins on human DCs and directly influences the production of the proinflammatory cytokine IL-6. Better insight in the carbohydrate recognition profile of C-type lectins will ultimately provide relevant information for the development of new drugs targeting specific fungal cell wall antigens. © 2008 by the American Society for Biochemistry and Molecular Biology, Inc.

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated proteins. The major class, the GlycosylPhosphatidylInositol (GPI)-anchored proteins are tethered to the wall by GPI-anchor remnants and include adhesins, glycosyltransferases, yapsins and superoxide dismutases. In silico analysis suggested that C. albicans possesses 115 putative GPI anchored proteins (GpiPs), almost twice the number reported for Saccharomyces cerevisiae. A global approach to characterise in silico predicted GpiPs has been initiated by generating a library of 45 mutants. This library was subjected to a screen for cell wall modifications by testing the cell wall integrity (SDS and Calcofluor White sensitivity) and response to caspofungin. We showed that, when caspofungin sensitivity was modified, in more than half of the cases the susceptibility can be correlated to the level of chitin and cell wall thickness: sensitive strains have low level of chitin and a thin cell wall. We also identified, for the first time, genes that when deleted lead to decreased caspofungin sensitivity: DFG5, PHR1, PGA4 and PGA62. The role of two unknown GpiPs, Pga31 and Pga62 in the cell wall structure and composition was clearly demonstrated during this study. © 2008 Elsevier Inc. All rights reserved.

The recognition of fungi is mediated by germline pattern recognition receptors (PRRs) such as Toll-like receptors and lectin receptors that interact with conserved structures of the microorganisms, the pathogen-associated molecular patterns (PAMPs). Subsequently, PRRs activate intracellular signals that collaborate for the efficient activation of the host defense. The specificity of these responses is achieved through the activation of a particular mosaic of PRRs, that is determined by the available fungal PAMPs and the innate immune cells involved. This will determine a divergence of the final type of reaction, and in this way the innate host defense has the capability to deliver tailored responses to each pathogen. © 2008 Elsevier Ltd. All rights reserved.

In the plant pathogen Phytophthora infestans, nuclear integration of inf1 transgenic DNA sequences results in internuclear gene silencing of inf1. Although silencing is regulated at the transcriptional level, it also affects transcription from other nuclei within heterokaryotic cells of the mycelium. Here we report experiments exploring the mechanism of internuclear gene silencing in P. infestans. The DNA methylation inhibitor 5-azacytidine induced reversion of the inf1-silenced state. Also, the histone deacetylase inhibitor trichostatin-A was able to reverse inf1 silencing. inf1-expression levels returned to the silenced state when the inhibitors were removed except in non-transgenic inf1-silenced strains that were generated via internuclear gene silencing, where inf1 expression was restored permanently. Therefore, inf1-transgenic seapences are required to maintain the silenced state. Prolonged culture of non-transgenic inf1-silenced strains resulted in gradual reactivation of inf1 gene expression. Nuclease digestion of inf1-silenced and non-silenced nuclei showed that inf1 sequences in silenced nuclei were less rapidly degraded than non-silenced inf1 sequences. Bisulfite sequencing of the endogenous inf1 locus did not result in detection of any cytosine methylation. Our findings suggest that the inf1-silenced state is based on chromatin remodelling. © 2008 SGM.

Cytosolic α-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble α-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin a during the preparation of cell homogenates enriched α-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-α1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 α1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that α1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound α1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of α-mannosidase activity in a kex2Δ null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound α1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic α1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble α1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell. © 2008 SGM.

Candida albicans is a common commensal and opportunistic pathogenic fungus. Although it normally reproduces clonally, several lines of evidence exist for genetic recombination and some form of sexual reproduction. We have sequenced seven regions of its mitochondrial genome in 36 strains and constructed haplotypes for the 66 polymorphic sites, which include single-nucleotide polymorphisms and insertion/deletions. Nineteen different haplotypes were observed. Strains with the same mitochondrial haplotype were found in different clades defined by nuclear multilocus sequence typing (MLST) and the UPGMA dendrograms constructed using either set of data were different in topology. There was no apparent correlation between mitochondrial haplotype and the source of the strain (geographical or anatomical). Examination of the mitochondrial haplotypes revealed substantial evidence for recombination between polymorphic sites. This suggests that the use of mitochondrial haplotypes in phylogenetic studies should be approached with caution. These results provide further evidence for recombination and genetic exchange in the biology of C. albicans.

Multilocus sequence typing of six Candida albicans colonies from primary isolation plates revealed instances of colony-to-colony microvariation and carriage of two strain types in single oropharyngeal and vaginal samples. Higher rates of colony variation in commensal samples suggest selection of types from mixed populations either in the shift to pathogenicity or the response to antifungal treatment.

Multi-locus sequencing data for 242 different isolates of Candida tropicalis generated a dendrogram showing 235 strains assigned to a single large recently evolved group which contained several small clonal clusters. Haplotype analysis of a representative strain subset revealed a high level of recombination events in an otherwise clonal population. Pairs of isolates from single sources showed non-identity attributable to loss of heterozygosity in some genes in a manner similar to that established for C. albicans.

Echinocandins are a new generation of novel antifungal agent that inhibit cell wall β(1,3)-glucan synthesis and are normally cidal for the human pathogen Candida albicans. Treatment of C. albicans with low levels of echinocandins stimulated chitin synthase (CHS) gene expression, increased Chs activity, elevated chitin content and reduced efficacy of these drugs. Elevation of chitin synthesis was mediated via the PKC, HOG, and Ca2+- calcineurin signalling pathways. Stimulation of Chs2p and Chs8p by activators of these pathways enabled cells to survive otherwise lethal concentrations of echinocandins, even in the absence of Chs3p and the normally essential Chs1p, which synthesize the chitinous septal ring and primary septum of the fungus. Under such conditions, a novel proximally offset septum was synthesized that restored the capacity for cell division, sustained the viability of the cell, and abrogated morphological and growth defects associated with echinocandin treatment and the chs mutations. These findings anticipate potential resistance mechanisms to echinocandins. However, echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for combination therapies with greatly enhanced cidal activity. © 2008 Walker et al.

Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF,MIP-1α andMIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk-and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Morphogenetic transitions between the yeast, pseudohyphal and true hyphal forms of the fungal pathogen Candida albicans are coordinated with the cell cycle. In true hyphae, large vacuoles are formed in subapical compartments that influence compartment size and hence cell cycle progression and branching frequency. Here, live-cell imaging techniques were applied to study the dynamic process of vacuolar segregation in the three different morphological forms of C. albicans. We show the presence of a filamentous vacuole segregation structure in pseudohyphae and true hyphae that differs from that of a stream of vacuole vesicles typical of vacuole inheritance in yeast cells. In growing hyphae, retrograde transport of the vacuoles led to apparent capture and adhesion of vacuoles to supapical septa, and septation events sometimes led to the bisection of large vacuoles that occupied sites of cytokinesis. Inhibitor studies also demonstrated that vacuole motility in C. albicans was actin dependent and microtubule independent. © 2008 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Fungal vacuoles have long been recognised as versatile organelles, involved in many aspects of protein turnover, cellular homeostasis, membrane trafficking, signalling and nutrition. Recent research has also revealed an expanding repertoire of physiological functions for fungal vacuoles that are vital for fungal growth, differentiation, symbiosis and pathogenesis. Vacuole-mediated long-distance nutrient transporting systems have been shown to facilitate mycelial foraging and long-distance communication in saprophytes and mycorrhizal fungi. Some hyphae of plant and human fungal pathogens can grow under severely nutrient-limited conditions by expanding the vacuolar space rather than synthesising new cytoplasm and organelles. Autophagy has been recognised as a crucial process in plant pathogens for the initiation of appressorium formation. These studies demonstrate the importance of fungal vacuoles as organelles that are essential for many of the attributes that define the activities and roles of fungi in their natural environments. Crown Copyright © 2008.

Candida albicans expresses specific virulence traits that promote disease establishment and progression. These traits include morphological transitions between yeast and hyphal growth forms that are thought to contribute to dissemination and invasion and cell surface adhesins that promote attachment to the host. Here, we describe the regulation of the adhesin gene ALS3, which is expressed specifically during hyphal development in C. albicans. Using a combination of reporter constructs and regulatory mutants, we show that this regulation is mediated by multiple factors at the transcriptional level. The analysis of ALS3 promoter deletions revealed that this promoter contains two activation regions: one is essential for activation during hyphal development, while the second increases the amplitude of this activation. Further deletion analyses using the Renilla reniformis luciferase reporter delineate the essential activation region between positions -471 and -321 of the promoter. Further 5′ or 3′ deletions block activation. ALS3 transcription is repressed mainly by Nrg1 and Tup1, but Rfg1 contributes to this repression. Efg1, Tec1, and Bcr1 are essential for the transcriptional activation of ALS3, with Tec1 mediating its effects indirectly through Bcr1 rather than through the putative Tec1 sites in the ALS3 promoter. ALS3 transcription is not affected by Cph2, but Cph1 contributes to full ALS3 activation. The data suggest that multiple morphogenetic signaling pathways operate through the promoter of this adhesin gene to mediate its developmental regulation in this major fungal pathogen. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Eukaryotic cells from fungal hyphae to neurites that grow by polarized extension must coordinate cell growth and cell orientation to enable them to exhibit growth tropisms and to respond to relevant environmental cues. Such cells generally maintain a tip-high Ca2+ cytoplasmic gradient, which is correlated with their ability to exhibit polarized tip growth and to respond to growth-directing extracellular signals [1-5]. In yeast and other fungi, the polarisome, exocyst, Arp2/3, and Spitzenkörper protein complexes collectively orchestrate tip growth and cell polarity, but it is not clear whether these molecular complexes also regulate cell orientation or whether they are influenced by cytoplasmic Ca2+ gradients. Hyphae of the human pathogenic fungus Candida albicans reorient their growth axis in response to underlying surface topography (thigmotropism) [6] and imposed electric fields (galvanotropism) [7]. The establishment and maintenance of directional growth in relation to these environmental cues was Ca2+ dependent. Tropisms were attenuated in media containing low Ca2+, or calcium-channel blockers, and in mutants where calcium channels or elements of the calcium signaling pathway were deleted. Therefore galvanotropism and thigmotropism may both be mediated by localized Ca2+ influx at sites of polarized growth via Ca2+ channels that are activated by appropriate environmental signals. © 2007 Elsevier Ltd. All rights reserved.

The shape and integrity of fungal cells is dependent on the skeletal polysaccharides in their cell walls of which β(1,3)-glucan and chitin are of principle importance. The human pathogenic fungus Candida albicans has four genes, CHS1, CHS2, CHS3 and CHS8, which encode chitin synthase isoenzymes with different biochemical properties and physiological functions. Analysis of the morphology of chitin in cell wall ghosts revealed two distinct forms of chitin microfibrils: short microcrystalline rodlets that comprised the bulk of the cell wall; and a network of longer interlaced microfibrils in the bud scars and primary septa. Analysis of chitin ghosts of chs mutant strains by shadow-cast transmission electron microscopy showed that the long-chitin microfibrils were absent in chs8 mutants and the short-chitin rodlets were absent in chs3 mutants. The inferred site of chitin microfibril synthesis of these Chs enzymes was corroborated by their localization determined in Chsp-YFP-expressing strains. These results suggest that Chs8p synthesizes the long-chitin microfibrils, and Chs3p synthesizes the short-chitin rodlets at the same cellular location. Therefore the architecture of the chitin skeleton of C. albicans is shaped by the action of more than one chitin synthase at the site of cell wall synthesis. © 2007 the Authors.

Research into the major fungal pathogen, Candida albicans has firmly entered the post-genomics era. The current challenge is to apply these technologies to the analysis of C. albicans infections. Initial studies, which focused on the expression of specific virulence genes, have supported the view that secreted hydrolases and adhesins are expressed in a niche-specific fashion during infection. However, genome-wide expression profiling has revealed that most infection-related changes in C. albicans gene expression reflect environmental adaptation. Initial contacts with the host and disease progression are clearly associated with metabolic and stress adaptation. These studies, together with analyses of C. albicans mutants, indicate that physiological fitness plays a central role in the pathogenicity of this fungus, alongside virulence factors. © 2007 Elsevier Ltd. All rights reserved.

We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then alpha/alpha types and was lowest for heterozygous a/alpha types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.

A multilocus sequence typing (MLST) scheme was devised for Aspergillus fumigatus. The system involved sequencing seven gene fragments and was applied to a panel of 100 isolates of A. fumigatus from diverse sources. Thirty different sequence types were found among the 100 isolates, and 93% of the isolates differed from the other isolates by only one allele sequence, forming a single clonal cluster as indicated by the eBURST algorithm. The discriminatory power of the MLST method was only 0.93. These results strongly indicate that A. fumigatus is a species of a relatively recent origin, with low levels of sequence dissimilarity. Typing methods based on variable numbers of tandem repeats offer higher levels of strain discrimination. Mating type data for the 100 isolates showed that 71 isolates were type MAT1-2 and 29 isolates were MAT1-1. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A 12 month survey of candidaemia in Scotland, UK, in which every Scottish hospital laboratory submitted all blood isolates of yeasts for identification, strain typing and susceptibility testing, provided 300 isolates from 242 patients, generating incidence data of 4.8 cases per 100 000 population per year and 5.9 cases per 100 000 acute occupied bed days; 27.9 % of cases occurred in intensive care units. More than half the patients with candidaemia had an underlying disease involving the abdomen, 78 % had an indwelling intravenous catheter, 62 % had suffered a bacterial infection within the 2 weeks prior to candidaemia and 37 % had undergone a laparotomy. Candida albicans was the infecting species in 50 % of cases, followed by Candida glabrata (21 %) and Candida parapsilosis (12 %). Seven cases of candidaemia were caused by Candida dubliniensis, which was more prevalent even than Candida lusitaniae and Candida tropicalis (six cases each). Among C. glabrata isolates, 55 % showed reduced susceptibility to fluconazole, but azole resistance among other species was extremely low. Multilocus sequence typing showed isolates with high similarity came from different hospitals across the country, and many different types came from the hospitals that submitted the most isolates, indicating no tendency towards hospital-specific endemic strains. Multiple isolates of C. albicans and C. glabrata from individual patients were of the same strain type with single exceptions for each species. The high prevalence of candidaemia in Scotland, relative to other population-based European studies, and the high level of reduced fluconazole susceptibility of Scottish C. glabrata isolates warrant continued future surveillance of invasive Candida infections. © 2007 SGM.

A multilocus sequence typing (MLST) scheme for Candida krusei was devised, based on sequencing of six gene fragments of the species. The existence of heterozygous results for each of the six fragments sequenced confirms that C. krusei is diploid for at least part of its genome. The C. krusei MLST scheme had a discriminatory index of 0.998, making this system ideal for strain typing of C. krusei clinical isolates. MLST data for 122 independent C. krusei isolates from a range of geographical sources were analyzed by eBURST, structure, and the unweighted-pair group method using average linkages to derive a population structure comprising four subtype strain clusters. There was no evidence of geographical associations with particular subtypes. Data for pairs of isolates from seven patients showed that each patient was colonized and/or infected with strain types that were indistinguishable by MLST. The C. krusei MLST database can be accessed online at http://pubmlst.org/ckrusei/.

Chitin is an essential component of the fungal cell wall and its synthesis is under tight spatial and temporal regulation. The fungal human pathogen Candida albicans has a four member chitin synthase gene family comprising of CHS1 (class II), CHS2 (class I), CHS3 (class IV) and CHS8 (class I). LacZ reporters were fused to each CHS promoter to examine the transcriptional regulation of chitin synthesis. Each CHS promoter had a unique regulatory profile and responded to the addition of cell wall damaging agents, to mutations in specific CHS genes and exogenous Ca2+. The regulation of both CHS gene expression and chitin synthesis was co-ordinated by the PKC, HOG MAP kinase and Ca2+/calcineurin signalling pathways. Activation of these pathways also resulted in increased chitin synthase activity in vitro and elevated cell wall chitin content. Combinations of treatments that activated multiple pathways resulted in synergistic increases in CHS expression and in cell wall chitin content. Therefore, at least three pathways co-ordinately regulate chitin synthesis and activation of chitin synthesis operates at both transcriptional and post-transcriptional levels. © 2007 the Authors.

Hyphal growth is prevalent during most Candida albicans infections. Current cell division models, which are based on cytological analyses of C. albicans, predict that hyphal branching is intimately linked with vacuolar inheritance in this fungus. Here we report the molecular validation of this model, snowing that a specific mutation that disrupts vacuolar inheritance also affects hyphal division. The armadillo repeat-containing protein Vac8p plays an important role in vacuolar inheritance in Saccharomyces cerevisiae. The VAC8 gene was identified in the C. albicans genome sequence and was resequenced. Homozygous C. albicans vac8Δ deletion mutants were generated, and their phenotypes were examined. Mutant vac8Δ cells contained fragmented vacuoles, and minimal vacuolar material was inherited by daughter cells in hyphal or budding forms. Normal rates of growth and hyphal extension were observed for the mutant hyphae on solid serum-containing medium. However, branching frequencies were significantly increased in the mutant hyphae. These observations are consistent with a causal relationship between vacuolar inheritance and the cell division cycle in the subapical compartments of C. albicans hyphae. The data support the hypothesis that cytoplasmic volume, rather than cell size, is critical for progression through G1. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

We typed 165 Candida albicans isolates from 44 different sources by multilocus sequence typing (MLST) and ABC typing of rRNA genes and determined their homozygosity or heterozygosity at the mating-type-like locus (MTL). The isolates represented pairs or larger sets from individual sources, which allowed the determination of strain diversity within patients. A comparison of replicate sequence data determined a reproducibility threshold for regarding isolates as MLST indistinguishable. For 36 isolate sets, MLST and ABC typing showed indistinguishable or highly related strain types among isolates from different sites or from the same site at different times from each patient. This observation included 11 sets with at least one isolate from a blood culture and a nonsterile site from the same patient. For one patient, strain replacement was evidenced in the form of two sets of isolates from different hospital admissions where the strain types within each set were nearly identical but where the two sets differed both by MLST and ABC typing. MLST therefore confirms the existing view of C. albicans strain carriage. Microvariation, evidenced as small differences between MLST types, resulted in most instances from a loss of heterozygosity at one or more of the sequenced loci. Among isolate sets that showed major strain type differences, some isolates could be excluded as likely examples of handling errors during storage. However, for a minority of isolates, intermittent differences in ABC type for tightly clustered MLST types and intermittent appearances of MTL homozygosity lead us to propose that some C. albicans isolates, or all isolates under yet-to-be-determined conditions, maintain a high level of genetic diversity by mechanisms such as recombination, gene conversion, or chromosomal ploidy change.

To establish an infection, the pathogen Candida albicans must assimilate carbon and grow in its mammalian host. This fungus assimilates six-carbon compounds via the glycolytic pathway, and two-carbon compounds via the glyoxylate cycle and gluconeogenesis. We address a paradox regarding the roles of these central metabolic pathways in C. albicans pathogenesis: the glyoxylate cycle is apparently required for virulence although glyoxylate cycle genes are repressed by glucose at concentrations present in the bloodstream. Using GFP fusions, we confirm that glyoxylate cycle and gluconeogenic genes in C. albicans are repressed by physiologically relevant concentrations of glucose, and show that these genes are inactive in the majority of fungal cells infecting the mouse kidney. However, these pathways are induced following phagocytosis by macrophages or neutrophils. In contrast, glycolytic genes are not induced following phagocytosis and are expressed in infected kidney. Mutations in all three pathways attenuate the virulence of this fungus, highlighting the importance of central carbon metabolism for the establishment of C. albicans infections. We conclude that C. albicans displays a metabolic program whereby the glyoxylate cycle and gluconeogenesis are activated early, when the pathogen is phagocytosed by host cells, while the subsequent progression of systemic disease is dependent upon glycolysis. © 2006 the Authors; Journal compilation © 2006 Blackwell Publishing Ltd.

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications. © 2005 Braun et al.

Immunoscreening of a Candida albicans expression library resulted in the isolation of a novel gene encoding a 32.9-kDa polypeptide (288 amino acids), with 27.7% homology to the product of Saccharomyces cerevisiae YGR106c, a putative vacuolar protein. Heterozygous mutants in this gene displayed an altered budding growth pattern, characterized by the formation of chains of buds, decreasingly in size towards the apex, without separation of the daughter buds. Consequently, this gene was designated ABG1. A conditional mutant for ABG1 with the remaining allele under the control of the MET3 promoter did not grow in the presence of methionine and cysteine, demonstrating that ABG1 was essential for viability. Western analysis revealed the presence of a major 32.9-kDa band, mainly in a particulate fraction (P40) enriched in vacuoles, and tagging with green fluorescent protein confirmed that Abg1p localized to the vacuole. Vacuole inheritance has been linked to the regulation of branching frequency in C. albicans. Under repressing conditions, the conditional mutant had an increased frequency of branching under hyphal inducing conditions and an altered sensitivity to substances that interfered with cell wall assembly. Repression of ABG1 in the conditional mutant strain caused disturbance of normal size and number of vacuoles both in yeast and mycelial cells and also in the asymmetric vacuole inheritance associated with the characteristic pattern of germ tubes and branching in C. albicans. These observations indicate that ABG1 plays a key role in vacuole biogenesis, cytokinesis, and hyphal branching. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Two new species, Candida orthopsilosis and C. metapsilosis, are proposed to replace the existing designations of C. parapsilosis groups II and III, respectively. The species C. parapsilosis is retained for group I isolates. Attempts to construct a multilocus sequence typing scheme to differentiate individual strains of C. parapsilosis instead revealed fixed DNA sequence differences between pairs of subgroups in four genes: COX3, L1A1, SADH, and SYA1. PCR amplicons for sequencing were obtained for these four plus a further seven genes from 21 group I isolates. For nine group II isolates, PCR products were obtained from only 5 of the 11 genes, and for two group III isolates PCR products were obtained from a different set of 5 genes. Three of the PCR products from group II and III isolates differed in size from the group I products. Cluster analysis of sequence polymorphisms from COX3, SADH, and SYA1, which were common to the three groups, consistently separated the isolates into three distinct sets. All of these differences, together with DNA sequence similarities

Objectives: to ascertain the effects of subinhibitory concentrations of several antifungal agents on a virulence factor: secreted proteinase (Sap) activity and expression of SAP genes in Candida albicans. Methods: Enzyme assays and growth measurements, GFP-SAP2 promoter constructs and fluorescence measurement, transcript profiling and RT-PCR. Results: for seven of eight C. albicans isolates tested, exposure to fluconazole gave an increase in Sap specific activity; for one isolate, resistant to azoles and flucytosine, fluconazole exposure led to a fall in Sap activity. A similar relationship between growth reduction and increased Sap activity was seen with C. albicans cells treated with subinhibitory concentrations of itraconazole, miconazole, flucytosine and caspofungin. Transcript profiling indicated antifungal exposure was associated with increased expression of mRNA from SAP2 and SAP9 genes; this was confirmed for fluconazole and caspofungin exposure by RT-PCR. Conclusions: Antifungal agents with three different mechanisms of action similarly generate a rise in expression of SAP2 and activity of the secreted Sap2 gene product, a known virulence factor, in most isolates of C. albicans. One isolate of the fungus showed an opposite response. © the Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

The genome sequence of the 'asexual' human pathogenic fungus Aspergillus fumigatus suggests it has the capability to undergo mating and meiosis. That this organism engages in clandestine sexual activity is also suggested by observations of two equally distributed complementary mating types in nature, the expression of mating type genes and evidence of recent genome recombination events.

The pathogen Candida albicans responds to amino acid starvation by activating pseudohyphal development and the expression of amino acid biosynthetic genes (GCN response). In Saccharomyces cerevisiae, the GCN response is dependent on Gcn2, which regulates the translation of the transcription factor Gcn4. Therefore, we examined the role of Gcn2 in C. albicans by using molecular, cellular, and genomic approaches. We show that C. albicans GCN2 encodes an eIF2α kinase, like its S. cerevisiae homologue. However, GCN4 appears to be regulated mainly at the transcriptional level in C. albicans. Furthermore, the inactivation of C. albicans Gcn2 only partially attenuates growth under amino acid starvation conditions and resistance to the histidine analogue 3-aminotriazole. Our comparison of the Gcn4 and Gcn2 regulons by transcript profiling reinforces the view that Gcn2 contributes to, but is not essential for, the activation of general amino acid control in C. albicans. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

To overview the gene content of the important pathogen Phytophthora infestans, large-scale cDNA and genomic sequencing was performed. A set of 75,757 high-quality expressed sequence tags (ESTs) from P. infestans was obtained from 20 cDNA libraries representing a broad range of growth conditions, stress responses, and developmental stages. These included libraries from P. infestans-potato and -tomato interactions, from which 963 pathogen ESTs were identified. To complement the ESTs, onefold coverage of the P. infestans genome was obtained and regions of coding potential identified. A unigene set of 18,256 sequences was derived from the EST and genomic data and characterized for potential functions, stage-specific patterns of expression, and codon bias. Cluster analysis of ESTs revealed major differences between the expressed gene content of mycelial and spore-related stages, and affinities between some growth conditions. Comparisons with databases of fungal pathogenicity genes revealed conserved elements of pathogenicity, such as class III pectate lyases, despite the considerable evolutionary distance between oomycetes and fungi. Thirty-seven genes encoding components of flagella also were identified. Several genes not anticipated to occur in oomycetes were detected, including chitin synthases, phosphagen kinases, and a bacterial-type FtsZ cell-division protein. The sequence data described are available in a searchable public database.

A system is described for typing isolates of the pathogenic fungus Candida tropicalis, based on sequence polymorphisms in fragments of six genes: ICL1, MDR1, SAPT2, SAPT4, XYR1, and ZWF1a. The system differentiated 87 diploid sequence types (DSTs) among a total of 106 isolates tested or 80 DSTs among 88 isolates from unique sources. Replicate isolates from the same source clustered together with high statistical similarity, with the exception of one isolate. However, a clade of very closely related isolates included replicate isolates from three different patients, as well as single isolates from eight other patients. This clade, provisionally designated clade 1, was one of three clusters of isolates with high statistical similarity. Five of six isolates in one cluster that may acquire clade status were resistant to flucytosine. This study adds C. tropicalis to Candida albicans and Candida glabrata as Candida species for which a multilocus sequence typing (MLST) system has been set up. The C. tropicalis MLST database can be accessed at http://pubmlst.org/ctropicalis/.

We submitted a panel of 416 isolates of Candida albicans from separate sources to multilocus sequence typing (MLST). The data generated determined a population structure in which four major clades of closely related isolates were delineated, together with eight minor clades comprising five or more isolates. By Fisher's exact test, a statistically significant association was found between particular clades and the anatomical source, geographical source, ABC genotype, decade of isolation, and homozygosity versus heterozygosity at the mating type-like locus (MTL) of the isolates in the clade. However, these associations may have been influenced by confounding variables, since in a univariate analysis of variance, only the clade associations with ABC type and anatomical source emerged as statistically significant, providing the first indication of possible differences between C. albicans strain type clades and their propensity to infect or colonize different anatomical locations. There were no significant differences between clades with respect to distributions of isolates resistant to fluconazole, itraconazole, or flucytosine. However, the majority of flucytosine-resistant isolates belonged to clade 1, and these isolates, but not flucytosine-resistant isolates in other clades, bore a unique mutation in the FUR1 gene that probably accounts for their resistance. A significantly higher proportion of isolates resistant to fluconazole, itraconazole, and flucytosine were homozygous at the MTL, suggesting that antifungal pressure may trigger a common mechanism that leads both to resistance and to MTL homozygosity. The utility of MLST for determining clade assignments of clinical isolates will form the basis for strain selection for future research into C. albicans virulence. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Zoospores of plant pathogenic oomycetes exhibit distinct swimming speeds and patterns under natural conditions. Zoospore swimming is influenced by ion homeostasis and changes in the ionic composition of media. Therefore, we used video microscopy to investigate swimming patterns of five oomycete species in response to changes in potassium homeostasis. In general, zoospore speed tended to be negatively correlated with zoospore size. Three Phytophthora species (Phytophthora palmivora, Phytophthora megakarya, and Phytophthora infestans) swam in straight patterns with speeds ranging from 50 to 250 μm/s whereas two Pythium species (Pythium aphanidermatum and Pythium dissotocum) swam at similar speeds ranging from 180 to 225 μm/s with a pronounced helical trajectory and varying amplitudes. High external concentrations of potassium salts reduced the swimming speed of Ph. palmivora and induced encystment. This was not observed for Py. aphanidermatum. Application of the potassium ionophores gramicidin, nigericin and valinomycin resulted in reduced swimming speeds and changes in the swimming patterns of the Phytophthora species. Therefore, potassium ions play a key role in regulating zoospore behavior. © 2004 Elsevier Inc. All rights reserved.

Publication of the complete diploid genome sequence of the yeast Candida albicans will accelerate research into the pathogenesis of Candida infections. Comparative genomic analysis highlights genes that may contribute to C. albicans survival and its fitness as a human commensal and pathogen. © 2004 BioMed Central Ltd.

The Candida albicans BNI4 gene was identified by homology to the Saccharomyces cerevisiae orthologue and encodes a predicted 1655 amino acid protein. In S. cerevisiae most cell-wall chitin is associated with primary septum formation and Bni4p is involved in tethering the Chs3p chitin synthase enzyme to the mother-bud neck by forming a bridge between a regulatory protein Chs4p and the septin Cdc10p. CaBni4p shows 20% overall identity to the ScBni4p, with 73% identity over the C-terminal 63 amino acids, which includes a putative protein phosphatase type 1 (PP1) binding domain. Northern blot analysis revealed a transcript of the expected size that was expressed in both yeast and hyphal growth forms. C. albicans has more chitin in its cell wall than S. cerevisiae, and again most chitin is synthesized by CaChs3p. The function of CaBNI4 was investigated by performing a targeted gene disruption using the 'Ura-blaster' method to delete amino acids 1120-1611 that are essential for function. The resulting Cabni4Δ/Cabni4Δ null mutants formed lemon-shaped yeast cells and had a 30% reduction in cell-wall chitin, reduced hyphal formation on solid serum-containing medium and increased sensitivity to SDS and increased resistance to Calcofluor White. The Cabni4Δ/Cabni4Δ null mutants were unaffected in chitin ring formation, but often exhibited displaced bud sites with more obvious but flattened birth scars. Therefore, unlike in S. cerevisiae, the Cabni4 mutant apparently alters chitin distribution throughout the cell wall and not exclusively at the bud-neck region. © 2004 SGM.

Uridine auxotrophy, based on disruption of both URA3 alleles in diploid Candida albicans strain SC5314, has been widely used to select gene deletion mutants created in this fungus by "Ura-blasting" and PCR-mediated disruption. We compared wild-type URA3 expression with levels in mutant strains where URA3 was positioned either within deleted genes or at the highly expressed RPS10 locus. URA3 expression levels differed significantly and correlated with the specific activity of Ura3p, orotidine 5′-monophosphate decarboxylase. Reduced URA3 expression following integration at the GCN4 locus was associated with an attenuation of virulence. Furthermore, a comparison of the SC5314 (URA3) and CAI-4 (ura3) proteomes revealed that inactivation of URA3 caused significant changes in the levels of 14 other proteins. The protein levels of all except one were partially or fully restored by the reintegration of a single copy of URA3 at the RPS10 locus. Transcript levels of genes expressed ectopically at this locus in reconstituted heterozygous mutants also matched the levels found when the genes were expressed at their native loci. Therefore, phenotypic changes in C. albicans can be associated with the selectable marker rather than the target gene. Reintegration of URA3 at an appropriate expression locus such as RPS10 can offset most problems related to the phenotypic changes associated with gene knockout methodologies.

A system has been developed for the quantitative analysis of gene expression within individual Candida albicans cells in infected tissue. The system is based on the plasmid pGFP, which contains the codon-optimized yeast enhanced green fluorescent protein (yEGFP; Cormack et al. 1997) cloned between a basal CaADH1 promoter and the ScCYC1 terminator on an integrating vector. Promoters were inserted into pGFP and GFP levels measured in individual cells by quantitative fluorescence microscopy. Analysis of pPCK1-GFP and pMET3-GFP fusions revealed that GFP folds rapidly following gene induction, and is turned over rapidly following gene repression. Hence, single cell fluorescence measurements are likely to reflect ongoing gene expression levels with reasonable accuracy. pACT1-GFP expression levels were relatively constant during growth of C. albicans in both yeast and hyphal forms, and during growth in vivo in the mouse model of systemic infection. Therefore, pACT1-GFP provides a useful control for this quantitative GFP-based system in future analyses of C. albicans molecular responses during fungal infections. Copyright © 2004 John Wiley & Sons, Ltd.

The possibility of sexual reproduction in the human pathogenic fungus Candida albicans is a question of great interest in medical mycology. Not only is it a fundamental biological issue, but it is also a potential mechanism for contributing to the phenotypic plasticity (and hence the virulence) of the organism. Molecular genotyping methods such as multi-locus sequence typing (MLST) are generating data that can shed light on this question. In the present study we have used MLST information to generate haplotypes that identify many different homologues of a chromosome within a collection of strains. Particular combinations of these haplotypes provide evidence for chromosomal segregation and intra-chromosome recombination. All of our observations of haplotype diversity could also be explained by other mechanisms, such as gene conversion or mitotic recombination, and the resolution of these issues will require a denser map of accurately localised markers. A common event observed in strain evolution is loss of heterozygosity at a particular marker. Our results contribute to the emerging picture of C. albicans as an organism whose primary means of reproduction is clonal, but with a small but important contribution from sexual reproduction, occurring in nature but not under commonly used laboratory conditions.

Chromosomal rearrangements are common in both clinical isolates and spontaneous mutants of Candida albicans. It appears that many of these rearrangements are caused by translocations around the major sequence repeat (MSR) that is present in all chromosomes except chromosome 3, suggesting that homologous recombination (HR) may play an important role in the survival of this organism. In order to gain information on these processes, we have cloned the homologue of RAD52, which in Saccharomyces cerevisiae is the only gene required for all HR events. CaRAD52 complemented poorly a rad52 mutant of S. cerevisiae. Two null Carad52Δ/Carad52Δ mutants were constructed by sequential deletion of both alleles and two reconstituted strains were obtained by reintegration of the gene. Characterization of these mutants indicated that HR plays an essential role in the repair of DNA lesions caused by both UV light and the radiomimetic compound methyl-methane-sulphonate (MMS), whereas the non-homologous end-joining pathway (NHEJ) is used only in the absence of Rad52p or after extensive DNA damage. Repair by HR is more efficient in exponentially growing than in stationary cells, probably because a larger number of cells are in late S or G2 phases of the cell cycle (and therefore, can use a sister chromatid as a substrate for recombinational repair), whereas stationary phase cells are mainly in G0 or G1, and only can be repaired using the chromosomal homologue. In addition, CaRad52p is absolutely required for the integration of linear DNA with long flanking homologous sequences. Finally, the absence of CaRad52p results in the lengthening of telomeres, even in the presence of an active telomerase, an observation not described in any other organism. This raises the possibility that both telomerase and homologous recombination may function simultaneously at C. albicans telomeres.

Chitin is an essential structural polysaccharicle in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHTchitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on, in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C, albicans during yeast-hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast-hypha morphogenesis in C. albicans. © 2004 SGM.

Immunoscreening of a Candida albicans cDNA library with a polyclonal germ-tube-specific antibody (pAb anti-gt) resulted in the isolation of a gene encoding a lysine/glutamic-acid-rich protein, which was consequently designated KER1. The nucleotide and deduced amino acid sequences of this gene displayed no significant homology with any other known sequence. KER1 encodes a 134 kDa lysine (14.5%)/glutamic acid (16-7%) protein (Ker1p) that contains two potential transmembrane segments. KER1 was expressed in a pH-conditional manner, with maximal expression at alkaline pH and lower expression at pH 4.0, and was regulated by RIM101. A Δker1/Δker1 null mutant grew normally but was hyperflocculant under germ-tube-inducing conditions, yet this behaviour was also observed in stationary-phase cells grown under other incubation conditions. Western blotting analysis of different subcellular fractions, using as a probe a monospecific polyclonal antibody raised against a highly antigenic domain of Ker1p (pAbanti-Ker1 p), revealed the presence of a 134 kDa band in the purified plasma-membrane fraction from the wild-type strain that was absent in the homologous preparation from Δker1/Δker1 mutant. The pattern of cell-wall protein and mannoprotein species released by digestion with β-glucanases, reactive towards pAbs anti-gt and anti-Ker1p, as well as against concanavalin A, was also different in the Δkerl/ΔKer1 mutant. Mutant strains also displayed an increased cell-surface hydrophobicity and sensitivity to Congo red and Calcofluor white. Overall, these findings indicate that the mutant strain was affected in cell-wall composition and/or structure. The fact that the ker1 mutant had attenuated virulence in systemic mouse infections suggests that this surface protein is also important in host-fungus interactions. © 2004 SGM.

The human fungal pathogen, Candida albicans can grow in at least three different morphologies: yeast, pseudohyphae and hyphae. Further morphological forms exist during colony switching, for example, opaque phase cells are oblong, rather than the oval shape of yeast cells. Pseudohyphae and hyphae are both elongated and sometimes there has been little attempt to distinguish between them, as both are 'filamentous forms' of the fungus. We review here the differences between them that suggest that they are distinct morphological states. We argue that studies on 'filamentous forms' should always include a formal analysis to determine whether the cells are hyphae or pseudohyphae and we suggest some simple experimental criteria that can be applied to achieve this.

This review discusses recent advances in research into plant pathogenic oomycetes with an emphasis on root-infecting species. We focus on aspects of host targeting, mycoparasitism, and the development of molecular techniques that enable functional dissection of key genes of this economically important group of pathogens, including genomics, proteomics and gene silencing. We have not incorporated aspects relating to host resistance, research carded out into downy mildews, and other phyloplane oomycetes, unless there is also a specific relevance to the root-oomycete research community. The analysis of the asexual life stages of these organisms from zoosporogenesis through zoospore liberation, host targeting, encystment, germination, and the formation of appressoria-like structures in the rhizosphere offer significant potential in the establishment of new approaches for the treatment of disease in these organisms. The advent of appropriate molecular tools is now enabling the molecular analysis of these developmental stages to begin in earnest and will stimulate research into an economically important but scientifically neglected group of organisms. © 2003 Elsevier Science Ltd. All rights reserved.

Clinical needs for novel antifungal agents have altered steadily with the rise and fall of AIDS-related mycoses, and the change in spectrum of fatal disseminated fungal infections that has accompanied changes in therapeutic immunosuppressive therapies. The search for new molecular targets for antifungals has generated considerable research using modern genomic approaches, so far without generating new agents for clinical use. Meanwhile, six new antifungal agents have just reached, or are approaching, the clinic. Three are new triazoles, with extremely broad antifungal spectra, and three are echinocandins, which inhibit synthesis of fungal cell wall polysaccharides - a new mode of action. In addition, the sordarins represent a novel class of agents that inhibit fungal protein synthesis. This review describes the targets and mechanisms of action of all classes of antifungal agents in clinical use or with clinical potential.

Candida albicans forms unconstricted hyphae in serum-containing medium that are divided into discrete compartments. Time-lapse photomicroscopy, flow cytometry, and a novel three-dimensional imaging system were used to demonstrate that the kinetics and cell cycle events accompanying hyphal development were correlated with dynamic changes in vacuole morphology and the pattern of vacuole inheritance. Apical cells of hyphae underwent continuous extension before and after the first cytokinesis event. However, the resulting mother cell and sub-apical compartments did not immediately reenter the cell cycle and instead underwent cell cycle arrest before reentering the cycle. Vacuole was inherited asymmetrically at cytokinesis so that the distal, arrested compartments inherited most vacuole and the growing apical cell inherited most cytoplasm. Hydroxyurea release experiments demonstrated that the arrested, vacuolated hyphal compartments were in the G1 phase of the cycle. The period of cell cycle arrest was decreased by the provision of assimilatable forms of nitrogen, suggesting that the hyphal cell cycle is regulated by nitrogen limitation that results in sup-apical cell cycle arrest. This pattern of growth is distinct from that of the synchronous, symmetrical development of pseudohyphae of C. albicans and other yeast species. These observations suggest that the cellular vacuole space correlates with alterations in the cell cycles of different cell types and that the total organelle space may influence size-regulated functions and hence the timing of the eukaryotic cell cycle.

In silico analysis of the genome sequence of the human pathogenic fungus Candida albicans identified an open reading frame encoding a putative fourth member of the chitin synthase gene family. This gene, named CaCHS8, encodes an 1105 amino acid open reading frame with the conserved motifs characteristic of class I zymogenic chitin synthases with closest sequence similarity to the non-essential C. albicans class I CHS2 gene. Although the CaCHS8 gene was expressed in both yeast and hyphal cells, homozygous chs8Δ null mutants had normal growth rates, cellular morphologies and chitin contents. The null mutant strains had a 25% reduction in chitin synthase activity and were hypersensitive to Calcofluor White. A chs2Δ chs8Δ double mutant had less than 3% of normal chitin synthase activity and had increased wall glucan and decreased mannan but was unaffected in growth or cell morphology. The C. albicans class I double mutant did not exhibit a bud-lysis phenotype as found in the class I chs1Δ mutant of Saccharomyces cerevisiae. Therefore, C. albicans has four chitin synthases with two non-essential class I Chs isoenzymes that contribute collectively to more than 97% of the in vitro chitin synthase activity. © 2003 Elsevier Science (USA). All rights reserved.

Several microbial pathogens augment their invasive potential by binding and activating human plasminogen to generate the proteolytic enzyme plasmin. Yeast cells and cell wall proteins (CWP) of the human pathogenic fungus Candida albicans bound plasminogen with a Kd of 70 ± 11 nM and 112 ± 20 nM respectively. Bound plasminogen could be activated to plasmin by mammalian plasminogen activators; no C. albicans plasminogen activator was detected. Binding of plasminogen to CWP and whole cells was inhibited by εACA, indicating that binding was predominantly to lysine residues. Candida albicans mutant strains defective in protein glycosylation did not show altered plasminogen binding, suggesting that binding was not mediated via a surface lectin. Binding was sensitive to digestion by basic carboxypeptidase, implicating C-terminal lysine residues in binding. Proteomic analysis identified eight major plasminogen-binding proteins in isolated CWP. Five of these (phosphoglycerate mutase, alcohol dehydrogenase, thioredoxin peroxidase, catalase, transcription elongation factor) had C-terminal lysine residues and three (glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and fructose bisphosphate aldolase) did not. Activation of plasminogen could potentially increase the capacity of this pathogenic fungus for tissue invasion and necrosis. Although surface-bound plasmin(ogen) degraded fibrin, no direct evidence for a role in invasion of endothelial matrix or in penetration and damage of endothelial cells was found.

A panel of 86 different Candida albicans isolates was subjected to multilocus sequence typing (MLST) in two laboratories to obtain sequence data for 10 published housekeeping gene fragments. Analysis of data for all possible combinations of five, six, seven, eight, and nine of the fragments showed that a set comprising the fragments AAT1a, ACC1, ADP1, MPIb, SYA1, VPS13, and ZWF1b was the smallest that yielded 86 unique diploid sequence types for the 86 isolates. This set is recommended for future MLST with C. albicans.

Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs. of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein of P. infestans. To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs, crnl and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora. Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including oomycetes, fungi, and nematodes.

We report the development of a simple model for assessing the ability of the fungal pathogen Candida albicans to invade the chorioallantoic membrane (CAM) of fertilized hens' eggs. Wild-type and mutant strains of C. albicans were inoculated onto CAM surfaces either as a liquid suspension or on a sterile filter disc. Invasion of the membrane led to death of the embryo due to damage of the CAM, which could be examined histologically to show cell distribution and morphology, and by RT-PCR for assessment of patterns of fungal gene expression in vivo. Prophylactic or co-administration of fluconazole with the inoculum protected the embryo from infection. Secretory aspartyl protease (Sap) mutant strains with reported attenuation of virulence were virulent in the CAM model. However, a C. albicans strain with mutations in two transcription factors Efg1 and Cph1 was unable to form hyphae on the CAM or to penetrate it. The chick CAM, therefore, represents an experimentally tractable and inexpensive alternative to rodent or tissue culture-based invasion models, and can be used to investigate fungal pathogenesis and the genetic regulation of infection and membrane penetration of C. albicans.

Multilocus sequence typing (MLST) was applied to 75 Candida albicans isolates, including 2 that were expected to be identical, 48 that came from diverse geographical and clinical sources, and 15 that were sequential isolates from two patients. DNA fragments (≈500 bp) of eight genes encoding housekeeping functions were sequenced, including four that have been described before for C. albicans MLST, and four new gene fragments, AAT1a, AAT1b, MPI, and ZWF1. In total, 87 polymorphic sites were found among 50 notionally different isolates, giving 46 unique sequence types, underlining the power of MLST to differentiate isolates for epidemiological studies. Additional typing information was obtained by detecting variations in size at the transcribed spacer region of the 25S rRNA gene and tests for homozygosity at the mating type-like (MTL) locus. The stability of MLST was confirmed in two sets of consecutive isolates from two patients. In each set the isolates were identical or varied by a single nucleotide. Reference strain SC5314 and a derived mutant, CAF2, gave identical MLST types. Heterozygous polymorphisms were found in at least one isolate for all but 16 (18.4%) of the variable nucleotides, and 35 (41%) of the 87 individual sequence changes generated nonsynonymous amino acids. Cloning and restriction digestion of a gene fragment containing heterozygous polymorphisms indicated that the heterozygosity was genuine and not the result of sequencing errors. Our data validate and extend previous MLST results for C. albicans, and we propose an optimized system based on sequencing eight gene fragments for routine MLST with this species.

Two-dimensional gel electrophoresis was used to analyse stage-specific proteins from Phytophthora palmivora, a pathogen of cocoa and other economically important tropical crops. Approximately 1% of proteins appeared to be specific for each of the mycelial, sporangial, zoospore, cyst and germinated cyst stages of the life-cycle. Three proteins excised from protein gels of P. palmivora were identified as isoforms of actin by database searches to public libraries of Phytophthora infestans. The protein profiles of parallel samples of P. palmivora and P. infestans demonstrated that 30% of proteins precisely co-migrated suggesting that proteomics may be used to examine changes in the specific stages in the life cycles of Phytophthora spp.

The "asexual" human fungal pathogen Candida albicans has recently been engineered to be able to mate. A paper in the August 9, 2002 issue of Cell (Miller and Johnson, 2002) shows that mating competence is increased dramatically when mating partners are in a rare switch variant cell type that does not normally occur at body temperature.

Continued value creation is paramount for the survival of firms competing in today's high velocity global business environment. This paper presents a conceptual framework for understanding how firms can create and capture value within a highly volatile and uncertain business environment by exploiting both performance gaps and opportunity gaps through the development and use of flexible supply chain architectures. The choice of flexible organizational architecture allows for the continued reconfiguration of the independent modular components of the supply chain so as to achieve optimal leverage of both the firms core competencies as well as their collaborative partners complementary resources. The case of Cellars of Canterbury, a New Zealand based International wine marketing and distribution cooperative enterprise provides empirical support.

Many fungal pathogens undergo morphological transformations during host invasion. However, the significance of this for fungal pathogenesis is not clear. Both yeast and hyphal cells have properties well suited to tissue invasion and evasion of the immune system. However, molecular control circuits that regulate morphogenesis also regulate the expression of other virulence traits. To establish the extent to which morphogenesis impacts on pathogenesis, it is necessary to characterise the morphology of the fungus at different stages and locations during the natural history of a disease and to untangle how gene expression is modulated at these stages. This review considers the role of morphogenesis in fungal infection and argues that no simple, universal relationship can be drawn between morphology and the invasive potential of a fungus. © 2002 Elsevier Science Ltd. All rights reserved.

Plant roots generate electrical currents and associated electrical fields as a consequence of electrogenic ion transport at the root surface. Here we demonstrate that the attraction of swimming zoospores of oomycete plant pathogens to plant roots is mediated in part by electrotaxis in natural root-generated electric fields. The zones of accumulation of anode- or cathode-seeking zoospores adjacent to intact and wounded root surfaces correlated with their in vitro electrotactic behavior. Manipulation of the root electrical field was reflected in changes in the pattern of zoospore accumulation and imposed focal electrical fields were capable of overriding endogenous signals at the root surface. The overall pattern of zoospore accumulation around roots was not affected by the presence of amino acids at concentrations expected within the rhizosphere, although higher concentrations induced encystment and reduced root targeting. The data suggest that electrical signals can augment or override chemical ones in mediating short-range tactic responses of oomycete zoospores at root surfaces.

The purpose of our study was to evaluate the effectiveness of a modified six-corner suspension in patients with a paravaginal defect and stress urinary incontinence (SUI) by observing changes in the POP (pelvic organ prolapse) stage, substage, and the cure rates in SUI. Forty-two women patients who had a paravaginal defect and stress urinary incontinence were treated by a modified six-corner suspension at the urogynecology clinic, Yonsei University Medical Center between January 1999 and March 2000. Each patient underwent a complete physical examination and a standardized urogynecologic interview that asked about age, hormone replacement, parity, urinary symptoms and previous gynecologic surgery. From the 42 patients who had the operation, 30 patients with 1-year follow-up made up the study group. Changes from stage III (n = 18, 60%) of the group to stage I (n = 6, 33.3%) or stage 0 (n = 12, 66.6%) were observed 3 months after surgery, and no further changes were observed up to 1 year after surgery, except in one case. Changes from stage IV (n = 12) to stage 0 (n = 3, 25%) or stage I (n = 6, 50%) or stage II (n = 6, 50%) were observed 3 months after surgery, but no further change was observed up to 1 year later. Changes from substage Aa (+2, +3) to - 3 (n = 27, 90%) and from substage Ba (+2,+3,+4,+5,+6,+7) to - 3 (n = 27, 90%) were observed 3 months after surgery. No further changes were observed up to 1 year. The average length of the genital hiatus was initially 4.95 cm and 2.5 cm 3 months after surgery. Little change (2.6 cm) was observed up to 1 year later. When the patients were assessed clinically by urodynamics and physical examination, none had urinary leakage symptoms up to 1 year after the operation. All patients had excellent functional results and no postoperative complaints of stress urinary incontinence. We observed that a modified six-corner suspension was surprisingly effective in patients having a paravaginal defect and a stress urinary incontinence.

Benjaminiella poitrasii is a zygomycetous, non-pathogenic dimorphic fungus. Chitin synthases are the membrane bound enzymes involved in the synthesis of chitin and are key enzymes in the cell wall metabolism. Multiplicity of these enzymes is a common occurrence. Here, we identify eight distinct CHS genes in B. poitrasii as confirmed through DNA sequence and Southern analysis. These genes are related to other fungal CHS genes. BpCHS1-4 are class I-III chitin synthases while BpCHS5-8 are class IV-V chitin synthases. These eight genes are differentially expressed during morphogenesis and under different growth conditions. Two of these genes viz. BpCHS2 and BpCHS3 appear to be specific to the mycelial growth form. These are the first B. poitrasii sequences to be reported. Based on CHS gene sequences, B. poitrasii chitin synthase genes place it with other zygomycetes on a fungal phylogenetic tree. © 2002 Elsevier Science (USA). All rights reserved.

In recent years it has become evident that the structural polysaccharide chitin is synthesized from a family of enzymes encoded by multiple CHS chitin synthase genes, and regulated by an array of ancillary gene products that influence CHS activation and localization. Considerable attention has therefore been given to elucidating the function of specific CHS gene products in individual fungi. In those fungi in which individual CHS genes have been deleted systematically, there is little evidence for redundancy of function in family members. Chs enzymes are now known that participate in lateral wall biosynthesis, septum synthesis and spore formation but the phenotype of some CHS gene mutations is subtle, and so the role of the corresponding isoenzymes remains obscure. Nonetheless, it has become clear that certain members of the CHS gene families of fungi are more important for growth, integrity and viability than others, and this knowledge has already led to the design of new classes of antifungal agents that are targeted against key enzyme activities. Future work in this area will help define how individual Chs enzymes are targeted to specific regions of the cell wall and at specific times of the cell cycle, and should facilitate the rational development of novel and highly specific antifungal agents.

CaCHS1 of the fungal pathogen Candida albicans encodes an essential chitin synthase that is required for septum formation, viability, cell shape and integrity. The CaCHS1 gene was inactivated by first disrupting one allele using the ura-blaster protocol, then placing the remaining allele under the control of the maltose-inducible, glucose-repressible MRP1 promoter. Under repressing conditions, yeast cell growth continued temporarily, but daughter buds failed to detach from parents, resulting in septumless chains of cells with constrictions defining contiguous compartments. After several generations, a proportion of the distal compartments lysed. The conditional Δchs1 mutant also failed to form primary septa in hyphae; after several generations, growth stopped, and hyphae developed swollen balloon-like features or lysed at one of a number of sites including the hyphal apex and other locations that would not normally be associated with septum formation. CHS1 therefore synthesizes the septum of both yeast and hyphae and also maintains the integrity of the lateral cell wall. The conditional mutant was avirulent under repressing conditions in an experimental model of systemic infection. Because this gene is essential in vitro and in vivo and is not present in humans, it represents an attractive target for the development of antifungal compounds.

Sophisticated molecular biological research has revealed many virulence attributes in at least four pathogenic fungi, but the future study of fungal virulence requires investigators to distinguish between molecules that directly interact with the host, molecules that regulate these, and molecules that are always required for fungal growth and survival, independent of the host.

Gene disruptions in the diploid opportunistic human fungal pathogen Candida albicans are usually created using multiple rounds of targeted integration called the 'ura-blaster' method. Resulting heterozygous and homozygous null mutants can be auxotrophic (Ura-) or prototrophic (Ura+) for uracil biosynthesis. Here we demonstrate that the Ura-status of otherwise isogenic mutants affected the adhesion of C. albicans. Moreover the effect of Ura-status on adhesion was also dependent on the null mutant background, the nature of the underlying surface and the carbon source for growth. Therefore the Ura-status is not neutral in determining adhesive properties of C. albicans mutants that are generated via the ura-blaster protocol. © 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Fragments of five genes encoding chitin synthase enzymes were identified in the dimorphic fungus Paracoccidioides brasiliensis by polymerase chain reaction (PCR) amplification of conserved CHS gene domains. These represent several classes of enzyme: PbrCHS1, class I; PbrCHS2, class II; PbrCHS3, class IV; and PbrCHS4 and PbrCHS5, class V. Expression of these genes during the temperature regulated dimorphic transition from yeast to mycelium and from mycelium to yeast was determined by Northern analysis. One gene (PbrCHS3) was not expressed at detectable levels. The others were regulated by morphology and/or by the growth phase of the organism. Despite the fact that yeast cells contain more chitin than hyphal cells, the levels of mRNA for PbrCHS1, PbrCHS2, PbrCHS4 and PbrCHS5 were higher in hyphal cells than in yeast cells. This supports observations in other fungi that transcript levels often do not correlate with chitin content and that post-transcriptional regulation of CHS gene expression is important for morphogenesis.

Phenotypic variability in pathogenic fungi has long been correlated with virulence, but specific genetic and molecular mechanisms are only recently being unraveled. Fungal morphogenesis, reflecting the expression of several regulated genes, and the capacity of the rising forms or phases to cause disease has been focused on at the XIVth Congress of the International Society for Human and Animal Mycology. Three experimental models of pathogenic fungi have been discussed. In Cryptococcus neoformans, phenotypic variability or switching represents controlled and programmed changes rather than random mutations. Evaluated phenotypic traits were the capsular polysaccharide, cell and colony morphology and virulence. In the dimorphic Paracoccidioides brasiliensis, the serine-thiol proteinase from the yeast phase cleaves the main components of the basal membrane, thus being potentially relevant in fungal dissemination. In Candida albicans, relationships between adhesion proteins and those of lymphocytes and neutrophils are related to fungal pathogenicity. Regulation of the directional growth of hyphae and its tropic responses are correlated with the invasive potential of C. albicans.

Restriction fragment length polymorphism (RFLP) was performed on 32 isolates of the pathogenic fungus Paracoccidioides brasiliensis from geographically separated regions of South America. The use of HinfI and HincII gave clear RFLP patterns, for which high discriminatory indices could be calculated. Computational analysis of the RFLP patterns for the 32 isolates suggested that at least five groups of strains existed, each of which was geographically distinct and corresponded closely with present country borders. These results underline the belief that P. brasiliensis infections are acquired from exogenous sources and that this fungus occupies specialist endemic niches within the natural environment. © 2000 ISHAM.

Evidence is presented that the growth medium used to prepare a Candida albicans challenge inoculum is a significant factor determining the ability of a fungus strain to gain an initial invasive hold immediately after injection into an animal host, and thus determining gross strain lethality. Three C. albicans strains, one known to be attenuated to virulence, were grown in two broth media and injected intravenously at different doses into female NMRI mice and male albino guinea pigs. For each fungus strain and challenge dose, survival was longer from inocula grown in a diluted, buffered peptone-based broth than from inocula grown in Sabouraud glucose broth. When animals were challenged intravenously with yeast doses adjusted to give the same mean survival time regardless of strain or growth medium, the progression of fungus tissue burdens (c.f.u.g-1) in kidneys, lungs, liver, spleen and brain samples was broadly similar for all three C. albicans strains but differed between the two animal hosts. The morphological form of C. albicans recovered from infected tissues differed at the level of both the fungus strain and the host tissue. Use of survival-standardized inocula provides a means of distinguishing differences in progression of experimental disseminated Candida infections that are related to the infecting strain from those related to the animal host.

Transgenic Phytophthora palmivora strains that produce green fluorescent protein (GFP) or β-glucuronidase (GUS) constitutively were obtained after stable DNA integration using a polyethylene-glycol and CaCl2-based transformation protocol. GFP and GUS production were monitored during several stages of the life cycle of P. palmivora to evaluate their use in molecular and physiological studies. 40% of the GFP transformants produced the GFP to a level detectable by a confocal laser scanning microscope, whereas 75% of the GUS transformants produced GUS. GFP could be visualised readily in swimming zoospores and other developmental stages of P. palmivora cells. For high magnification microscopic studies, GFP is better visualised and was superior to GUS. In contrast, for macroscopic examination, GUS was superior. Our findings indicate that both GFP and GUS can be used successfully as reporter genes in P. palmivora. Copyright (C) 1999 Federation of European Microbiological Societies.

Candida albicans undergoes reversible morphogenetic transitions between budding, pseudohyphal and hyphal growth forms that promote the virulence of this pathogenic fungus. The regulatory networks that control morphogenesis are being elucidated; however, the primary signals that trigger morphogenesis remain obscure, and the physiological outputs of these networks are complex. Copyright (C) 1999 Elsevier Science Ltd.

Ten years ago, when molecular genetic methods were being applied vigorously to viruses, bacterial pathogens and eukaryotic parasites, there seemed to be a partial paralysis in applying them to infectious fungi; this state of affairs was more than apparent in the composition of the symposia at the ISHAM conference in 1987. Since then, however, things have changed. The ISHAM conference held in Italy in 1997 was replete with studies utilizing molecular genetic techniques to answer questions related to epidemiology, pathogenesis, drug development and typing. In the symposium Advances in Molecular Genetics of Fungal Pathogens, several new applications of molecular biology to fungal pathogenesis were reviewed. Although the presentations in this symposium covered only a fraction of the molecular methods now being applied to Candida pathogenesis, they nevertheless provided a intriguing view of what is in store for us in the coming years.

The aspartate proteinase inhibitor pepstatin a has been shown previously to reduce the adherence of Candida albicans yeast cells to human surfaces. This suggests that in addition to their presumed function facilitating tissue penetration, the secreted aspartate proteinases (Saps) of this fungal pathogen may have auxiliary roles as cellular adhesins. We therefore examined the relative adherence of yeast cells of a parental wild-type strain of C. albicans in relation to yeast cells of strains harbouring specific disruptions in various members of the SAP gene family in an otherwise isogenic background. The adhesiveness of Δsap1, Δsap22 and Δsap3 null mutants and a triple Δsap4-6 disruptant was examined on three surfaces - glass coated with poly-L-lysine or a commercial cell-free basement membrane preparation (Matrigel) and on human buccal epithelial cells. Pepstatin a reduced adherence to all surfaces. Adherence of the each of the single SAP null mutants to these three substrates was either reduced or not affected significantly compared to that of the parental strain. The adherence of the Δsap4-6 mutant was reduced on poly-L-lysine and Matrigel, but increased on buccal cells. The results suggest that in addition to a primary enzymatic role, various SAPs may also act singly or synergistically to enhance the adhesiveness to C. albicans cells to certain human tissues.

Candida dubliniensis is a recently identified species which is implicated in oral candidosis in HIV-infected and AIDS patients. The species shares many phenotypic characteristics with, and is phylogenetically closely related to, Candida albicans. In this study the phylogenetic relationship between these two species was investigated and a comparison of putative virulence factors was performed. Four isolates of C. dubliniensis from different clinical sources were chosen for comparison with two reference C. albicans strains. First, the distinct phylogenetic position of C. dubliniensis was further established by the comparison of the sequence of its small rRNA subunit with representative Candida species. The C. dubliniensis isolates formed true unconstricted hyphae under most induction conditions tested but failed to produce true hyphae when induced using N-acetylglucosamine. Oral C. dubliniensis isolates were more adherent to human buccal epithelial cells than the reference C. albicans isolates when grown in glucose and equally adherent when grown in galactose. The C. dubliniensis isolates were sensitive to fluconazole, itraconazole, ketoconazole and amphotericin B. Homologues of seven tested C. albicans secretory aspartyl proteinase (SAP) genes were detected in C. dubliniensis by Southern analysis. In vivo virulence assays using a systemic mouse model suggest that C. dubliniensis is marginally less virulent than C. albicans. These data further confirm the distinct phenotypic and genotypic nature of C. dubliniensis and suggest that this species may be particularly adapted to colonization of the oral cavity.

There is an immediate need for identification of new antifungal targets in opportunistic pathogenic fungi like Candida albicans. In the past, efforts have focused on synthesis of chitin and glucan, which confer mechanical strength and rigidity upon the cell wall. This paper describes the molecular analysis of CaMNT1, a gene involved in synthesis of mannoproteins, the third major class of macromolecule found in the cell wall. CaMNT1 encodes an α- 1,2-mannosyl transferase, which adds the second mannose residue in a tri- mannose oligosaccharide structure which represents O-linked mannan in C. albicans. The deduced amino acid sequence suggests that CaMnt1p is a type II membrane protein residing in a medial Golgi compartment. The absence of CaMnt1p reduced the ability of C. albicans cells to adhere to each other, to human buccal epithelial cells, and to rat vaginal epithelial cells. Both heterozygous and homozygous Camnt1 null mutants of C. albicans showed strong attenuation of virulence in guinea pig and mouse models of systemic candidosis, which, in guinea pigs, could be attributed to a decreased ability to reach and/or adhere internal organs. Therefore, correct CaMnt1p-mediated O-linked mannosylation of proteins is critical for adhesion and virulence of C. albicans.

Immunoscreening of a Candida albicans cDNA library with a monoclonal antibody (mAb 4C12) recognizing an epitope present in high-molecular-weight mannoprotein (HMWM) components specific for the mycelial cell walls (a 180 kDa component and a polydispersed 260 kDa species) resulted in the isolation of the gene CaCYC3 encoding for cytochrome c haem lyase (CCHL). The CaCYC3 gene was transcribed preferentially in mycelial cells in which two mRNA transcripts of 0.8 and 1 kb were found. The nucleotide and the deduced amino acid sequences of this gene displayed 45% homology and 46% identity, respectively, to the Saccharomyces cerevisiae CYC3 gene and shared common features with other reported genes encoding for CCHL. The CaCYC3 gene restored the respiratory activity when transformed in a S. cerevisiae cyc3- mutant strain. A C. albicans CYC3 null mutant was constructed after sequential disruption using the hisG::URA3::hisG ('ura-blaster') cassette. Null mutant cells were unable to use lactate as a sole carbon source and had a reduced ability to form germ tubes. Western immunoblotting analysis of subcellular fractions from wild-type and null mutant strains demonstrated the presence of two gene products, a 33 kDa mitochondrial protein and a 40kDa cell wall-associated moiety reacting with antibodies against CCHL, in both yeast cells and germ tubes. mAb 4C12 still reacted with the CaCYC3 null mutant (by immunofluorescence and immunoblotting) but showed an altered pattern of immunoreactivity against cell wall HMWM species, indicating a relationship between these moieties and the CaCYC3 gene products. The results suggest that the CaCYC3 gene encodes two proteins, one targeted to the mitochondria and the other to the cell wall.

The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino- terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus. The nucleotide sequence of CHS2 and its flanking regions has been submitted to GenBank under Accession Number Y09231.

Candida albicans has three genes encoding chitin synthase enzymes. In wild-type strains, the expression of CHS2 and CHS3 peaked 1-2 h after the induction of hyphal growth, whilst mRNA levels in a non-germinative strain, CA2, remained low under the same conditions, CHS1 gene expression did not peak during germ tube formation but remained at low levels in both yeast and hyphal growth. The pattern of gene expression did not predict the changes in measured chitin synthase activities or changes in chitin content during dimorphic transition. Chitin synthase activity increased steadily, and did not peak shortly after germ tube induction, and activity profiles were similar in germ-tube-competent and germ-tube-negative strains. The phenotype of a Δchs2 null mutant suggested that CHS2 encoded the major enzyme activity in vitro and was largely responsible for elevated chitin synthase activities in microsomal preparations from hyphal cells compared to yeast cells. However, CaChs3p was responsible for synthesis of most chitin in both yeast and hyphae. Three independent chitin assays gave markedly different estimates of the relative chitin content of yeast and hyphae and wild-type and chs mutants. Only one of the methods gave a significantly higher chitin content for hyphal compared to yeast cell walls and a lower chitin content for hyphae of the Δchs2 null mutant compared to the parental strain.

The direction of growth of hyphae of the pathogenic fungus Candida albicans responds thigmotropically to surface contours by following scratches, ridges and grooves and by penetrating pores. Here it is shown that the thigmotropic response to ridges is attenuated by GdCl3 and verapamil [blockers of stretch-activated (SA) ion channels and L-type calcium channels, respectively]. At low concentrations, both compounds reduced the percentage of hyphae reorienting on contact with a ridge without markedly affecting hyphal extension rate, suggesting a possible role for SA or other calcium channels in the transduction of the thigmotropic response. In addition, patch-clamp recordings demonstrated SA channel activity in the plasma membrane of both yeast and hyphal cells of C. albicans. Two distinct SA channels with conductances of 54 pS and 20-25 pS in 200 mM KCl were observed in protoplasts from yeast cells and one channel of 51 pS was found in protoplasts from hyphal cells.

Secreted Aspartat Proteinases (Sap) are among those factors of the human pathogen Candida albicans, which promote infections in the immunocompromised host. Sap isoenzymes are encoded by at least nine different genes (SAP1-9), which are differentially regulated in vitro. RT-PCR analysis during experimental infections and from patient samples confirmed the expression of SAP genes in vivo. However, while Sap2 is the dominant isoenzyme under culture conditions, other SAP genes are also expressed during infections. In order to investigate the role of single isoenzymes during the pathogenesis of candidosis, mutants were produced which habour deletions in SAP1, SAP2, SAP3 and SAP4-6. Although only SAP2 and SAP4-6 mutants showed a strong reduction of proteolytic activity in vitro, all SAP mutants were significantly attenuated in systemic infections. In addition, SAP2, SAP3 and SAP4-6 mutants were clearly more sensitive to neutrophilic leucozytes compared to the wild type SC5314. These investigations show that several proteinase isoenzymes are likely to be involved in the pathogenesis of candidosis. © Blackwell Wissenschafts-Verlag GmbH.

A new project to map the genome of the pathogenic fungus, Candida albicans, has been started. The entire genome was cloned as 5088 cosmids, stored in individual microtiter plate wells. DNA was prepared and fingerprinted using restriction digestion, fluorescent labeling, and analysis on an ABI sequencer. These data are being used to construct contigs of the genome. Simultaneously, a DNA pooling system has been set up, suitable for PCR-based isolation of cosmids containing any known gene. Ultimately, these approaches will lead to the creation of a physically based map of the C. albicans genome, providing the means to localize precisely all the genes, act as a substrate for genome sequencing projects, and provide probes for future studies of genome rearrangement and comparative genomics.

Secreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multigene family with at least nine members (SAP1 to SAP9) and are considered putative virulence factors important for the pathogenicity of this human pathogen. The role of Sap isoenzymes in the virulence of C. albicans has not yet been clearly established, and therefore, using recent progress in the genetics of this yeast, we have constructed a panel of isogenic yeasts, each with a disruption of one or several SAP genes. We focused on the construction of a C. albicans strain in which three related SAP genes (SAP4, SAP5, and SAP6) were disrupted. Growth of the Δsap4,5,6 triple homozygous null mutant DSY459 in complex medium was not affected, whereas, interestingly, growth in a medium containing protein as the sole nitrogen source was severely impaired compared to the growth of the wild-type parent strain SC5314. Since the presence of Sap2 is required for optimal growth on such medium, this suggests that Sap4, Sap5, or Sap6 plays an important role for the process of induction of SAP2. When guinea pigs and mice were injected intravenously with DSY459, their survival time was significantly longer than that of control animals infected with the wild-type SC5314. Attenuated virulence of DSY459 was followed by a significant reduction of yeast cells in infected organs. These data suggest that the group of Sap4, Sap5, and Sap6 isoenzymes is important for the normal progression of systemic infection by C. albicans in animals.

The Aspergillus fumigatus chsE (AfchsE) gene was isolated from an A. fumigatus DNA library on the basis of hybridization to a segment of Saccharomyces cerevisiae CHS3 (ScCHS3). The amino acid sequence derived from AfchsE is 28% identical with ScCHS3 and 80% identical with the product of Aspergillus nidulans chsD (AnchsD). A mutant strain constructed by disruption of AfchsE has reduced levels of mycelial chitin, periodic swellings along the length of hyphae, and a block in conidiation that can be partially restored by growth in osmotic stabilizer. This phenotype is different from that reported for an AnchsD mutant, in which germinating conidia and hyphal tips undergo lysis and the colonial growth rate is significantly reduced. Despite the defects associated with the AfchsE- strain, its virulence was not significantly reduced when compared with the wild-type parental strain in a mouse model of pulmonary aspergillosis.

Contact-sensing or thigmotropism is the directional growth response of cells in relation to topographical guidance cues. Thigmotropism is thought to play a major role in the location of infectable sites on plants by phytopathogenic fungi and has recently been shown to be a property of hyphae in the human pathogenic fungus Candida albicans. Here we show that hyphae of the dermatophytes Epidermophyton floccosum, Microsporum canis and Trichophyton mentagrophytes reorientate their direction of growth in response to grooves and pores of membrane substrata as did hyphae of the saprophytes Mucor mucedo and Neurospora crassa. This suggests that the thigmotropic behaviour of hyphae is not a specific property of pathogens, but rather a general feature of the growth of fungal hyphae that must forage for nutrients on surfaces and within solid materials.

Contact-sensing or thigmotropism is the directional growth response of cells in relation to topographical guidance cues. Thigmotropism is thought to play a major role in the location of infectable sites on plants by phytopathogenic fungi and has recently been shown to be a property of hyphae in the human pathogenic fungus Candida albicans. Here we show that hyphae of the dermatophytes Epidermophyton floccosum, Microsporum canis and Trichophyton mentagrophytes reorientate their direction of growth in response to grooves and pores of membrane substrata as did hyphae of the saprophytes Mucor mucedo and Neurospora crassa. This suggests that the thigmotropic behaviour of hyphae is not a specific property of pathogens, but rather a general feature of the growth of fungal hyphae that must forage for nutrients on surfaces and within solid materials.

Secreted aspartyl proteinases (Saps), encoded by a gene family with at least nine members (SAP1 to SAP9), are one of the most discussed virulence factors produced by the human pathogen Candida albicans. In order to study the role of each Sap isoenzyme in pathogenicity, we have constructed strains which harbor mutations at selected SAP genes. SAP1, SAP2, and SAP3, which are regulated differentially in vitro, were mutated by targeted gene disruption. The growth rates of all homozygous null mutants were similar to those of the isogenic wild-type parental strain (SC5314) in complex and defined media. In medium with protein as the sole source of nitrogen, sap1 and sap3 mutants grew with reduced growth rates but reached optical densities similar to those measured for SC5314. In contrast, sap2 null mutants tended to clump, grew poorly in this medium, and produced the lowest proteolytic activity. Addition of ammonium ions reversed such growth defects. These results support the view that Sap2 is the dominant isoenzyme. When sap1, sap2, and sap3 mutants were injected intravenously in guinea pigs and mice, the animals had increased survival rates compared to those of control animals infected with SC5314. However, reduction of proteolytic activity in vitro did not correlate directly with the extent of attenuation of virulence observed for all Sap-deficient mutants. These data suggest that SAP1, SAP2, and SAP3 all contribute to the overall virulence of C. albicans and presumably all play important roles during disseminated infections.

The clinical pathogen Candida albicans is a budding yeast that is capable of forming a range of polarized and expanded cell shapes from pseudohyphae to true nonconstricted hyphae. Filamentous forms consist of contiguous uninucleated compartments that are partitioned by septa. It has long been held that the so-called "dimorphic transition" from a budding to a filamentous form may aid the fungus to penetrate epithelia and may therefore be a virulence factor. This review summarized new information regarding the physiology and ecology of hyphal growth in C. albicans. New evidence has demonstrated that hyphae of C. albicans have a sense of touch so that they grow along grooves and through pores (thigmotropism). This may aid infiltration of epithelial surfaces during tissue invasion. Hyphae are also aerotropic and can form helices when contacting solid surfaces. Growing evidence supports the view that hyphal growth is a response to nutrient deprivation, especially low nitrogen and that filamentous growth enables the fungus to forage for nutrients more effectively. Further insights into the growth of C. albicans have come from the analysis of genes and mutations of Saccharomyces which have begun to reveal the molecular mechanisms underlying the mechanisms of bud site selection, cell polarity and signal transduction pathways that lead to pseudohyphal development in this and other organisms. For example, it is now clear that a MAP-kinase cascade, homologous to the mating pathway in Saccharomyces, regulates filamentous growth in both fungi. However, this must be only one of several overlapping or separate signal transduction pathways for hyphal development because filamentous growth still occurs in mutants of Candida and Saccharomyces which are blocked in this pathway. Cell cycle analyses have shown that hyphal phase cell cycle of Candida is distinct from that in budding and pseudohyphal formation and so pseudohyphal growth of Saccharomyces is not a true model of germ tube growth in Candida. Pseudohyphal growth in both Candida and Saccharomyces involves synchronous division of mother cells and their daughters. In contrast, during germ tube growth of Candida, cytoplasm is unequally partitioned at cytokinesis so that apical cells inherit more cytoplasm and sub-apical cells have a single nucleus but are extensively vacuolated. As a result, apical cells grow and divide while sub-apical cells are apparently arrested in the cell cycle until they can regenerate sufficient cytoplasm to re-enter the cell cycle. Although current studies still fall short of verifying the status of yeast-hypha dimorphism as a virulence factor, they suggest that the cell biology of germ tube growth of C. albicans is well suited for the invasive growth of the fungus in vivo.

The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein. Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TTG. C. albicans cells expressing this construct contained CFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C. albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain CFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.

Germ tube production of Candida albicans is favoured by and is adapted to, conditions of nutrient deprivation, particularly low nitrogen. Hyphal development in media containing low serum concentrations is characterised by the formation of sparsely branched hyphae in which uninucleated, highly vacuolated intercalary compartments are arrested in the cell cycle, probably in G1. Re-entry into the cell cycle is accelerated by enrichment of the medium for nutrients and is preceded by reduction of the vacuolar space and concomitant increase in the size of the cytoplasmic volume. Hyphae exhibit contact guidance (thigmotropism) which may facilitate the invasion of tissues through local wounds, invaginations or sites of weakened surface integrity. Thus hyphal growth is adapted for nutrient scavenging and penetration of irregular surfaces. The genetics of dimorphism and virulence has been investigated through studies of differential gene expression and by specific manipulation of gene expression by overexpression and the creation of null mutants. The expression of many genes is regulated during dimorphism however few of these play active roles in the regulation of morphogenesis. Disruption of genes encoding certain secreted aspartyl proteinases (SAP2) and a key step in the assembly of O-linked mannosylation (MNT1) led to attenuation of virulence. Manipulation of the expression of a hyphal specific gene HYR1 did not affect germ tube growth while overexpression of a constitutively expressed Candida gene HST7 stimulated pseudohyphal growth in Saccharomyces. Therefore, analysis and manipulation of the normal temporal pattern of gene expression is yielding valuable information regarding the regulation of morphogenesis and pathogenesis in this fungus. © 1996, the Japanese Society for Medical Mycology. All rights reserved.

A wide variety of prokaryotic and eukaryotic cells exhibit a multidrug resistance (MDR) phenotype, indicating that resistance to potentially toxic compounds is mediated by their active efflux from the cell. We have sought to determine whether resistance to azoles in some strains of Candida species may be due in part to active drug efflux. Rhodamine 123 (Rh123) is a fluorescent compound that is transported by a wide variety of MDR cell types. We have shown that certain azole-resistant strains of Candida albicans, C. glabrata, and C. krusei accumulate less Rh123 than azole-susceptible ones. In C. albicans, Rh123 accumulation was growth phase and temperature dependent and was increased by proton uncouplers and by reserpine, an MDR modulator. This is consistent with an energy-dependent efflux mechanism for Rh123, mediated by an MDR transporter. In C. glabrata, but not in C. albicans, there was competition between Rh123 and fluconazole for efflux. Thus, in C. glabrata, Rh123 and fluconazole appear to be transported via a common MDR-like transporter, whereas in C. albicans, the Rh123 transporter dries not appear to transport azoles.

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65(PAK) family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid 'Spider' media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.

The Candida albicans ADH1 gene encodes an alcohol dehydrogenase which is immunogenic during infections in humans. The ADH1 gene was isolated and sequenced, and the 5'- and 3'-ends of its mRNA were mapped. The gene encodes a 350 amino acid polypeptide with strong homology (70.5-85.2% identity) to alcohol dehydrogenases from Saccharomyces cerevisiae, Kluyveromyces lactis and Schizosaccharomyces pombe. The cloned C. albicans ADH1 gene was shown to be functional through complementation of adh mutations and efficient production of active alcohol dehydrogenase in S. cerevisiae. Northern analysis of C. albicans RNA revealed that ADH1 mRNA levels were regulated in response to carbon source and during batch growth. During growth on glucose, ADH1 mRNA levels rose to maximum levels during late exponential growth phase and declined to low levels in stationary phase. The ADH1 mRNA was relatively abundant during growth on galactose, glycerol, pyruvate, lactate or succinate, and less abundant during growth on glucose or ethanol. Alcohol dehydrogenase levels did not correlate closely with ADH1 mRNA levels under the growth conditions studied, suggesting either that this locus is controlled at both transcriptional and post-transcriptional levels, or that other differentially regulated ADH loci exist in C. albicans.

Two genes, designated chsC and chsG were isolated from DNA libraries of the opportunistic fungal pathogen, Aspergillus fumigatus. The genes were characterized with respect to their nucleotide sequences and mutant phenotypes. The complete deduced amino acid sequences of chsC and chsG show that the products of both genes are Class III zymogen-type enzymes. A mutant strain constructed by disruption of chsC is phenotypically indistinguishable from the wild-type strain, but chsG and chsC chsG strains have reduced colony radial growth rate and chitin synthase activity, conidiate poorly and produce highly branched hyphae. Despite these defects, the double-mutant strain retained the ability to cause pulmonary disease in neutropenic mice. However, in comparison to the wild-type strain, there was a decrease in mortality and delay in the onset of illness in mice inoculated with the double-mutant strain, which was associated with smaller and more highly branched fungal colonies in lung tissue.

A hyphally regulated gene (HYR1) from the dimorphic human pathogenic fungus Candida albicans was isolated and characterized. Northern (RNA) analyses showed that the HYR1 mRNA was induced specifically in response to hyphal development when morphogenesis was stimulated by serum addition and temperature elevation, increases in both culture pH and temperature, or N- acetylglucosamine addition. The HYR1 gene sequence revealed a 937-codon open reading frame capable of encoding a protein with an N-terminal signal sequence, a C-terminal glycosylphosphatidylinositol-anchoring domain, 17 potential N glycosylation sites, and a large domain rich in serine and threonine (51% of 230 residues). These features are observed in many yeast cell wall proteins, but no homologs are present in the databases. In addition, Hyr1p contained a second domain rich in glycine, serine, and asparagine (79% of 239 residues). The HYR1 locus in C. albicans CAI4 was disrupted by 'Ura-blasting,' but the resulting homozygous Δhyr1/Δhyr1 null mutant displayed no obvious morphological phenotype. The growth rates for yeast cells and hyphae and the kinetics of germ tube formation in the null mutant were unaffected. Aberrant expression of HYR1 in yeast cells, when an ADH1-HYR1 fusion was used, did not stimulate hyphal formation in C. albicans or pseudohyphal growth in Saccharomyces cerevisiae. HYR1 appears to encode a nonessential component of the hyphal cell wall.

Yeast-like fungi such as Saccharomyces cerevisiae exhibit a range of cell types differing in cell shape, gene expression and growth pattern. Signal transduction pathways mediate transitions between different cell types. Nutritional signals induce rounded yeast-form cells either to enter invasive growth as elongated filamentous cells or to arrest to prepare for stationary phase, conjugation, or meiosis. An emerging theme is that development critically depends upon differential regulation of vegetative functions, including cytoskeletal organization and cell cycle progression, as much as on the expression of cell type specific gene products. © 1995.

Reid, B. Morris, B. M. and Gow, N. A. R. 1995. Calcium-dependent, genus-specific, autoaggregation of zoospores of phytopathogenic fungi. Experimental Mycology, 19, 202-213. Dense populations of zoospores of Phytophthora palmivora, Pythium catenulatum, and Pythium dissotocum formed multicell clumps, or autoaggregates. Autoaggregation was the result of active taxis and was shown to be density-dependent, calcium-requiring, and influenced by pH. In addition, autoaggregation appeared to be species-specific, since aggregates of Ph. palmivora did not attract zoospores of three Pythium species and aggregates of Py. catenulatum did not attract Ph. palmivora zoospores. Aggregation centers generated a calcium ion gradient and induced chemotropic growth of germ tubes emerging from zoospore cysts. Autoaggregation also functions to enhance zoospore accumulation at plant root surfaces, thereby increasing inoculum potential for infection. In the absence of roots, autoaggregation may enhance zoospore population survival. © 1995 Academic Press. All rights reserved.

Electrical currents associated with the plant‐fungus interaction between roots and an arbuscular mycorrhizal (AM) fungus Gigaspora margarita were measured with a one‐dimensional vibrating probe. Uninfected roots of clover (Trifolium repens L. cv. New Zealand White) and carrot (Daucus carota L. cv. Nantes) had an inwardly directed positive electrical current at the apical meristematic and elongation zones and an outward flow at the mature non‐growing regions. The current profile of endomycorrhizal colonized roots did not differ markedly in magnitude or pattern compared with non‐colonized roots of an equivalent stage of development. A small current was measured circulating around and through single mycorrhizal azygospores of G. margarita. Germ tubes emerged within the zone of outward current and an inward current was found at the hyphal tip. When roots were colonized by the fungus an inward current was induced at the point of hyphal contact with the host root. This current declined over a period of 6 d. Because hyphal penetration of plant roots by AM fungi does not rupture the plant host membrane we propose that the host current is not the result of wounding but is induced by fungal elicitors at the infection court. Copyright © 1995, Wiley Blackwell. All rights reserved

The Candido albicans clone cDNA10 was isolated on the basis that it encodes a protein which is immunogenic during infections in humans (R. K. Swoboda, G. Bertram, H. Hollander, D. Greenspan, J. S. Greenspan, N. A. R. Gow, G. W. Gooday, and A. J. P. Brown, Infect. Immun. 61:4263-4271, 1993). cDNA10 was used to isolate its cognate gene, and both the cDNA and gene were sequenced, revealing a major open reading frame with the potential to encode a basic protein of 256 amino acids with a predicted molecular weight of 29 kDa. Over its entire length, the open reading frame showed strong homology at both the nucleic acid (75 to 78%) and amino acid (79 to 81%) levels to two Saccharomyces cerevisiae genes encoding the 40S ribosomal protein, Rp10. Therefore, our C. albicans gene was renamed RP10. Northern (RNA) analyses in C. albicans 3153 revealed that RP10 expression is regulated in a manner very similar to that of S. cerevisiae ribosomal genes. The level of the RP10 mRNA decreased upon heat shock (from 25 to 45°C) and was tightly regulated during growth. Maximal levels of the mRNA were reached during mid-exponential phase before they decreased to negligible levels in stationary phase. The level of the RP10 mRNA was induced only transiently during the yeast-to-hyphal morphological transition but did not appear to respond to hyphal development per se.

Candida albicans HSP90 sequences were isolated by screening cDNA and genomic libraries with a probe derived from the Saccharomyces cerevisiae homolog, HSP82, which encodes a member of the heat shock protein 90 family of molecular chaperones. Identical sequences were obtained for the 2,197-bp overlap of the eDNA and gene sequences, which were derived from C. albicans 3153A and ATCC 10261, respectively. The C. albicans HSP90 gene contained no introns, and it showed strung homology (61 to 79% identity) to HSP90 sequences from other fungi, vertebrates, and plants. The C-terminal portion of the predicted Hsp90 amino acid sequence was identical to the 47-kDa protein which is thought to be immunoprotective during C. albicans infections (R. C. Matthews, J. Med. Microbiol. 36:367-370, 1992), confirming that this protein represents the C-terminal portion of the 81-kDa Hsp90 protein. Quantitative Northern (RNA) analyses revealed that C. albicans HSP90 mRNA was heat shock inducible and that its levels changed during batch growth, with its maximum levels being reached during the mid-exponential growth phase. HSP90 mRNA levels increased transiently during the yeast-to-hyphal transition but did not correlate directly with germ tube production per se. These data do not exclude a role for Hsp90 in the dimorphic transition. Southern blotting revealed only one HSP90 locus in the diploid C. albicans genome. Repeated attempts to disrupt both alleles and generate a homozygous C. albicans Δhsp90/Δhsp90 null mutant were unsuccessful. These observations suggest the existence of a single HSP90 locus which is essential for viability in C. albicans.

Zoospores of the plant pathogen Phytophthora palmivora use a number of tactic responses to target specific infectible regions of host roots. Although the dominant one is believed to be chemotaxis, it has been shown that zoospores of oomycetes may also use the exogenous proton/electrical currents generated by plant roots for guidance. Since these proton currents also generate significant pH gradients in the rhizosphere, the tactic response of zoospores to changes in pH was examined. Using 'swim-in' capillary tests, zoospores of P. palmivora were found to be repelled by solutions of high pH and attracted to solutions of low pH, relative to a control at neutrality. This in vitro tactic response was generally consistent with the measured pH at sites of zoospore accumulation around intact and wounded roots. However, the endogenous pH gradient around host roots could be abolished with buffers and this treatment did not affect the extent or pattern of zoospore accumulation. Therefore, detection of root-generated pH gradients is unlikely to have a major role in the homing response of zoospores towards plant roots.

In the dimorphic fungus Candida albicans, the CHS2 gene encodes a chitin synthase that is expressed preferentially in the hyphal form. Gene disruption of CHS2 in this diploid asexual fungus was achieved by the 'ura-blaster' protocol described for Saccharomyces [Alani, E. Cao, L. and Kleckner, N. (1987) Genetics 116, 541-545]. This involves the sequential disruption of multiple alleles by integrative transformation with URA3 as a single selectable marker. After disrupting the first CHS2 allele, the Ura- phenotype was recovered through cis recombination between repeated hisG sequences that flanked the URA3 marker in the disruption cassette, which was then used again to disrupt further CHS2 alleles. This method of gene disruption is well suited to the mutational analysis of this genetically recalcitrant human pathogen. Three rounds of disruption were required, suggesting that the strain SGY243 is triploid for the CHS2 locus. The resulting homozygous Δchs2::hisG null mutants were viable and made germ tubes with a normal morphology. The germ tubes were formed more slowly than parental strains in serum-containing medium and the germinating cells had a 40% reduction in their chitin content compared to germ tubes of the parent strain. The chitin content of the yeast form was not affected. A prototrophic strain of the chs2 null mutant was not attenuated significantly in its virulence when tested in normal and immunosuppressed mice.

Directional growth in response to electric fields (galvanotropism) is known for eukaryotic cells as diverse as fibroblasts, neurons, algae, and fungal hyphae. The mechanism is not understood, but all proposals invoke actin either directly or indirectly. We applied electric fields to bacteria (which are inherently free of actin) to determine whether actin was essential for galvanotropism. Field-treated (but not control) Enterobacter cloacae and Escherichia coli cells curved rapidly toward the anode. The response was both field strength and pH dependent. The direction of curvature was reversed upon reversal of field polarity. The directional growth was not due to passive bending of the cells or to field-induced gradients of tropic substances in the medium. Field-treated Bacillus subtilis cells also curved, but the threshold was much higher than for E. cloacae or E. coli. Since the curved morphology must reflect spatial differences in the rates of cell wall synthesis and degradation, we looked for regions of active wall growth. Experiments in which the cells were decorated with latex beads revealed that the anode-facing ends of cells grew faster than the cathode-facing ends of the same cells. Inhibitors of cell wall synthesis caused spheroplasts to form on the convex regions of field-treated cells, suggesting that the initial curvature resulted from enhanced growth of cathode-facing regions. Our results indicate that an electric field modulates wall growth spatially and that the mechanism may involve differential stimulation of wall growth in both anode- and cathode-facing regions. Electric fields may therefore serve as valuable tools for studies of bacterial wall growth. Use of specific E. coli mutants may allow dissection of the galvanotropic mechanism at the molecular level.

The opportunistic fungal pathogen Candida albicans produces secretory aspartyl proteinases, which are believed to be virulence factors in infection. We have studied the in vitro expression of seven known members of the SAP gene family in a range of strains and serotypes by Northern analysis. SAP1 and SAP3 were regulated during phenotypic switching between the white and opaque forms of the organism. The SAP2 mRNA, which was the dominant transcript in the yeast form, was found to be autoinduced by peptide products of Sap2 activity and to be repressed by amino acids. The expression of the closely related SAP4‐SAP6 genes was observed only at neutral pH during serum‐induced yeast to hyphal transition. No SAP7 mRNA was detected under any of the conditions or in any of the strains tested. Our data suggest that the various members of the SAP gene family may have distinct roles in the colonization and invasion of the host. Copyright © 1994, Wiley Blackwell. All rights reserved

The levels of pyruvate kinase (PYK), alcohol dehydrogenase (ADH1), phosphoglycerate kinase (PGK1) and phosphoglycerate mutase (GPM1) mRNAs were measured during batch growth and during the yeast‐to‐hyphal transition in Candida albicans. The four mRNAs behaved in a similar fashion. PYK1, ADH1, PGK1 and GPM1 mRNA levels were shown to increase dramatically during the exponential growth phase of the yeast form, and then to decrease to relatively low levels in the stationary phase. The dimorphic transition was induced using two sets of conditions: (i) an increase in temperature (from 25°C to 37°C) combined with the addition of serum to the medium; and (ii) an increase in temperature (from 25°C to 37°C) and an increase in pH of the growth medium (from pH 4.5 to pH 6.5). Additional cultures were analysed to control for the addition of serum, and for changes in temperature or pH. Immediately following dilution of late‐exponential cells into fresh media the levels of all four glycolytic mRNAs decreased rapidly in contrast to the ACT1 mRNA control, the level of which increased under most conditions. The recovery of glycolytic mRNA levels depended on the culture conditions, but there was no direct correlation with the formation of germ tubes, with the addition of serum to the medium, the Increase in culture temperature, the medium pH, or the glucose concentration. This indicates that the changes in glycolytic gene expression that accompany the dimorphic transition in C. albicans reflect the underlying physiological status of the cells during morphogenesis and not alterations to cell shape. Copyright © 1994, Wiley Blackwell. All rights reserved

When grown on a range of surfaces in conditions favouring hyphal growth, hyphae of Candida albicans grew in a right-handed helical fashion. This phenomenon was observed with eight strains and with two nutrient media. It is suggested that this is a result of rotation of the hyphal apex as it extends, which on some surfaces results in a helical hyphal wall, but which in a liquid results in a straight hypha. The consequence is that on a surface, a helically growing hypha will be exposed to a more diverse environment than a straight hypha. This phenomenon may have significance in the colonization of tissue by C. albicans.

When grown on a range of surfaces in conditions favouring hyphal growth, hyphae of Candida albicans grew in a right-handed helical fashion. This phenomenon was observed with eight strains and with two nutrient media. It is suggested that this is a result of rotation of the hyphal apex as it extends, which on some surfaces results in a helical hyphal wall, but which in a liquid results in a straight hypha. The consequence is that on a surface, a helically growing hypha will be exposed to a more diverse environment than a straight hypha. This phenomenon may have significance in the colonization of tissue by C. albicans. © 1994 Informa UK Ltd all rights reserved: reproduction in whole or part not permitted.

Candida albicans is a fungus that commonly infects the mucosal surface of humans. The hyphal growth form of this fungus may initiate the primary invasion of the host. Here we show that hyphae respond thigmotropically and morphologically to cues such as the presence of a surface, pores, grooves and ridges. Growth on some firm surfaces elicits a helical growth response. Hyphae follow grooves and ridges of inert substrates and penetrate pores of filtration membranes. Our in vitro experiments suggest that thigmotropism may enhance the ability of a hypha to invade epithelial of a host at sites of weakened integrity.

The level of the TEF3 mRNA, which encodes the fungal-specific translation elongation factor 3 (EF-3), was measured during the yeast-to-hyphal transition in Candida albicans. In contrast to a previous report, TEF3 mRNA levels were shown to change during dilution into fresh medium, increasing only transiently when dimorphism was induced by either (i) an increase in growth temperature (from 25°C to 37°C) combined with the addition of 10% (v/v) bovine calf serum to the medium, or (ii) an increase in growth temperature (from 25°C to 37°C) combined with an increase in the pH of the medium (from pH 4.5 to 6.5). TEF3 mRNA levels also increased in control cultures under conditions where germ tubes were not formed, but they remained elevated in contrast to cultures undergoing morphological changes. TEF3 mRNA levels were not significantly affected by heat-shock, but were tightly regulated during batch growth of the yeast form, reaching maximal levels in exponential phase. Therefore, the changes in TEF3 expression that accompany the dimorphic transition in C. albicans appear to reflect the underlying physiological changes that occur during morphogenesis and are not a response to morphogenesis per se. For this reason TEF3 mRNA measurement cannot be used as a loading control in Northern analyses of dimorphic gene regulation. Comparison of TEF3 mRNA levels with the abundance of the EF-3 polypeptide indicated that the synthesis of this essential translation factor might be subject to post-transcriptional regulation.

Galvanotropism is the directional growth response of organisms to externally applied electrical fields. The galvanotropic response of hyphae of Aspergillus nidulans, Neurospora crassa and Coprinus cinereus was pH-dependent. Alignment of hyphae of N. crassa towards the anode increased with increasing external pH. In contrast, hyphae of A. nidulans and C. cinereus became increasingly cathodotropic in media of more alkaline pH. Galvanotropism of each fungus had an isoelectric point at which the hyphae were unresponsive to the field. It is suggested that the electrical field changes the distribution of certain proteins in the cell membrane thereby inducing polarity. External pH may influence galvanotropism by altering the net charge on such proteins. The anodotropic response of Aspergillus fumigatus was dependent on exogenous calcium ions, decreasing in media with low external Ca2+. This suggests that Ca2+-channels may be involved in the regulation of galvanotropism. A model is presented that accounts for the various anodotropic, cathodotropic movements of different fungi and the pH and Ca2+-dependency of their responses to electrical fields. This involves an interplay between (i) the electromobilization of proteins in the cell membrane and (ii) electrical field-induced perturbation of calcium ion transport in the determination of the extent and direction of galvanotropism. © 1994, British Mycological Society. All rights reserved.

The calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ) and R24571 inhibited the yeast-to-mycelial transition of Candida albicans in vitro. This dimorphic switch is thought to potentiate the invasion of host tissues in superficial and systemic candidosis. Growth of six strains of C. albicans and a range of other pathogenic and saprophytic fungi was inhibited by TFP in vitro with an MIC (minimum inhibitory concentration) of. 25 mg kg-1 dose-1, too large to be considered of therapeutic value. This is in contrast to previous reports describing protective effects of TFP against candidiasis. Therefore, although TFP can inhibit growth and dimorphism of C. albicans in vitro, we conclude that this calmodulin antagonist is unsuitable as a chemotherapeutic agent.

The calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ) and R24571 inhibited the yeast-to-mycelial transition of Candida albicans in vitro. This dimorphic switch is thought to potentiate the invasion of host tissues in superficial and systemic candidosis. Growth of six strains of C. albicans and a range of other pathogenic and saprophytic fungi was inhibited by TFP in vitro with an MIC (minimum inhibitory concentration) of 25 mg kg-1 dose-1, too large to be considered of therapeutic value. This is in contrast to previous reports describing protective effects of TFP against candidiasis. Therefore, although TFP can inhibit growth and dimorphism of C. albicans in vitro, we conclude that this calmodulin antagonist is unsuitable as a chemotherapeutic agent. © 1993 Informa UK Ltd all rights reserved: reproduction in whole or part not permitted.

A cDNA library was made with mRNA from Candida albicans grown under conditions favoring the hyphal form. The library was screened for sequences that encode immunogenic proteins by using pooled sera from five patients with oral candidiasis and five uninfected patients. Most of these patients were human immunodeficiency virus positive. From 40,000 cDNA clones screened, 83 positive clones were identified. of these, 10 clones were chosen at random for further analysis. None of these 10 cDNAs were derived from a multigene family. The 5' and 3' ends of all 10 clones were analyzed by DNA sequencing. Two cDNAs were separate isolates of a sequence with strong homology to pyruvate kinase genes from other fungi (59 to 73%) and humans (60%). A third cDNA had strong sequence homology to the Saccharomyces cerevisiae and Kluyveromyces lactis alcohol dehydrogenase genes (68 to 73%). A fourth cDNA was homologous (81%) to an S. cerevisiae protein of unknown function. The functions of the remaining six C. albicans cDNAs are not known. A more detailed analysis of the clones encoding glycolytic enzymes revealed that sera from few patients recognized them as antigens. Therefore, although glycolytic enzymes constitute a group of C. albicans proteins that are immunogenic during oral and esophageal infections, their detection cannot be exploited as an accurate marker of infection.

Not all aspects of the interactions between fungal pathogens and their hosts are mediated by specific chemical signals, but until recently the functional significance of nonchemical cues (such as local electrical fields, pH gradients and host surface topography) was obscure. Recent studies of the mechanisms underlying fungal tropisms and taxes to such cues have revealed novel architectural and physiological properties of the fungal cells that contribute to their success as pathogens. © 1993.

Hyphal development in the dimorphic pathogenic fungus Candida albicans is thought to facilitate the primary invasion of surface epithelia during superficial infections. When mycelia were grown on Nuclepore membrane filters that were placed over serum-containing agar, the hyphae grew over the membrane surface and through the pores thereby crossing to the other side of the membrane. Hyphae that did not contact the lip of a pore did not enter it. The response was likely to be due to contact guidance (thigmotropism) and not chemotropism towards the nutrients since hyphae growing on the underside of the membrane also entered the pores then grew away from the underlying nutrient agar. The response therefore seems to be due to sensation of the substrate topography, and tropic movement in relation to changes in contour. This behaviour may enable the hyphae to penetrate epithelia at microscopic wound sites, membrane invaginations and other foci where the integrity of the epithelium is weak.

Hyphal development in the dimorphic pathogenic fungus Candida albicans is thought to facilitate the primary invasion of surface epithelia during superficial infections. When mycelia were grown on Nuclepore membrane filters that were placed over serum-containing agar, the hyphae grew over the membrane surface and through the pores thereby crossing to the other side of the membrane. Hyphae that did not contact the lip of a pore did not enter it. The response was likely to be due to contact guidance (thigmotropism) and not chemotropism towards the nutrients since hyphae growing on the underside of the membrane also entered the pores then grew away from the underlying nutrient agar. The response therefore seems to be due to sensation of the substrate topography, and tropic movement in relation to changes in contour. This behaviour may enable the hyphae to penetrate epithelia at microscopic wound sites, membrane invaginations and other foci where the integrity of the epithelium is weak. © 1992 Informa UK Ltd all rights reserved: reproduction in whole or part not permitted.

Plant roots generate electrical fields in the rhizosphere as a consequence of their ion transport activities. We show here that zoospores of the plant pathogen Phytophthora palmivora exhibit anodal electrotaxis in electrical fields ≥0.5 V m−1 comparable in size to the physiological fields around roots. An experimental protocol for applying weak electrical fields and quantifying electrotaxis is described. In this system, zoospore suspensions are isolated from the electrodes and their products using agarose bridges. Therefore, electrotaxis was not due to movement or trapping of zoospores in chemical, oxygen, pH or inhibitor gradients established by electrolysis. The electrophoretic and electroosmotic mobilities of encysted zoospores were measured. These forces did not influence the distribution of zoospores in electrotactic experiments at physiological field strengths. The electrotactic response saturated at fields above 10 V m−1 was inhibited in media of osmotic strength below 400 Osmol m−3, was maximal at pH 7.5 and increased at high zoospore densities. These data suggest that electrotaxis may be a useful adjunct to chemotaxis in root targeting by zoospores. Copyright © 1992, Wiley Blackwell. All rights reserved

Electrical currents are generated by tip-growing cells and have been proposed to be important in the establishment and maintenance of cell polarity. Unlike the situation in all other fungi thus far examined, the electrical current of Allomyces macrogynus normally flows out of the hyphal tip, not into it. In this organism, as with another chytrid Blastocladiella emersonii, the electrical current enters the rhizoids. Extracellular ion-selective microelectrodes detected local acidification of the medium around hyphal apices, but no local accumulation of K+ or Ca2+ at this site. These results suggest that the outward current is generated by H+ efflux, not K+ or Ca2+ efflux. The hyphae of this fungus were found to be weakly chemotropic, in contrast to the rhizoids which were strongly chemotropic. Rhizoid growth was also directed strongly towards the anode of an applied electrical field; the hyphae were directed to the cathode. Chemotropism and galvanotropism are therefore unrelated. We suggest that, as with other chytridomycetes, the rhizoid of A. macrogynus is an organ specializing in nutrient transport. Since transport is often coupled to proton symport the inward electrical current at rhizoids may be the consequence of localized proton-driven solute transport. In hyphae of the oomycete Achlya bisexualis inward current, localized nutrient transport and chemotropic behaviour again are correlated. We conclude that for water moulds, inward currents indicate zones of localized nutrient transport. The current is therefore incidental to the process of tip growth. © 1992, British Mycological Society. All rights reserved.

Abstract. Profiles of self‐generated ion currents associated with the growing primary root tips of intact Hordeum vulgare L. and Trifolium repens L. (nonnodulated) seedlings were measured using a highly sensitive vibrating electrode in media containing NH+4 or NO‐3, and compared to control roots growing in nitrogen free media. Under these three nutrient regimes, positive current entered the root at regions corresponding to the meristematic tissues and main elongation zones of root tips and left from the mature root tissues. Mapping the surface of the roots with a pH‐sensitive microelectrode revealed regions of external alkalinity where positive electrical current entered the root, and external acidity where positive current exited. The correlation between pH‐profile and the pattern of ion current generation in these experiments suggests that H+ ions were responsible for carrying the bulk of the root‐generated current. Assimilation of NHJ results in net H+ extrusion while assimilation of NO‐3, results in net OH‐3 efflux. Growth on NH+4, as compared to growth on NO‐3, stimulated the magnitude of the electrical current but did not affect significantly the growth rate of the roots. However, despite the differing stresses on internal pH regulation that arise due to growth on the two exogenous forms of combined nitrogen, the current profiles were qualitatively similar under the different conditions that were examined. The role of the circulating proton current is not yet known; however, the constancy of the current profile under different nutrient regimes sustains the hypothesis that the current may have a role in the regulation of root polarity. Copyright © 1991, Wiley Blackwell. All rights reserved

The rates of germ tube formation from growing and non-growing yeast cells of Candida albicans were investigated using a protocol for dimorphism regulated by temperature and pH. Stationary-phase cells formed germ tubes less rapidly than yeast cells that were preincubated in fresh growth medium prior to induction of dimorphism by an upshift in temperature or pH. On the basis of experiments using inhibitors of macromolecular biosyntheses it is suggested that the accelerated growth kinetics required de novo RNA and protein biosynthesis, but not DNA synthesis. The results suggest that metabolically active yeast cells are better able to undergo dimorphism than non-growing cells. © 1991.

This chapter discusses circulating ionic current on microorganisms such as bacteria, fungi, protozoa, and algae. Ionic currents are carried by charged particles, their flow creates an electrical current and a voltage gradient around the cell. These circulating ionic currents are carried by a variety of inorganic ions, most commonly protons or calcium ions. The ionic currents are often studied by measuring the extracellular component of the current using an ultrasensitive vibrating micro-electrode called a vibrating probe. Ionic currents are very common in nature. They are found during the normal course of growth and development, and are associated with initiation and maintenance of growth and with regrowth and damage repair. Studies of fungi and of certain photosynthetic algae show that some currents may arise from the necessity to localize electrogenic nutrient transport to support heterotrophic or autotrophic growth, respectively. This leads to the conclusion that the current need not necessarily be related to control of cellular polarity and of development. © 1989, Academic Press Limited

Yeast cells of Candida albicans which had been attached to polylysine-coated microscope slides were induced to form buds or germ tubes in the presence of external electrical fields. The sites of budding and germ tube formation and the growth of germ tubes and hyphal branches were polarized preferentially towards the cathode. Buds were not converted to pseudohyphae or germ tubes by the field and the field had no effect on the positioning of nuclei or septa in the yeast cell or germ tube. Buds were less polarized than germ tubes at any given applied voltage. The polarization of buds reached a peak at an electrical field of 12 mV per cell. Polarization of germ tubes was biphasic, increasing rapidly with increasing field strengths up to 5 mV per cell, and then more slowly in stronger fields. An electrical field was only required for a fraction of the time taken for germ tubes to start to form, so cells retained a memory of experiencing an electrical field which influenced the selection of sites of evagination. Increasing the electrical field delayed the time of germ tube evagination and inhibited the rate of germ tube extension. Unlike previous findings with other filamentous fungi, germ tubes grew unidirectionally towards the cathode for extended periods and did not deviate to a perpendicular orientation. This result suggests that the septal pore of the filamentous form may have high electrical resistance and would act as an effective barrier to solute transport between intercalary compartments.

Non-dimorphic variants of Candida albicans, which were unable to form germ tubes or mature hyphae in media containing amino acids, glucose and salts or N-acetylglucosamine or serum, were prepared from two hyphal positive laboratory strains using a physical separation method. The hyphal-minus phenotype was stable and may be due to mutations or phenotypic variation. The variant strains maintained their internal pH within narrower bounds as compared to their parental wild-types. When exposed to conditions that normally supported the induction of germ tubes the cytoplasmic pH of the wild type strains increased from 6.8 to over pH 8.0 within 5 min while in the variants the rise in internal pH was only about 0.3 pH units. The wild type strains acidified the growth medium more rapidly than the variants. The results suggest that the control of internal pH is directly or indirectly associated with the regulation of dimorphism. The variants had unaltered cell volumes and specific growth rates. The hyphal-minus phenotype was however fully reversible since revertants occurred spontaneously on serum containing agar. © 1989 Springer-Verlag.

The electrical currents associated with developing primary root tips of Triticum aestivum L. cv. Slejpner, Avena sativa L. cv. Victory I, Lolium perenne L. cv. Melle, Vigna radiata (L.) Wilczek, Arachis hypogaea L. Pisum sativum L. cv. Keluedon Wonder, Lonchocarpus leucanthus Burk, Dalbergia nigra Fr. Allen and Picea abies (L.) Karst. (U. K. Forestry Commission Number: 85/498/B). and that of the adventitious root tips of Fragaria vesca L. Solanum tuberosum L. cv. Maris Piper and Neptunia plena Lindl. were examined with a vibrating electrode. Current was found consistently to enter the meristematic and elongating tissues of all the intact growing roots examined. Mature non‐growing root regions were responsible for generating the outword limb of the current loop. Peak inward current densities ranged between 2 mA m−2 (Lolium) and 28 mA m−2 (Arachis). The point at which the inward current reversed to outward current also varied between species. These results, which are derived from 5 taxonomically diverse families (Graminae, Leguminosae, Rosaceae, Solanaceae and Pinaceae) extend the range of different species that have been shown to generate ion currents that transverse, in a highly polar manner, the growing regions of their root systems. This supports the correlations between endogenous current generation and root development. Copyright © 1989, Wiley Blackwell. All rights reserved

In contrast to all filamentous fungi examined to date, vegetative hyphae of Allomyces macrogynus, whether extending or not, produced an outward flow of positive electrical current, at a maximum of 0.16 μA cm-2 around 40 μm behind the apex, as measured with a vibrating probe. Inward currents of up to 0.55 μA cm-2 were recorded around the rhizoids. Increases in outward current were observed in hyphae pre-grown under oxygen deficiency and then allowed to widen backwards to the hyphal base in sufficient oxygen. When spores were germinated in an applied electrical field they produced rhizoids predominantly towards the anode. Hyphae were produced initially towards the cathode but later bent around towards the anode. Experiments with a range of chemicals provided no evidence for the involvement of calcium in vegetative growth and development in A. macrogynus. Polyoxin and nikkomycin, inhibitors of chitin synthesis, had no effect on swimming zoospores, but inhibited wall formation of cysts, rhizoids and forward and backward growing hyphae. © 1988 Springer-Verlag.

Weak acids were used to measure the internal pH of yeast cells of Candida albicans that had been induced to form buds or germ tubes. Under conditions that supported germ tube formation the internal pH rose from around 6.8 to over 8.0 after 30 min in two different induction media. Internal pH measured by 31P NMR confirmed this pattern and also showed that the internal pH fell to around 7.0 prior to the outgrowth of germ tubes. Conditions which led to budding induced less cytoplasmic alkalinization. This alkalinization was brought about when cells were inoculated into media of neutral pH and at an increased temperature. Increasing the temperature of the medium augmented the alkalinization of the cytoplasm induced by raising the external pH. Strains of C. albicans defective in the ability to produce germ tubes did not show this dramatic cytoplasmic alkalinization under conditions which normally supported filamentous growth. The raising of internal pH may be due to the activation of the plasma membrane proton-pumping ATPase since diethylstilboestrol inhibited the cytoplasmic alkalinization and germ tube formation without causing irreversible loss of cell viability. The results show that the induction of the dimorphic transition in this organism is accompanied by a steep rise in internal pH. It is not known whether these changes are the cause or consequence of morphogenesis.

Abstract Growing roots of Nicotiana tabacum var. Havana generate transcellular ion currents which traverse developing and wounded tissues. Positive current of around 10 mA m−2 enters meristematic and elongating cells at the tip of primary roots. The growing tips of first order laterals are also traversed by a similar positive current with a density of around 2.0 mA m−2, as are immature laterals emerging at the primary root surface. These self‐generated ion currents flow basipetally through developing tissues and leave from mature non‐elongating tissue. A large positive current of around 70 mA m−2 also enters induced wound sites on the primary root surface. Motile zoospores of the fungal pathogen Phytophthora parasitica var. nicotianae have been reported to associate preferentially with these regions of the root. This might suggest that electrotaxis may be part of the mechanism by which zoospores locate root regions susceptable to fungal infection. Copyright © 1988, Wiley Blackwell. All rights reserved

The cytology of nuclear division and septation in the yeast and hyphal phases of Candida albicans growing at 37 degrees C has been studied by fluorescence microscopy after staining of specimens with 4'6-diaminido-2-phenylindole (DAPI) and Calcofluor. Yeast and hyphal cells replicated their nuclei at about 18 min after the emergence of a bud or germ-tube. The site of nuclear division coincided with the future location of the septum in both forms. This occurred at the junction of the bud and parent yeast cell or 6.0 micron from the parent yeast in germ tubes which were formed in medium containing serum. The filamentous forms of a range of clinical and laboratory strains grown in a variety of germ tube-inducing media were all extensively vacuolated. Germ tube extension in all of these media was linear. It is suggested that there is little biosynthesis of cytoplasm during the initial stages of germ tube growth in this organism and that this accounts for the development of the large vacuoles and the linear growth kinetics.

A vibrating probe was used to detect and measure electrical currents at the surface of excised leaves and isolated leaf epidermis from Commelina commnunis. Currents of up to 4.0 μA cm-2 moving out from the leaf surface were observed when the stomata were open. When the stomata were almost closed no current was detected and when they were fully closed a small inwardly directed current was observed. There appeared to be a linear relationship between current and stomatal aperture. The current was stimulated by fusicoccin and eliminated by increasing the external pH suggesting that it was brought about by a flow of H+ from the leaf surface. © 1986 Oxford University Press.

A model based on morphological and ultrastructural evidence is presented which illustrates a novel and hitherto undescribed pattern of germ tube formation and hyphal growth in early and mature colonies of Candida albicans. Accordingly, most of the cytoplasm within the parent yeast cell migrates into and forward with the extending germ tubes and leaves behind an extensively vacuolated yeast cell. Growing hyphae similarly are subtended by migrating slugs of protoplasm and leave behind vacuolated intercalary compartments. The vacuolated cell compartments apparently must first regenerate their protoplasmic contents before producing branches or secondary germ tubes. This model is used to explain certain unusual features of the growth kinetics of the filamentous form of this organism. © 1984 Informa UK Ltd all rights reserved: reproduction in whole or part not permitted.

Achlya, like other tip-growing organisms, generates an endogenous electrical current such that positive charge flows into the hyphal apex and exits from the trunk. The present study is concerned with the mechanism of current generation by hyphae growing in a defined, complete medium. The intensity of the current, measured in the extracellular medium with a vibrating probe, was unaffected by the removal of all the inorganic constituents of the growth medium. However, an increase in the external pH or the deletion of amino acids abolished the current. Removal of methionine alone diminished the current by two thirds. Hyphae also generated a longitudinal pH gradient in the extracellular medium; the region surrounding the tip was more alkaline than the bulk medium, whereas the region around the trunk was relatively acidic. These findings suggest that a flux of protons, dependent upon amino acids in the medium, carries current into the tip and creates the surrounding alkaline zone. The proton current appears to result from the transport of amino acids rather than their metabolism. Conditions that abolished the current also inhibited methionine uptake but had little effect on the respiratory rate. The findings imply a connection between the proton current and chemiosmotic energy transduction. We propose that protons flow into the hyphal tip through amino acid/proton symporters that are preferentially localized there. The proton flux energizes the uptake of amino acids into the growing zone and may also contribute to the polarization of hyphal growth.

Observations of chitin preparations from hyphae of Candida albicans suggest that the septum is made of two juxtaposed plates rich in chitin microfibrils. Thus the trilaminate appearance of sections of septa seen in electron micrographs can be interpreted as two plates of densely staining chitin-rich material separated by a lightly staining area. This model may be applicable to septa of other fungi. © 1983.

Chitin microfibrils exposed by chemical extraction of hyphal walls of Candida albicans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidiodes brasiliensis, Coprinus cinereus and Mucor mucedo were of variable morphology but gave identical infrared spectra and behaved as pure chitin in chromatographic analyses. The microfibrils of the four dimorphic fungi studied were shorter than those in the moulds C. cinereus and M. mucedo but were similar to those reported for the yeast Saccharomyces cerevisiae. In C. albicans the microfibrils in the septal plates of hyphae were predominantly tangentially orientated and were longer than those in the lateral walls. Microfibrils produced by chitin synthase in vitro were very much longer than any observed from hyphal preparations. © 1983 Springer-Verlag.

The growth and development of mycelia of the dimorphic fungus C. albicans in serum-containing medium is described. Initially, colonies are undifferentiated (all hyphae in the mycelium having approximately the same diameter, extension rate, apical and intercalary compartment lengths) whereas older mycelia differentiate at the colony margin to produce leading hyphae that are wider, faster growing and have longer apical and intercalary compartment lengths than the branches they subtend. Early colony development exhibits unusual features; germ tube extension is linear (not exponential as in other fungi) and there is a prolonged delay between septation and the onset of branch formation. The subsequent patterns of growth and branching are similar in all other respects to those of other mycelial moulds. Mycelia have septa that delimit single nuclei with compartments. The septa do not prevent cytoplasmic flow and consequently allow the peripheral growth zone to span several compartments. From these results the authors conclude that filamentous growth of C. albicans in this medium is best described as truly mycelial.

During germination of yeast cells of C. albicans in liquid or solid serum-containing media the parent yeast cell and then sub-apical regions of the emerging germ tube became extensively vacuolated. Intercalary compartments were often almost entirely vacuolated, while the apices of germ tubes and branches maintained a high cell solids content. This length of non-vacuolate hypha may correspond to the growth zone of the organism. These observations may explain the observed unexpected linear growth of these germ tubes and the delay between septation and branch formation during filamentous growth.

The ultrastructure of the septum in the mycelial phase of the dimorphic fungus Candida albicans has been studied both in thin sections of fixed material and in shadow casts of the chemically purified chitinous wall layer. The septum has a 25-nm central micropore which would not allow the passage of nuclei or mitochondria, thus delimiting these organelles within the mycelial compartments without preventing cytoplasmic continuity. © 1980, Springer-Verlag New York Inc. All rights reserved.