This chapter discusses the basic concepts related to transcript analysis. mRNA abundance measurements provide a useful tool to study gene regulation. Currently, most groups use such measurements to investigate the expression of a small number of specific genes. However, with the advent of genomic sequencing projects and the completion of the yeast genome sequence, genomic transcript analyses are being exploited to generate global pictures of gene regulation. By necessity, genome-scale transcript analyses are focusing mainly on the open reading frames (ORFs) themselves—that is, coding sequences. However, the secondary and tertiary structures formed by these sequences and their 5′-leader and 3′-trailer regions exert strong influences on gene expression at post-transcriptional levels. Hence, these issues are an integral part of transcript analysis. The chapter discusses classical methods for the analysis of specific mRNAs. It describes new technologies that are being developed for gene expression analysis and focuses mainly on mRNA abundance measurements. Methods for the analysis of transcript structure and stability are also discussed in the chapter.

Previous work has implicated the Hog1 stress-activated protein kinase (SAPK) in osmotic and oxidative stress responses in the human pathogen Candida albicans. In this study, we have characterized the role of Hog1 in mediating these and other stress responses in C. albicans. We provide evidence that a SAPK-dependent core stress response exists in this pathogen. The Hog1 SAPK is phosphorylated and it accumulates in the nucleus in response to diverse stress conditions. In addition, we have identified Hog1-regulated genes that are induced in response to stress conditions that activate Hog1. These analyses reveal both activator and repressor functions for the Hog1 SAPK. Our results also demonstrate that stress cross-protection, a classical hallmark of the core stress response, occurs in C. albicans between stresses that activate the Hog1 SAPK. Importantly, we find that the core stress response in C. albicans has adapted to the environmental niche of this human pathogen. This niche specificity is reflected by the specific environmental conditions that drive the Hog1-regulated core stress response in C. albicans and by differences in the molecular circuitry that control this response.

Real-time monitoring of the spatial and temporal progression of infection/gene expression in animals will contribute greatly to our understanding of host-pathogen interactions while reducing the number of animals required to generate statistically significant data sets. Sensitive in vivo imaging technologies can detect low levels of light emitted from luciferase reporters in vivo, but the existing reporters are not optimal for fungal infections. Therefore, our aim was to develop a novel reporter system for imaging Candida albicans infections that overcomes the limitations of current luciferase reporters for this major fungal pathogen. This luciferase reporter was constructed by fusing a synthetic, codon-optimized version of the Gaussia princeps luciferase gene to C. albicans PGA59, which encodes a glycosylphosphatidylinositol-linked cell wall protein. Luciferase expressed from this PGA59-gLUC fusion (referred to as gLUC59) was localized at the C. albicans cell surface, allowing the detection of luciferase in intact cells. The analysis of fusions to strong (ACT1 and EFT3), oxidative stress-induced (TRX1, TRR1, and IPF9996), and morphogenesis-dependent (HWP1) promoters confirmed that gLUC59 is a convenient and sensitive reporter for studies of gene regulation in yeast or hyphal cells, as well as a flexible screening tool. Moreover, the ACT1-gLUC59 fusion represented a powerful tool for the imaging of disease progression in superficial and subcutaneous C. albicans infections. gLUC59 and related cell surface-exposed luciferase reporters might find wide applications in molecular biology, cell biology, pathobiology, and high-throughput screens.

We have tested the clones used in the European Yeast Chromosome III Sequencing Programme for possible artefacts that might have been introduced during cloning or passage through Escherichia coli. Southern analysis was performed to compare the BamHI, EcoRI, HindIII and PstI restriction pattern for each clone with that of the corresponding locus on chromosome III in the parental yeast strain. In addition, further enzymes were used to compare the restriction maps of most clones with the map predicted by the nucleotide sequence (Oliver et al. 1992). Only four of 506 6‐bp restriction sites predicted by the sequence were not observed experimentally. No significant cloning artefacts appear to disrupt the published sequence of chromosome III. The restriction patterns of six yeast strains have also been compared. In addition to two previously identified sites of Ty integration on chromosome III (Warmington et al. 1986; Stucka et al. 1989; Newlon et al. 1991), a new polymorphic site involving Ty retrotransposition (the Far Right‐Arm transposition Hot‐Spot, FRAHS) has been identified close to CRY1. On the basis of simple restriction polymorphisms, the strains S288C, AB972 and W303‐1b are closely related, while XJ24‐24a and J178 are more distant relatives of S288C. A polyploid distillery yeast is heterozygous for many polymorphisms, particularly on the right arm of the chromosome.

Messenger RNA (mRNA) translation is an essential step in eukaryotic gene expression that contributes to the regulation of this process. We describe a deterministic model based on ordinary differential equations that describe mRNA translation in Saccharomyces cerevisiae. This model, which was parameterized using published data, was developed to examine the kinetic behaviour of translation initiation factors in response to amino acid availability. The model predicts that the abundance of the eIF1–eIF3–eIF5 complex increases under amino acid starvation conditions, suggesting a possible auxiliary role for these factors in modulating translation initiation in addition to the known mechanisms involving eIF2. Our analyses of the robustness of the mRNA translation model suggest that individual cells within a randomly generated population are sensitive to external perturbations (such as changes in amino acid availability) through Gcn2 signalling. However, the model predicts that individual cells exhibit robustness against internal perturbations (such as changes in the abundance of translation initiation factors and kinetic parameters). Gcn2 appears to enhance this robustness within the system. These findings suggest a trade-off between the robustness and performance of this biological network. The model also predicts that individual cells exhibit considerable heterogeneity with respect to their absolute translation rates, due to random internal perturbations. Therefore, averaging the kinetic behaviour of cell populations probably obscures the dynamic robustness of individual cells. This highlights the importance of single-cell measurements for evaluating network properties. Copyright \circledC 2010 John Wiley & Sons, Ltd.

A transcriptome analysis was performed of Saccharomyces cerevisiae undergoing isoamyl alcohol-induced filament formation. In the crucial first 5 h of this process, only four mRNA species displayed strong and statistically significant increases in their levels of more than 10-fold. Two of these (YEL071w/DLD3 and YOL151w/GRE2) appear to play important roles in filamentation. The biochemical activities ascribed to these two genes (d-lactate dehydrogenase and methylglyoxal reductase, respectively) displayed similarly timed increases to those of their respective mRNAs. Mutants carrying dld3 mutations displayed reduced filamentation in 0.5% isoamyl alcohol and needed a higher concentration of isoamyl alcohol to effect more complete filament formation. Hence, DLD3 seems to be required for a full response to isoamyl alcohol, but is not absolutely essential for it. Mutants carrying gre2 mutations were derepressed for filament formation and formed large, invasive filaments even in the absence of isoamyl alcohol. These results indicate a previously unsuspected and novel role for the GRE2 gene product as a suppressor of filamentation by virtue of encoding isovaleraldehyde reductase activity.

The protective capacity of a parenterally administered β-glucan-conjugate vaccine formulated with the human-compatible MF59 adjuvant was assessed in a murine model of vaginal candidiasis. To monitor infection, an in vivo imaging technique exploiting genetically engineered, luminescent Candida albicans was adopted, and compared with measurements of colony forming units. The vaccine conferred significant protection, and this was associated with production of serum and vaginal anti-β-glucan IgG antibodies. Vaginal IgG molecules were the likely mediators of protection as inferred by the efficacy of passive transfer of immune vaginal fluid and passive protection by an anti-β-1,3-glucan mAb. Overall, the in vivo imaging technique was more reliable than vaginal CFU counts in assessing the extent and duration of the vaginal infection, and the consequent protection level.

Fungal APSES proteins regulate morphogenetic processes, including filamentation and differentiation. The human fungal pathogen Candida albicans contains two APSES proteins: the regulator Efg1p and its homologue Efh1p, described here. Overexpression of EFG1 or EFH1 led to similar phenotypes, including pseudohypha formation and opaque-white switching. An efh1 deletion generated no phenotype under most conditions but caused hyperfilamentation in an efg1 background under embedded or hypoxic conditions. This suggests cooperation of these APSES proteins in the suppression of an alternative morphogenetic signaling pathway. Genome-wide transcriptional profiling revealed that EFG1 and EFH1 regulate partially overlapping sets of genes associated with filament formation. Unexpectedly, Efg1p not only regulates genes involved in morphogenesis but also strongly influences the expression of metabolic genes, inducing glycolytic genes and repressing genes essential for oxidative metabolism. Using one- and two-hybrid assays, we further demonstrate that Efg1p is a repressor, whereas Efh1p is an activator of gene expression. Overall, the results suggest that Efh1p supports the regulatory functions of the primary regulator, Efg1p, and indicate a dual role for these APSES proteins in the regulation of fungal morphogenesis and metabolism.

The evolutionarily conserved heat shock transcription factor Hsf1 plays a central role in thermal adaptation in the major fungal pathogen of humans, Candida albicans. Hsf1 becomes hyperphosphorylated in response to heat shock and activates the transcription of genes with heat shock elements (HSEs) in their promoters, these genes contributing to thermal adaptation. However, the relevance of Hsf1 activation to C. albicans virulence is not clear as this pathogen is thought to be obligately associated with warm blooded animals, and this issue has not been tested because HSF1 is essential for viability in C. albicans. In this study, we demonstrate that the HSE regulon is active in C. albicans cells infecting the kidney. We also show the CE2 region of Hsf1 is required for activation and that the phosphorylation of specific residues in this domain contributes to Hsf1 activation. C. albicans HSF1 mutants that lack this CE2 region are viable. However, they are unable to activate HSE-containing genes in response to heat shock, and they are thermosensitive. Using this HSF1 CE2 deletion mutant we demonstrate that Hsf1 activation, and hence thermal adaptation, contributes significantly to the virulence of C albicans.

The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H+- ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H+-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak- acid stress. The weak acid did not appear to affect the synthesis of ATP because CO2 production and O2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one- third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth.

Novel luciferase reporters have been developed that allow real-time monitoring of infections by the fungal pathogens Candida albicans and Aspergillus fumigatus. Although these reporters still suffer limitations in the context of invasive infections, they provide unprecedented tools to monitor superficial infections and the efficacy of antifungal drugs or vaccines. In particular, the sensitivity and ease of detection of the cell-surface Gaussia princeps luciferase developed for C. albicans should make it a powerful tool for functional genomics studies in this and other pathogenic fungi.

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.

Candida glabrata is considered a major opportunistic fungal pathogen of humans. The capacity of this yeast species to cause infections is dependent on the ability to grow within the human host environment and to assimilate the carbon sources available. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources, such as carboxylic acids, contributes to the virulence of this fungus. Transcriptional studies on C. glabrata cells identified a similar response, upon nutrient deprivation. In this work, we aimed at analyzing biofilm formation, antifungal drug resistance, and phagocytosis of C. glabrata cells grown in the presence of acetic acid as an alternative carbon source. C. glabrata planktonic cells grown in media containing acetic acid were more susceptible to fluconazole and were better phagocytosed and killed by macrophages than when compared to media lacking acetic acid. Growth in acetic acid also affected the ability of C. glabrata to form biofilms. The genes ADY2a, ADY2b, FPS1, FPS2, and ATO3, encoding putative carboxylate transporters, were upregulated in C. glabrata planktonic and biofilm cells in the presence of acetic acid. Phagocytosis assays with fps1 and ady2a mutant strains suggested a potential role of FPS1 and ADY2a in the phagocytosis process. These results highlight how acidic pH niches, associated with the presence of acetic acid, can impact in the treatment of C. glabrata infections, in particular in vaginal candidiasis.

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose-grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate-grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose-grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate-grown cells. We identified mating and pheromone-regulated proteins that were exclusive to lactate-grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa-specific and other niche-specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.

Neutrophils are key players during Candida albicans infection. However, the relative contributions of neutrophil activities to fungal clearance and the relative importance of the fungal responses that counteract these activities remain unclear. We studied the contributions of the intra- and extracellular antifungal activities of human neutrophils using diagnostic Green Fluorescent Protein (GFP)-marked C. albicans strains. We found that a carbohydrate starvation response, as indicated by up-regulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus. Similarly, the nitrosative stress response was only observed in internalised fungal cells. In contrast, the response to oxidative stress was observed in both phagocytosed and non-phagocytosed fungal cells, indicating that oxidative stress is imposed both intra- and extracellularly. We assessed the contributions of carbohydrate starvation, oxidative and nitrosative stress as antifungal activities by analysing the resistance to neutrophil killing of C. albicans mutants lacking key glyoxylate cycle, oxidative and nitrosative stress genes. We found that the glyoxylate cycle plays a crucial role in fungal resistance against neutrophils. The inability to respond to oxidative stress (in cells lacking superoxide dismutase 5 or glutathione reductase 2) renders C. albicans susceptible to neutrophil killing, due to the accumulation of reactive oxygen species (ROS). We also show that neutrophil-derived nitric oxide is crucial for the killing of C. albicans: a yhb1¿/¿ mutant, unable to detoxify NO•, was more susceptible to neutrophils, and this phenotype was rescued by the nitric oxide scavenger carboxy-PTIO. The stress responses of C. albicans to neutrophils are partially regulated via the stress regulator Hog1 since a hog1¿/¿ mutant was clearly less resistant to neutrophils and unable to respond properly to neutrophil-derived attack. Our data indicate that an appropriate fungal response to all three antifungal activities, carbohydrate starvation, nitrosative stress and oxidative stress, is essential for full wild type resistance to neutrophils.

The distinctive properties of satellite DNA molecules containing the genes for ribosomal RNA in Physarum polycephalum permits their identification in total, unfractionated nuclear DNA in the foldback form, after denaturation and fast annealing. Using the electron microscope the location and properties of three characteristic regions containing tandemly-repeated, inverted sequences have been investigated. At least two additional regions, also containing tandem repeats, are shown to be present and located towards each end of the rDNA molecule, at a site adjacent to the segment coding for the 26 S rRNA. All the regions which contain tandem repeats are composed of sequences which, within experimental error, appear to share a common unit repeat length of about 90 nucleotides.

Strife concerning the accessibility of essential trace elements, such as transition metals, represents an important aspect of the dynamic interaction between a pathogenic fungus and its mammalian host. The host defends itself against infection by sequestering these essential micronutrients away from the invading pathogen via a phenomenon termed “nutritional immunity”. In turn, the fungus employs an array of tactics (scavenging and storage) to hoard micronutrients and support growth when these resources are scarce. In addition, micronutrient limitation triggers the expression of virulence determinants that can aggravate disease

The HST7 gene of Candida albicans encodes a protein with structural similarity to MAP kinase kinases. Expression of this gene in Saccharomyces cerevisiae complements disruption of the Ste7 MAP kinase kinase required for both mating in haploid cells and pseudohyphal growth in diploids. However, Hst7 expression does not complement loss of either the Pbs2 (Hog4) MAP kinase kinase required for response to high osmolarity, or loss of the Mkk1 and Mkk2 MAP kinase kinases required for proper cell wall biosynthesis. Intriguingly, HST7 acts as a hyperactive allele of STE7; expression of Hst7 activates the mating pathway even in the absence of upstream signaling components including the Ste7 regulator Ste11, elevates the basal level of the pheromone-inducible FUS1 gene, and amplifies the pseudohyphal growth response in diploid cells. Thus Hst7 appears to be at least partially independent of upstream activators or regulators, but selective in its activity on downstream target MAP kinases. Creation of Hst7/Ste7 hybrid proteins revealed that the C-terminal two-thirds of Hst7, which contains the protein kinase domain, is sufficient to confer this partial independence of upstream activators.

Most biotechnology companies that exploit microbial fermentations are actively seeking methods of improving the productivity or quality of their fermentations. On the one hand, strain manipulation is becoming easier, as molecular tool boxes grow ever more refined and microbial genome sequence information expands exponentially. On the other hand, the molecular manipulation of metabolic pathways appears to be becoming more complex, as many attempts to increase the flux through specific pathways have met with hmited success. Although such problems were predicted many years ago by metabolic control analysis (MCA), many molecular biologists remain blissfully unaware of this area. Therefore, a recent European meeting*,which brought together industrial and academic researchers with expertise ranging from metabolic modelling to molecular genetics, was particularly timely.

Deadly infections from opportunistic fungi have risen in frequency, largely because of the at-risk immunocompromised population created by advances in modern medicine and the HIV/AIDS pandemic. This review focuses on dynamics of the fungal polysaccharide cell wall, which plays an outsized role in fungal pathogenesis and therapy because it acts as both an environmental barrier and as the major interface with the host immune system. Human fungal pathogens use architectural strategies to mask epitopes from the host and prevent immune surveillance, and recent work elucidates how biotic and abiotic stresses present during infection can either block or enhance masking. The signaling components implicated in regulating fungal immune recognition can teach us how cell wall dynamics are controlled, and represent potential targets for interventions designed to boost or dampen immunity.

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.

In the pathogenic fungus, Candida albicans, Nrgl down-regulates the expression of morphogenetic genes and is presumed to act as a transcriptional repressor. In contrast, Gcn4 up-regulates amino acid biosynthetic genes and is presumed to be a transcriptional activator. However, these presumptions remain to be tested directly. A classic approach has been to use a one-hybrid assay that exploits the Escherichia coli lexA protein fusions. However in C albicans, the alternate decoding of CUG as serine prevents the expression of heterologous genes such as lexA, which contain numerous CUG codons. Therefore, we have developed a one-hybrid system, based on the Staphylococcus aureus lexA gene, as a tool for one-hybrid analyses of transcription factors in C. dalbicans. Using this one-hybrid system we have confirmed directly the positive and negative transcriptional activities of Nrgl and Gcn4 in C. albicans. (c) 2005 Elsevier Inc. All rights reserved.

Summary the major fungal pathogen Candida albicans has the metabolic flexibility to assimilate a wide range of nutrients in its human host. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources contributes to its virulence. JEN1 encodes a monocarboxylate transporter in C. albicans and we show that its paralogue, JEN2, encodes a novel dicarboxylate plasma membrane transporter, subjected to glucose repression. A strain deleted in both genes lost the ability to transport lactic, malic and succinic acids by a mediated mechanism and it displayed a growth defect on these substrates. Although no significant morphogenetic or virulence defects were found in the double mutant strain, both JEN1 and JEN2 were strongly induced during infection. Jen1-GFP (green fluorescent protein) and Jen2-GFP were upregulated following the phagocytosis of C. albicans cells by neutrophils and macrophages, displaying similar behaviour to an Icl1-GFP fusion. In the murine model of systemic candidiasis approximately 20-25% of C. albicans cells infecting the kidney expressed Jen1-GFP and Jen2-GFP. Our data suggest that Jen1 and Jen2 are expressed in glucose-poor niches within the host, and that these short-chain carboxylic acid transporters may be important in the early stages of infection.

Heat shock protein 90 (HSP90) is an essential, abundant and ubiquitous eukaryotic chaperone that has crucial roles in protein folding and modulates the activities of key regulators. The fungal Hsp90 interactome, which includes numerous client proteins such as receptors, protein kinases and transcription factors, displays a surprisingly high degree of plasticity that depends on environmental conditions. Furthermore, although fungal Hsp90 levels increase following environmental challenges, Hsp90 activity is tightly controlled via post-translational regulation and an autoregulatory loop involving heat shock transcription factor 1 (Hsf1). In this Review, we discuss the roles and regulation of fungal Hsp90. We propose that Hsp90 acts as a biological transistor that modulates the activity of fungal signalling networks in response to environmental cues via this Hsf1–Hsp90 autoregulatory loop.

Nutritional immunity - the withholding of nutrients by the host - has long been recognised as an important factor that shapes bacterial-host interactions. However, the dynamics of nutrient availability within local host niches during fungal infection are poorly defined. We have combined laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS), MALDI imaging and immunohistochemistry with microtranscriptomics to examine iron homeostasis in the host and pathogen in the murine model of systemic candidiasis. Dramatic changes in the renal iron landscape occur during disease progression. The infection perturbs global iron homeostasis in the host leading to iron accumulation in the renal medulla. Paradoxically, this is accompanied by nutritional immunity in the renal cortex as iron exclusion zones emerge locally around fungal lesions. These exclusion zones correlate with immune infiltrates and haem oxygenase 1-expressing host cells. This local nutritional immunity decreases iron availability, leading to a switch in iron acquisition mechanisms within mature fungal lesions, as revealed by laser capture microdissection and qRT-PCR analyses. Therefore, a complex interplay of systemic and local events influences iron homeostasis and pathogen-host dynamics during disease progression.

Candida albicans is a major fungal pathogen of humans. It regulates its morphology in response to various environmental signals, but many of these signals are poorly defined. We show that amino acid starvation induces filamentous growth in C.albicans. Also, starvation for a single amino acid (histidine) induces CaHIS4, CaHIS7, CaARO4, CaLYS1 and CaLYS2 gene expression in a manner reminiscent of the GCN response in Saccharomyces cerevisiae. These morphogenetic and GCN-like responses are both dependent upon CaGcn4, which is a functional homologue of S.cerevisiae Gcn4. Like ScGcn4, CaGcn4 activates the transcription of amino acid biosynthetic genes via the GCRE element, and CaGcn4 confers resistance to the histidine analogue, 3-aminotriazole. CaGcn4 interacts with the Ras-cAMP pathway to promote filamentous growth, but the GCN-like response is not dependent upon morphogenetic signalling. CaGcn4 acts as a global regulator in C.albicans, co-ordinating both metabolic and morphogenetic responses to amino acid starvation.

Candida albicans is more pathogenic than Candida dubliniensis. However, this disparity in virulence is surprising given the high level of sequence conservation and the wide range of phenotypic traits shared by these two species. Increased sensitivity to environmental stresses has been suggested to be a possible contributory factor to the lower virulence of C. dubliniensis. In this study, we investigated, in the first comparison of C. albicans and C. dubliniensis by transcriptional profiling, global gene expression in each species when grown under conditions in which the two species exhibit differential stress tolerance. The profiles revealed similar core responses to stresses in both species, but differences in the amplitude of the general transcriptional responses to thermal, salt and oxidative stress. Differences in the regulation of specific stress genes were observed between the two species. In particular, ENA21, encoding a sodium ion transporter, was strongly induced in C. albicans but not in C. dubliniensis. In addition, ENA21 was identified in a forward genetic screen for C. albicans genomic sequences that increase salt tolerance in C. dubliniensis. Introduction of a single copy of CaENA21 was subsequently shown to be sufficient to confer salt tolerance upon C. dubliniensis.

Pathogenic Candida species remain a significant medical problem despite the availability of antifungal therapies. Two key issues must be addressed to improve the treatment of life-threatening systemic Candida infections. First, advanced diagnostic tools are required to facilitate the early identification of these infections, when therapeutic intervention is more likely to be effective. Second, improved antifungal therapies are needed. These therapies, which might include combinations of antifungals, need to be less toxic to the patient and more potent in killing a broader range of Candida species. Recent advances in unravelling the genomics of these species should facilitate efforts to achieve these goals. We discuss the contribution of genomics to the development of novel antifungals and new diagnostic tools.

In budding yeast, Tup1 and Ssn6/Cyc8 form a corepressor that regulates a large number of genes. This Tup1-Ssn6 corepressor appears to be conserved from yeast to man. In the pathogenic fungus Candida albicans, Tup1 regulates cellular morphogenesis, phenotypic switching, and metabolism, but the role of Ssn6 remains unclear. We show that there are clear differences in the morphological and invasive phenotypes of C. albicans ssn6 and tup1 mutants. Unlike Tup1, Ssn6 depletion promoted morphological events reminiscent of phenotypic switching rather than filamentous growth. Transcript profiling revealed minimal overlap between the Ssn6 and Tup1 regulons. Hypha-specific genes, which are repressed by Tup1 and Nrg1, were not derepressed in ssn6 cells under the conditions studied. In contrast, the phase specific gene WH11 was derepressed in ssn6 cells, but not in tup1 or nrg1 cells. Hence Ssn6 and Tup1 play distinct roles in C. albicans. Nevertheless, both Ssn6 and Tup1 were required for the Nrg1-mediated repression of an artificial NRE promoter, and lexA-Nrg1 mediated repression in the C. albicans one-hybrid system. These observations are explained in models that are generally consistent with the Tup1-Ssn6 paradigm in budding yeast.

The human fungal pathogen Candida glabrata appears to utilise unique stealth, evasion and persistence strategies in subverting the onslaught of host immune response during systemic infection. However, macrophages actively deprive the intracellular fungal pathogen of glucose, and therefore alternative carbon sources probably support the growth and survival of engulfed C. glabrata. The present study aimed to investigate the role of the glyoxylate cycle gene ICL1 in alternative carbon utilisation and its importance for the virulence of C. glabrata. The data showed that disruption of ICL1 rendered C. glabrata unable to utilise acetate, ethanol or oleic acid. In addition, C. glabrata icl1∆ cells displayed significantly reduced biofilm growth in the presence of several alternative carbon sources. It was also found that ICL1 is crucial for the survival of C. glabrata in response to macrophage engulfment. Disruption of ICL1 also conferred a severe attenuation in the virulence of C. glabrata in the mouse model of invasive candidiasis. In conclusion, a functional glyoxylate cycle is essential for C. glabrata to utilise certain alternative carbon sources in vitro and to display full virulence in vivo. This reinforces the view that antifungal drugs that target fungal Icl1 have potential for future therapeutic intervention.

Candida albicans is a normal resident of the human gastrointestinal and urogenital tracts and also a prevalent fungal pathogen. During both commensalism and infection, it must match the immunological defenses of its host while adapting to environmental cues and the local nutrient status. C. albicans regularly colonizes glucose-poor niches, thereby depending upon alternative carbon sources for growth. However, most studies of host immune responses to C. albicans have been performed on fungal cells grown on glucose, and the extent to which alternative physiologically relevant carbon sources impact innate immune responses has not been studied. The fungal cell wall is decorated with multifarious pathogen-associated molecular patterns and is the main target for recognition by host innate immune cells. Cell wall architecture is both robust and dynamic, and it is dramatically influenced by growth conditions. We found that growth of C. albicans cells on lactate, a nonfermentative carbon source available in numerous anatomical niches, modulates their interactions with immune cells and the resultant cytokine profile. Notably, lactate-grown C. albicans stimulated interleukin-10 (IL-10) production while decreasing IL-17 levels, rendering these cells less visible to the immune system than were glucose-grown cells. This trend was observed in clinical C. albicans isolates from different host niches and from different epidemiological clades. In addition, lactate-grown C. albicans cells were taken up by macrophages less efficiently, but they were more efficient at killing and escaping these phagocytic cells. Our data indicate that carbon source has a major impact upon the C. albicans interaction with the innate immune system.

Nutritional immunity is a process whereby an infected host manipulates essential micronutrients to defend against an invading pathogen. We reveal a dynamic aspect of nutritionalimmunity during infection that involves copper assimilation. Using a combination of laser ablation inductively coupled mass spectrometry (LA-ICP MS) and metal mapping, immunohistochemistry, and gene expression profiling from infected tissues, we show that readjustments in hepatic, splenic and renal copper homeostasis accompany disseminated Candidaalbicans infections in the mouse model. Localized host-imposed copper poisoning manifests itself as a transient increase in copper early in the kidney infection. Changes in renal copper are detected by the fungus, as revealed by gene expression profiling and fungal virulence studies. The fungus responds by differentially regulating the Crp1 copper efflux pump (higher expression during early infection and down-regulation late in infection) and the Ctr1 copper importer (lower expression during early infection, and subsequent up-regulation late in infection) to maintain copper homeostasis during disease progression. Both Crp1 and Ctr1 are required for full fungal virulence. Importantly, copper homeostasis influences other virulence traits—metabolic flexibility and oxidative stress resistance. Our study highlights the importance of copper homeostasis for host defence and fungal virulence during systemic disease.

Thermal adaptation is essential in all organisms. In yeasts, the heat shock response is commanded by the heat shock transcription factor Hsf1. Here we have integrated unbiased genetic screens with directed molecular dissection to demonstrate that multiple signalling cascades contribute to thermal adaptation in the pathogenic yeast Candida albicans. We show that the molecular chaperone heat shock protein 90 (Hsp90) interacts with and down-regulates Hsf1 thereby modulating short term thermal adaptation. In the longer term, thermal adaptation depends on key MAP kinase signalling pathways that are associated with cell wall remodelling: the Hog1, Mkc1 and Cek1 pathways. We demonstrate that these pathways are differentially activated and display cross talk during heat shock. As a result ambient temperature significantly affects the resistance of C. albicans cells to cell wall stresses (Calcofluor White and Congo Red), but not osmotic stress (NaCl). We also show that the inactivation of MAP kinase signalling disrupts this cross talk between thermal and cell wall adaptation. Critically, Hsp90 coordinates this cross talk. Genetic and pharmacological inhibition of Hsp90 disrupts the Hsf1-Hsp90 regulatory circuit thereby disturbing HSP gene regulation and reducing the resistance of C. albicans to proteotoxic stresses. Hsp90 depletion also affects cell wall biogenesis by impairing the activation of its client proteins Mkc1 and Hog1, as well as Cek1, which we implicate as a new Hsp90 client in this study. Therefore Hsp90 modulates the short term Hsf1-mediated activation of the classic heat shock response, coordinating this response with long term thermal adaptation via Mkc1- Hog1- and Cek1-mediated cell wall remodelling.

Posttranslational modifications of proteins play critical roles in the control of cellular differentiation, development, and environmental adaptation. In particular, the covalent attachment of the small ubiquitin-like modifier, SUMO, to target proteins (sumoylation) regulates cell cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Here we combine proteomic, molecular, and cellular approaches to examine the roles of sumoylation in the major fungal pathogen of humans, Candida albicans. Using an N-terminally FLAG-tagged SUMO, 31 sumoylated proteins were identified in C. albicans with roles in stress responses (e. g. Hsp60, Hsp70 family members, Hsp104), the cytoskeleton and polarized growth (e. g. Tub1, Cct7, Mlc1), secretion, and endocytosis (e. g. Lsp1, Sec24, Sec7). The output from this proteomic screen was entirely consistent with the phenotypes of C. albicans mutants in which the single SUMO-encoding locus (SMT3) was inactivated or down-regulated. C. albicans smt3/smt3 cells displayed defects in growth, morphology, cell separation, nuclear segregation, and chitin deposition, suggesting important roles for sumoylation in cell cycle control. Smt3/smt3 cells also displayed sensitivity to thermal, oxidative, and cell wall stresses as well as to the antifungal drug caspofungin. Mutation of consensus sumoylation sites in Hsp60 and Hsp104 affected the resistance of C. albicans to thermal stress. Furthermore, signaling via the cell integrity pathway was defective in C. albicans smt3/smt3 cells. These observations provide mechanistic explanations for many of the observed phenotypic effects of Smt3 inactivation upon C. albicans growth and environmental adaptation. Clearly sumoylation plays key roles in fundamental cellular processes that underpin the pathogenicity of this medically important fungus.

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.

Proteomics, the study of the protein complement of a biological system, is generating increasing quantities of data from rapidly developing technologies employed in a variety of different experimental workflows. Experimental processes, e.g. for comparative 2D gel studies or LC-MS/MS analyses of complex protein mixtures, involve a number of steps: from experimental design, through wet and dry lab operations, to publication of data in repositories and finally to data annotation and maintenance. The presence of inaccuracies throughout the processing pipeline, however, results in data that can be untrustworthy, thus offsetting the benefits of high-throughput technology. While researchers and practitioners are generally aware of some of the information quality issues associated with public proteomics data, there are few accepted criteria and guidelines for dealing with them. In this article, we highlight factors that impact on the quality of experimental data and review current approaches to information quality management in proteomics. Data quality issues are considered throughout the lifecycle of a proteomics experiment, from experiment design and technique selection, through data analysis, to archiving and sharing.

Glutamine:fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase) catalyzes the first step of the hexosamine pathway required for the biosynthesis of cell wall precursors. The Candida albicans GFA1 gene was tinned by complementing a gfa1 mutation of Saccharomyces cerevisiae (previously known as gcn1-1; W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). GFA1 encodes a predicted protein of 713 amino acids and is homologous to the corresponding gene from S. cerevisiae (72% identity at the nucleotide sequence level) as well as to the genes encoding glucosamine- 6-phosphate synthases in bacteria and vertebrates. In cell extracts, the C. albicans enzyme was 4-fold more sensitive than the S. cerevisiae enzyme to UDP-N-acetylglucosamine (an inhibitor of the mammalian enzyme) and 2.5-fold more sensitive to N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (a glutamine analog and specific inhibitor of glucosamine-6-phosphate synthase). Cell extracts from the S. cerevisiae gfa1 strain transformed with the C. albicans GFA1 gene exhibited sensitivities to glucosamine-6-phosphate synthase inhibitors that were similar to those shown by the C. albicans enzyme. Southern hybridization indicated that a single GFA1 locus exists in the C. albicans genome. Quantitative Northern (RNA) analysis showed that the expression of GFA1 in C. albicans is regulated during growth: maximum mRNA levels were detected during early log phase. GFA1 mRNA levels increased following induction of the yeast-to-hyphal-form transition, but this was a response to fresh medium rather than to the morphological change.

Fungal pathogens must assimilate local nutrients to establish an infection in their mammalian host. We focus on carbon, nitrogen, and micronutrient assimilation mechanisms, discussing how these influence host-fungus interactions during infection. We highlight several emerging trends based on the available data. First, the perturbation of carbon, nitrogen, or micronutrient assimilation attenuates fungal pathogenicity. Second, the contrasting evolutionary pressures exerted on facultative versus obligatory pathogens have led to contemporary pathogenic fungal species that display differing degrees of metabolic flexibility. The evolutionarily ancient metabolic pathways are conserved in most fungal pathogen, but interesting gaps exist in some species (e.g. Candida glabrata). Third, metabolic flexibility is generally essential for fungal pathogenicity, and in particular, for the adaptation to contrasting host microenvironments such as the gastrointestinal tract, mucosal surfaces, bloodstream, and internal organs. Fourth, this metabolic flexibility relies on complex regulatory networks, some of which are conserved across lineages, whereas others have undergone significant evolutionary rewiring. Fifth, metabolic adaptation affects fungal susceptibility to antifungal drugs, and also presents exciting opportunities for the development of novel therapies.

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 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.

Eukaryotic cells have evolved mechanisms to sense and adapt to dynamic environmental changes. Adaptation to thermal insults, in particular, is essential for their survival. The major fungal pathogen of humans, Candida albicans, is obligately associated with warm-blooded animals and hence occupies thermally buffered niches. Yet during its evolution in the host it has retained a bona fide heat shock response whilst other stress responses have diverged significantly. Furthermore the heat shock response is essential for the virulence of C. albicans. With a view to understanding the relevance of this response to infection we have explored the dynamic regulation of thermal adaptation using an integrative systems biology approach. Our mathematical model of thermal regulation, which has been validated experimentally in C. albicans, describes the dynamic autoregulation of the heat shock transcription factor Hsf1 and the essential chaperone protein Hsp90. We have used this model to show that the thermal adaptation system displays perfect adaptation, that it retains a transient molecular memory, and that Hsf1 is activated during thermal transitions that mimic fever. In addition to providing explanations for the evolutionary conservation of the heat shock response in this pathogen and the relevant of this response to infection, our model provides a platform for the analysis of thermal adaptation in other eukaryotic cells.

Corrigendum

Post-translational modifications of proteins play key roles in eukaryotic growth, differentiation and environmental adaptation. In model systems the ubiquitination of specific proteins contributes to the control of cell cycle progression, stress adaptation and metabolic reprogramming. We have combined molecular, cellular and proteomic approaches to examine the roles of ubiquitination in Candida albicans, because little is known about ubiquitination in this major fungal pathogen of humans. Independent null (ubi4/ubi4) and conditional (MET3p-UBI4/ubi4) mutations were constructed at the C. albicans polyubiquitin-encoding locus. These mutants displayed morphological and cell cycle defects, as well as sensitivity to thermal, oxidative and cell wall stresses. Furthermore, ubi4/ubi4 cells rapidly lost viability under starvation conditions. Consistent with these phenotypes, proteins with roles in stress responses (Gnd1, Pst2, Ssb1), metabolism (Acs2, Eno1, Fba1, Gpd2, Pdx3, Pgk1, Tkl1) and ubiquitination (Ubi4, Ubi3, Pre1, Pre3, Rpt5) were among the ubiquitination targets we identified, further indicating that ubiquitination plays key roles in growth, stress responses and metabolic adaptation in C. albicans. Clearly ubiquitination plays key roles in the regulation of fundamental cellular processes that underpin the pathogenicity of this medically important fungus. This was confirmed by the observation that the virulence of C. albicans ubi4/ubi4 cells is significantly attenuated.

In Saccharomyces cerevisiae, the (C2H2)(2) zinc finger transcription factors Msn2 and Msn4 play central roles in responses to a range of stresses by activating gene transcription via the stress response element (STRE; CCCCT). The pathogen Candida albicans displays stress responses that are thought to help it survive adverse environmental conditions encountered within its human host. However, these responses differ from those in S. cerevisiae, and hence we predicted that the roles of Msn2- and Msn4-like proteins might have been functionally reassigned in C. albicans. C. albicans has two such proteins: CaMsn4 and Mull (for Msn2- and Msn4-like). CaMSN4, but not MNL1, weakly complemented the inability of an S. cerevisiae msn2 msn4 mutant to activate a STRE-lacZ reporter. Also, the disruption of CaMsn4 and Mull had no discernible effect upon the resistance of C. albicans to heat, osmotic, ethanol, nutrient, oxidative, or heavy-metal stress or upon the stress-activated transcriptome in C. albicans. Furthermore, although Capl-dependent activation of a Yap response element-luciferase reporter was observed, a STRE reporter was not activated in response to stresses in C. albicans. Ectopic expression of CaMsn4 or Mull did not affect the cellular or molecular responses of C. albicans to stress. Under the conditions tested, the putative activation and DNA binding domains of CaMsn4 did not appear to be functional. These data suggest that CaMsn4 and Mull do not contribute significantly to stress responses in C. albicans. The data are consistent with the idea that stress signaling in this fungus has diverged significantly from that in budding yeast.

The Saccharomyces cerevisiae pyruvate kinase gene (PYK1) was transformed into yeast using the multicopy vector pJDB207. Growth rates and PYK1 gene expression levels varied considerably amongst the transformants. Yeast transformants expressing the PYK1 gene at high levels formed small colonies compared with those expressing the gene at relatively low levels. Slow-growing transformants ’reverted’ at high frequency to more rapid growth, and this correlated with decreases in PYK1 gene copy number and PYK1 mRNA abundance. This apparent selection against PYK1 over-expression was disrupted by the introduction of a stop codon at the 5’-end of the PYK1 coding region, thus confirming that the growth effects were mediated by the PYK1 gene. However, massive overproduction of pyruvate kinase in yeast, using multiple copies of a PGK:PYK gene fusion, had no significant effect upon cell growth. This suggests that the deleterious effect upon the host yeast cell is mediated by abnormally high levels of the wild-type gene or PYK1 mRNA, rather than by increased pyruvate kinase levels.

We have characterized CaNrg1 from Candida albicans, the major fungal pathogen in humans. CaNrg1 contains a zinc finger domain that is conserved in transcriptional regulators from fungi to humans. It is most closely related to ScNrg1, which represses transcription in a Tup1-dependent fashion in Saccharomyces cerevisiae. Inactivation of CaNrg1 in C.albicans causes filamentous and invasive growth, derepresses hypha-specific genes, increases sensitivity to some stresses and attenuates virulence. A tup1 mutant displays similar phenotypes. However, unlike tup1 cells, nrg1 cells can form normal hyphae, generate chlamydospores at normal rates and grow at 42 degreesC. Transcript profiling of 2002 C.albicans genes reveals that CaNrg1 represses a subset of CaTup1-regulated genes, which includes known hypha-specific genes and other virulence factors. Most of these genes contain an Nrg1 response element (NRE) in their promoter. CaNrg1 interacts specifically with an NRE in vitro. Also, deletion of two NREs from the ALS8 promoter releases it from Nrg1-mediated repression. Hence, CaNrg1 is a transcriptional repressor that appears to target CaTup1 to a distinct set of virulence-related functions, including yeast-hypha morphogenesis.

Candida albicans is a major opportunistic pathogen of humans. The pathogenicity of this fungus depends upon its ability to deal effectively with the host defenses and, in particular, the oxidative burst of phagocytic cells. We have explored the activation of the oxidative stress response in C. albicans in ex vivo infection models and during systemic infection of a mammalian host. We have generated C. albicans strains that contain specific green fluorescent protein (GFP) promoter fusions and hence act as biosensors of environmental oxidative stress at the single-cell level. Having confirmed that CTA1-, TRX1-, and TTR1/GRX2-GFP reporters respond specifically to oxidative stress, and not to heat shock, nitrosative, or osmotic stresses, we used these reporters to show that individual C. albicans cells activate an oxidative stress response following phagocytosis by neutrophils, but not by macrophages. Significantly, only a small proportion of C. albicans cells (about 4%) activated an oxidative stress response during systemic infection of the mouse kidney. The response of these cells was generally equivalent to exposure to 0.4 mM hydrogen peroxide in vitro. We conclude that most C. albicans cells are exposed to an oxidative stress when they come into contact with neutrophils in the bloodstream of the host but that oxidative killing is no longer a significant threat once an infection has been established in the kidney.

A network of conserved proteases known as the intramitochondrial quality control (IMQC) system is central to mitochondrial protein homeostasis and cellular health. IMQC proteases also appear to participate in establishment of signaling cues for mitochondrion-to-nucleus communication. However, little is known about this process. Here, we show that in Saccharomyces cerevisiae, inactivation of the membrane-bound IMQC protease Oma1 interferes with oxidative-stress responses through enhanced production of reactive oxygen species (ROS) during logarithmic growth and reduced stress signaling via the TORC1-Rim15-Msn2/Msn4 axis. Pharmacological or genetic prevention of ROS accumulation in Oma1-deficient cells restores this defective TOR signaling. Additionally, inactivation of the Oma1 ortholog in the human fungal pathogen Candida albicans also alters TOR signaling and, unexpectedly, leads to increased resistance to neutrophil killing and virulence in the invertebrate animal model Galleria mellonella. Our findings reveal a novel and evolutionarily conserved link between IMQC and TOR-mediated signaling that regulates physiological plasticity and pancellular oxidative-stress responses.

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.

During interactions with its mammalian host, the pathogenic yeast Candida albicans is exposed to a range of stresses such as superoxide radicals and cationic fluxes. Unexpectedly, a non-biased screen of transcription factor deletion mutants revealed that the phosphate-responsive transcription factor, Pho4, is vital for the resistance of C. albicans to these diverse stresses. RNA-Seq analysis indicated that Pho4 does not induce stress-protective genes directly. Instead, we show that loss of Pho4 impacts on metal cation toxicity, accumulation, and bioavailability. We demonstrate that pho4Δ cells are sensitive to metal and non-metal cations, and that Pho4-mediated polyphosphate synthesis mediates manganese resistance. Significantly, we show that Pho4 is important for mediating copper bioavailability to support the activity of the copper/zinc superoxide dismutase Sod1, and that loss of Sod1 activity contributes to the superoxide sensitivity of pho4Δ cells. Consistent with the key role of fungal stress responses in countering host phagocytic defences, we also report that C. albicans pho4Δ cells are acutely sensitive to macrophage-mediated killing, and display attenuated virulence in animal infection models. The novel connections between phosphate metabolism, metal homeostasis and superoxide stress resistance, presented in this study, highlights the importance of metabolic adaptation in promoting C. albicans survival in the host.

BACKGROUND: Microbes must sense environmental stresses, transduce these signals and mount protective responses to survive in hostile environments. In this study we have tested the hypothesis that fungal stress signalling pathways have evolved rapidly in a niche-specific fashion that is independent of phylogeny. To test this hypothesis we have compared the conservation of stress signalling molecules in diverse fungal species with their stress resistance. These fungi, which include ascomycetes, basidiomycetes and microsporidia, occupy highly divergent niches from saline environments to plant or mammalian hosts. RESULTS: the fungi displayed significant variation in their resistance to osmotic (NaCl and sorbitol), oxidative (H2O2 and menadione) and cell wall stresses (Calcofluor White and Congo Red). There was no strict correlation between fungal phylogeny and stress resistance. Rather, the human pathogens tended to be more resistant to all three types of stress, an exception being the sensitivity of Candida albicans to the cell wall stress, Calcofluor White. In contrast, the plant pathogens were relatively sensitive to oxidative stress. The degree of conservation of osmotic, oxidative and cell wall stress signalling pathways amongst the eighteen fungal species was examined. Putative orthologues of functionally defined signalling components in Saccharomyces cerevisiae were identified by performing reciprocal BLASTP searches, and the percent amino acid identities of these orthologues recorded. This revealed that in general, central components of the osmotic, oxidative and cell wall stress signalling pathways are relatively well conserved, whereas the sensors lying upstream and transcriptional regulators lying downstream of these modules have diverged significantly. There was no obvious correlation between the degree of conservation of stress signalling pathways and the resistance of a particular fungus to the corresponding stress. CONCLUSION: Our data are consistent with the hypothesis that fungal stress signalling components have undergone rapid recent evolution to tune the stress responses in a niche-specific fashion.

Flexibility in carbon metabolism is pivotal for the survival and propagation of many human fungal pathogens within host niches. Indeed, flexible carbon assimilation enhances pathogenicity and affects the immunogenicity of Candida albicans. Over the last decade, Candida glabrata has emerged as one of the most common and problematic causes of invasive candidiasis. Despite this, the links between carbon metabolism, fitness, and pathogenicity in C. glabrata are largely unexplored. Therefore, this study has investigated the impact of alternative carbon metabolism on the fitness and pathogenic attributes of C. glabrata. We confirm our previous observation that growth on carbon sources other than glucose, namely acetate, lactate, ethanol, or oleate, attenuates both the planktonic and biofilm growth of C. glabrata, but that biofilms are not significantly affected by growth on glycerol. We extend this by showing that C. glabrata cells grown on these alternative carbon sources undergo cell wall remodeling, which reduces the thickness of their β-glucan and chitin inner layer while increasing their outer mannan layer. Furthermore, alternative carbon sources modulated the oxidative stress resistance of C. glabrata as well as the resistance of C. glabrata to an antifungal drug. In short, key fitness and pathogenic attributes of C. glabrata are shown to be dependent on carbon source. This reaffirms the perspective that the nature of the carbon sources available within specific host niches is crucial for C. glabrata pathogenicity during infection

A general system has been devised for the in vivo construction of Candida albicans integrative vectors in Saccharomyces cerevisiae. The system is especially useful for the integration of genes in C. albicans that cannot be propagated in Escherichia coli, possibly because of their toxic effects. The ligation of S. cerevisiae 2 μ sequences to a C. albicans integrative vector permits in vivo maintenance and gap repair cloning within S. cerevisiae. After the vector assembly, it can be purified from S. cerevisiae or amplified by PCR and then used for transformation of C. albicans. The S. cerevisiae 2 μ sequence is completely removed by linearization prior to C. albicans transformation, such that no unwanted DNA is transferred in the final construct. The system was successfully used to clone and reintegrate the C. albicans JEN2 gene, which encodes a membrane protein that is apparently toxic to E. coli. Three popular C. albicans integrative vectors, CIp10, CIp20 and CIp30, are now available in versions that permit gap repair in S. cerevisiae. GenBank Accession Nos CIp10-2 μ (GU550119), CIp20-2 μ (GU550120) and CIp30-2 μ (GU550121).

The LPD1 gene of Saccharomyces cerevisiae, encoding lipoamide dehydrogenase (LPDH), is subject to catabolite repression. The promoter of this gene contains a number of motifs for DNA-binding transcriptional activators, including three which show strong sequence homology to the core HAP2/HAP3/HAP4 binding motif. Here we report that transcription of LPD1 requires HAP2, HAP3 and HAP4 for release from glucose repression. In the wild-type strain, specific activity of LPDH was increased 12-fold by growth on lactate, 10-fold on glycerol and four- to five-fold on galactose or raffinose, compared to growth on glucose. In hap2, hap3 and hap4 null mutants, the specific activities of LPDH in cultures grown on galactose and raffinose showed only slight induction above the basal level on glucose medium. Similar results were obtained upon assaying for β-galactosidase production in wild-type, or hap2, hap3 or hap4 mutant strains carrying a single copy of the LPD1 promoter fused in frame to the lacZ gene of Escherichia coli and integrated at the URA3 locus. Transcript analysis in wild-type and hap2 mutants confirmed that the HAP2 protein regulates LPD1 expression at the level of transcription in the same way as it does for the CYC1 gene. Site-directed mutagenesis of the putative HAP2/HAP3/HAP4 binding site at -204 relative to the ATG start codon showed that this element was required for full derepression of the LPD1 gene on non-fermetable substrates.

Posttranslational modifications of proteins drive a wide variety of cellular processes in eukaryotes, regulating cell growth and division as well as adaptive and developmental processes. With regard to the fungal kingdom, most information about posttranslational modifications has been generated through studies of the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, where, for example, the roles of protein phosphorylation, glycosylation, acetylation, ubiquitination, sumoylation, and neddylation have been dissected. More recently, information has begun to emerge for the medically important fungal pathogens Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, highlighting the relevance of posttranslational modifications for virulence. We review the available literature on protein modifications in fungal pathogens, focusing in particular upon the reversible peptide modifications sumoylation, ubiquitination, and neddylation.

We have exploited a modular cat reporter system (Vega Laso, M. R. Zhu, D. Sagliocco, F. A. Brown, A. J. P. Tuite, M. F. and McCarthy, J. E. G. (1993) J. Biol. Chem. 268, 6453-6462) to investigate the relationship between mRNA structure, translation, and stability in the yeast Saccharomyces cerevisiae. The stability of the cat mRNA was not influenced by changes in the length and nucleotide sequence of the 5’-leader, but was affected by the formation of stable 5’-secondary structures (>-15 kcal · mol-1). Cat mRNA stability changed only slightly when the CYC1 3’-trailer was replaced with PGK1 sequences, and was influenced by some secondary structures in the 3’- trailer. Secondary structures formed by interactions between the 5’-leader and 3’-trailer increased the stability of the cat mRNA. However, all of the cat mRNAs studied were intrinsically unstable, having half-lives between 4 and 14 min. The translatability of the cat mRNAs did not correlate with their half-life, and their decay was not blocked by cycloheximide. Therefore, the rapid degradation of the cat mRNA does not seem to depend on translational elongation and is not related in any obvious way to the rate of translational initiation. Furthermore, sequences in the 3’-trailer do not program the rapid decay of the cat mRNA. We discuss the implications of these data in the light of current models of mRNA degradation pathways.

The resistance of Candida albicans to many stresses is dependent on the stress-activated protein kinase (SAPK) Hog1. Hence we have explored the role of Hog1 in the regulation of transcriptional responses to stress. DNA microarrays were used to characterize the global transcriptional responses of HOG1 and hog1 cells to three stress conditions that activate the Hog1 SAPK: osmotic stress, oxidative stress, and heavy metal stress. This revealed both stress-specific transcriptional responses and a core transcriptional response to stress in C. albicans. The core transcriptional response was characterized by a subset of genes that responded in a stereotypical manner to all of the stresses analyzed. Inactivation of HOG1 significantly attenuated transcriptional responses to osmotic and heavy metal stresses, but not to oxidative stress, and this was reflected in the role of Hog1 in the regulation of C. albicans core stress genes. Instead, the Capl transcription factor plays a key role in the oxidative stress regulation of C. albicans core stress genes. Our data show that the SAPK network in C. albicans has diverged from corresponding networks in model yeasts and that the C. albicans SAPK pathway functions in parallel with other pathways to regulate the core transcriptional response to stress.

All organisms have evolved mechanisms that protect them against environmental stress. The major fungal pathogen of humans, Candida albicans, has evolved robust stress responses that protect it against human immune defences and promote its pathogenicity. However, C. albicans is unlikely to be exposed to heat shock as it is obligatorily associated with warm-blooded animals. Therefore, we examined the role of the heat shock transcription factor (Hsf1) in this pathogen. We show that C. albicans expresses an evolutionarily conserved Hsf1 (orf19.4775) that is phosphorylated in response to heat shock, induces transcription via the heat shock element (HSE), contributes to the global transcriptional response to heat shock, and is essential for viability. Why has Hsf1 been conserved in this obligate animal saprophyte? We reasoned that Hsf1 might contribute to medically relevant stress responses. However, this is not the case, as an Hsf1-specific HSE-lacZ reporter is not activated by oxidative, osmotic, weak acid or pH stress. Rather, Hsf1 is required for the expression of essential chaperones in the absence of heat shock (e.g. Hsp104, Hsp90, Hsp70). Furthermore, Hsf1 regulates the expression of HSE-containing genes in response to growth temperature in C. albicans. Therefore, the main role of Hsf1 in this pathogen might be the homeostatic modulation of chaperone levels in response to growth temperature, rather than the activation of acute responses to sudden thermal transitions.

The pathogenicity of the clinically important yeast, Candida albicans, is dependent on robust responses to host-imposed stresses. These stress responses have generally been dissected in vitro at 30°C on artificial growth media that do not mimic host niches. Yet host inputs, such as changes in carbon source or temperature, are known to affect C. albicans stress adaptation. Therefore, we performed screens to identify novel regulators that promote stress resistance during growth on a physiologically relevant carboxylic acid and at elevated temperatures. These screens revealed that, under these ’non-standard’ growth conditions, numerous uncharacterised regulators are required for stress resistance in addition to the classical Hog1, Cap1 and Cta4 stress pathways. In particular, two transcription factors (Sfp1 and Rtg3) promote stress resistance in a reciprocal, carbon source-conditional manner. SFP1 is induced in stressed glucose-grown cells, whereas RTG3 is upregulated in stressed lactate-grown cells. Rtg3 and Sfp1 regulate the expression of key stress genes such as CTA4, CAP1 and HOG1 in a carbon source-dependent manner. These mechanisms underlie the stress sensitivity of C. albicans sfp1 cells during growth on glucose, and rtg3 cells on lactate. The data suggest that C. albicans exploits environmentally contingent regulatory mechanisms to retain stress resistance during host colonisation. This article is protected by copyright. All rights reserved.

Stress adaptation is critical for the survival of microbes in dynamic environments, and in particular, for fungal pathogens to survive in and colonise host niches. Proteomic analyses have the potential to significantly enhanceour understanding of these adaptive responses by providing insight into post-transcriptional regulatory mechanisms that contribute to the outputs, as well as testing presumptions about the regulation of protein levels based on transcript profiling. Here, we used label-free, quantitative mass spectrometry to re-examine the response of the major fungal pathogen of humans, Candida albicans, to osmotic stress. of the 1,262 proteins that were identified, 84 were down-regulated in response to 1M NaCl, reflecting the decrease in ribosomebiogenesis and translation that often accompanies stress. The 64 up-regulated proteins included central metabolic enzymes required for glycerol synthesis, a key osmolyte for this yeast, as well as proteins with functions during stress. These data reinforce the view that adaptation to salt stress involves a transientreduction in ribosome biogenesis and translation together with the accumulation of the osmolyte, glycerol. The specificity of the response to salt stress is highlighted by the small proportion of quantified C. albicans proteins (5%) whose relative elevated abundances were statistically significant.

Fungal species display an extraordinarily diverse range of lifestyles. Nevertheless, the survival of each species depends on its ability to sense and respond to changes in its natural environment. Environmental changes such as fluctuations in temperature, water balance or pH, or exposure to chemical insults such as reactive oxygen and nitrogen species exert stresses that perturb cellular homeostasis and cause molecular damage to the fungal cell. Consequently, fungi have evolved mechanisms to repair this damage, detoxify chemical insults, and restore cellular homeostasis. Most stresses are fundamental in nature, and consequently, there has been significant evolutionary conservation in the nature of the resultant responses across the fungal kingdom and beyond. For example, heat shock generally induces the synthesis of chaperones that promote protein refolding, antioxidants are generally synthesized in response to an oxidative stress, and osmolyte levels are generally increased following a hyperosmotic shock. In this article we summarize the current understanding of these and other stress responses as well as the signaling pathways that regulate them in the fungi. Model yeasts such as Saccharomyces cerevisiae are compared with filamentous fungi, as well as with pathogens of plants and humans. We also discuss current challenges associated with defining the dynamics of stress responses and with the elaboration of fungal stress adaptation under conditions that reflect natural environments in which fungal cells may be exposed to different types of stresses, either sequentially or simultaneously.

Morphogenesis between yeast and hyphal growth is a characteristic associated with virulence in Candida albicans and involves changes in the cell wall. In Saccharomyces cerevisiae, the transcription factor pair Ace2p and Swi5p are key regulators of cell wall metabolism. Here, we have characterized the CaACE2 gene, which encodes the only C. albicans homologue of S. cerevisiae ACE2 and SWI5. Deleting CaACE2 results in a defect in cell separation, increased invasion of solid agar medium and inappropriate pseudohyphal growth, even in the absence of external inducers. The mutant cells have reduced adherence to plastic surfaces and generate biofilms with distinctly different morphology from wild-type cells. They are also avirulent in a mouse model. Deleting CaACE2 has no effect on expression of the chitinase gene CHT2, but expression of CHT3 and the putative cell wall genes CaDSE1 and CaSCW11 is reduced in both yeast and hyphal forms. The CaAce2 protein is localized to the daughter nucleus of large budded cells at the end of mitosis. C. albicans Ace2p therefore plays a major role in morphogenesis and adherence and resembles S. cerevisiae Ace2p in function.

The yeast TRP3 gene encodes a bifunctional protein with anthranilate synthase II and indoleglycerol‐phosphate synthase activities. Replacing ten consecutive non‐preferred codons in the indoleglycerol‐phosphate synthase region of the TRP3 gene with synonymous preferred codons (to create the TRP3pr gene; translational pause replaced) causes a 1.5‐fold reduction in relative indoleglycerol‐phosphate synthase activity [Crombie, T. Swaffield, J. C. & Brown, A. J. P. (1992) J. Mol. Biol. 228, 7–12]. Here, we report that both the anthranilate synthase II and indoleglycerol‐phosphate synthase domains are affected to similar extents when the translational pause is removed. Also, structural modelling of the yeast indoleglycerol‐phosphate synthase domain against the X‐ray crystal structure of indoleglycerol‐phosphate synthase from Escherichia coli indicates that the translational pause lies in a region of structural divergence between similar structures. To probe the role of cytoplasmic heat‐shock protein 70 (Hsp70) chaperones in Trp3 protein folding, anthranilate synthase and indoleglycerol‐phosphate synthase activities were measured in ssa and ssb mutants. Neither indoleglycerol‐phosphate synthase nor anthranilate synthase were affected significantly in the ssb mutant. However, depletion of Hsp70 proteins encoded by the SSA genes led to decreased anthranilate synthase and indoleglycerol‐phosphate synthase activities from the TRP3 gene, suggesting that both domains depend to some extent upon the SSA chaperone family. The data are consistent with roles for both the translational pause and Ssa chaperones in Trp3 protein folding in vivo.

The FBP1 and PCK1 genes encode the gluconeogenic enzymes fructose‐1,6‐bisphosphatase and phosphoenolpyruvate carboxykinase, respectively. In the yeast, Saccharomyces cerevisiae, the corresponding mRNAs are present at low levels during growth on glucose, but are present at elevated levels during growth on gluconeogenic carbon sources. We demonstrate that the levels of the FBP1 and PCK1 mRNAs are acutely sensitive to the addition of glucose to the medium and that the levels of these mRNAs decrease rapidly when glucose is added to the medium at a concentration of only 0.005%. At this concentration, glucose blocks FBP1 and PCK1 transcription, but has no effect on iso‐1 cytochrome c (CYCI) mRNA levels. Glucose also increases the rate of degradation of the PCK1 mRNA approximately twofold, but only has a slight effect upon FBP1 mRNA turnover. We show that the levels of the FBP1 and PCK1 mRNAs are also sensitive to other environmental factors. The levels of these mRNAs decrease transiently in response to a decrease of the pH from pH 7.5 to pH 6.5 in the medium, or to a mild temperature shock (from 24°C to 36°C). The latter response appears to be mediated by accelerated mRNA decay.

Candida albicans is the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed the C. albicans TATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a single C. albicans TBP1 locus is expressed at similar levels in the yeast and hyphal forms of this fungus. The TBP1 open reading frame is 716 bp long and encodes a functional TBP of 27 kDa. C. albicans TBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal Δspt15 mutation in Saccharomyces cerevisiae. The predicted amino acid sequences of TBPs from C. albicans and other organisms reveal a striking pattern of C-terminal conservation and N- terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.

The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.

We report the sequence of a 7·5 kb region lying between the CRY1 and MAT loci of chromosome III from Saccharomyces cerevisiae. This region lies in the overlap between two major contigs used for the generation of the complete nucleotide sequence of this chromosome. Comparison of this sequence with those reported previously for this overlap [Thierry et al. (1990) Yeast 6, 521; Jia et al. (1991) Yeast 7, 413] reveals 38 nucleotide differences, 45% of which generate changes in the amino acid sequences of the four genes in this region (YCR591, YCR592, YCR521 and YCR522). These differences appear to reflect true sequence polymorphisms between the two yeast strains used to generate the clones used in the sequencing project. Three of the four genes in this region display weak homologies to proteins in the PIR database. Some properties of YCR521 are analogous to those of ribosomal protein genes. However, the functions of all four genes remain obscure.

The influence of 5’-secondary structure formation and 5’-leader length upon mRNA translation in yeast has been analyzed using a closely related set of cat mRNAs (Vega Laso, M. R. Zhu, D. Sagliocco, F. A. Brown, A. J. P. Tuite, M. F. and McCarthy, J. E. G. (1993) J. Biol. Chem. 268, 6453-6462). A cat mRNA with a relatively short unstructured 5’-leader (22 bases) had a ribosome loading about half that of a cat mRNA with an unstructured 5’- leader of 77 bases. The introduction of 5’-secondary structures at various positions throughout the 5’-leader of the cat mRNA inhibited translation initiation, the degree of inhibition being largely dependent upon the thermodynamic stability of the structure. Each mRNA carrying a 5’-secondary structure had a biphasic polysome distribution, indicating that the mRNA molecules were distributed between untranslated and well translated subpopulations. This suggests that once 5’-secondary structures are unwound, they reform slowly relative to the rate of translation initiation in yeast. Untranslated mRNA accumulated in 43 S preinitiation complexes, even when there were only 5 bases between the 5’-cap and the base of the hairpin. The data are consistent with the scanning hypothesis (Kozak, M. (1989) J. Cell. Biol. 108, 229-241) and suggest that 40 S ribosomal subunits bind to mRNA early in the scanning process, probably before mRNA unwinding has taken place.

Gordon Research Conferences (GRCs) are famous for fostering insight through wide-ranging discussions of cutting-edge science. In addition to the hallmark GRC scientific program, participants in the 2018 GRC on Molecular and Cellular Fungal Biology gained powerful insights into ways to better support women in science. Since 2016, GRC conference organizers have had the option of including the GRC Power HourTM discussion session focusing on the challenges that women in science face (https://www.grc.org/the-power-hour/). 2018 was the first year that the GRC on Molecular and Cellular Fungal Biology included the Power Hour and it was such a rousing success that it is sure to be continued in the future.

Despite being obligately associated with warm-blooded animals, Candida albicans expresses a bona fide heat shock response that is regulated by the evolutionarily conserved, essential heat shock transcription factor Hsf1. Hsf1 is thought to play a fundamental role in thermal homeostasis, adjusting the levels of essential chaperones to changes in growth temperature, for example in febrile patients. Hsf1 also regulates the expression of Hsp90, which controls the yeast-hypha transition in C. albicans, and we argue, might also control morphogenesis in other fungal pathogens of humans.

The European Functional Analysis Network (EUROFAN) is systematically analysing the function of novel Saccharomyces cerevisiae genes revealed by genome sequencing. As part of this effort our consortium has performed a detailed transcript analysis for 250 novel ORFs on chromosome XIV. All transcripts were quantified by Northern analysis under three quasi-steady-state conditions (exponential growth on rich fermentative, rich non-fermentative, and minimal fermentative media) and eight transient conditions (glucose derepression, glucose upshift, stationary phase, nitrogen starvation, osmo-stress, heat-shock, and two control conditions). Transcripts were detected for 82% of the 250 ORFs, and only one ORF did not yield a transcript of the expected length (YNL285w). Transcripts ranged from low (62%), moderate (16%) to high abundance (2%) relative to the ACI mRNA, the levels of 73% of the 206 chromosome XIV transcripts detected fluctuated in response to the transient states tested. However, only a small number responded strongly to the transients: eight ORFs were induced upon glucose upshift: five were repressed by glucose; six were induced in response to nitrogen starvation three were induced in stationary phase; five were induced by osmo-stress; four were induced by heat-shock. These data provide useful clues about the general function of these ORFs and add to our understanding of gene regulation on a genome-wide basis. Copyright (C) 1999 John Wiley & Sons, Ltd.

The effect of growth on the efficiency of utilization of poly(A)‐containing mRNA for translation has been investigated in microplasmodia of Physarum polycephalum. Measurement of the relative proportions of poly(A)‐rich mRNA in polysomal and post‐polysomal fractions isolated by sucrose density gradient centrifugation reveals that newly synthesized poly(A)‐rich mRNA is present in increasing proportions in the polysomal region during exponential growth. However, the proportion of long‐lived poly(A)‐rich mRNA observed in actively‐translating polysomes declines as starvation approaches. The ribonuclease content and morphology of the microplasmodia were monitored during growth and starvation in an effort to relate this phenomenon to the onset of spherulation.

The regulation of glycolytic genes in response to carbon source in the yeast Saccharomyces cerevisiae has been studied. When the relative levels of each glycolytic mRNA were compared during exponential growth on glucose or lactate, the various glycolytic mRNAs were found to be induced to differing extents by glucose. No significant differences in the stabilities of the PFK2, PGK1, PYK1, or PDC1 mRNAs during growth on glucose or lactate were observed. PYK::lacZ and PGK::lacZ fusions were integrated independently into the yeast genome at the ura3 locus. The manner in which these fusions were differentially regulated in response to carbon source was similar to that of their respective wild-type loci. Therefore, the regulation of glycolytic mRNA levels is mediated at the transcriptional level. When the mRNAs are ordered with respect to the glycolytic pathway, two peaks of maximal induction are observed at phosphofructokinase and pyruvate kinase. These enzymes (i) catalyze the two essentially irreversible steps on the pathway, (ii) are the two glycolytic enzymes that are circumvented during gluconeogenesis and hence are specific to glycolysis, and (iii) are encoded by mRNAs that we have shown previously to be coregulated at the translational level in S. cerevisiae (P. A. Moore, A. J. Bettany, and A. J. P. Brown, NATO ASI Ser. Ser. H Cell Biol. 49:421-432, 1990). This differential regulation of glycolytic mRNA levels might therefore have a significant influence upon glycolytic flux in S. cerevisiae.

The expression of many genes is regulated at multiple levels. For most genes, the predominant control is at transcription, and this is usually inferred from changes in mRNA levels that respond to specific stimuli. However, changes in mRNA stability may contribute to observed variation m mRNA abundance, and many such changes in mRNA stability probably remain undetected. The relative contributions of post-transcriptional regulatory circuits can only be detected by careful RNA analysis.

The rate at which a protein is synthesized is influenced by the stability of its mRNA and the efficiency of translation. mRNA degradation is associated with translation, but the link between these processes is unclear. In this article, Alistair Brown discusses this relationship in the light of a model of translation that involves three distinct cellular phases: a primary phase in which the translation of an mRNA molecule starts before its export from the nucleus is complete, a secondary phase that begins when nuclear export is complete and that may involve circular polyribosomal complexes, and a terminal phase involving residual translation once mRNA degradation is initiated.

The expression of some Saccharomyces cerevisiae genes is induced as cells enter stationary phase. Their mRNAs are translated during a period in the growth cycle when the translational apparatus is relatively inert, thereby raising the possibility that these mRNAs compete effectively for a limiting pool of translation factors. To test this idea, the translation of mRNAs carrying different 5’-leaders was compared during exponential growth and after entry into stationary phase upon glucose starvation. Closely related sets of lacZ mRNAs, carrying 5’-leaders from the PYK1, PGK1, RpL3, Rp29, HSP12, HSP26 or TH14 mRNAs, were studied. These mRNAs displayed differing translational efficiencies during exponential growth, but their relative translatabilities were not significantly affected by entry into stationary phase, indicating that they compete just as effectively under these conditions. Polysome analysis revealed that the wild-type PYK1, ACT1 and HSP26 mRNAs are all translated efficiently during stationary phase, when the translational apparatus is relatively inert. Also, significant levels of the translation initiation factors eIF-2α, eIF-4E and eIF-4A were maintained during the growth cycle. These data are consistent with the idea that, while translational activity decreases dramatically during entry into stationary phase, yeast cells maintain excess translational capacity under these conditions.

Pneumocystis jirovecii kills hundreds of thousands of immunocompromised patients each year. Yet many aspects of the biology of this obligate pathogen remain obscure because it is not possible to culture the fungus in vitro independently of its host. Consequently, our understanding of Pneumocystis pathobiology is heavily reliant upon bioinformatic inferences. We have exploited a powerful combination of genomic and phylogenetic approaches to examine the evolution of transcription factors in Pneumocystis species. We selected protein families (Pfam families) that correspond to transcriptional regulators and used bioinformatic approaches to compare these families in the seven Pneumocystis species that have been sequenced to date with those from other yeasts, other human and plant pathogens, and other obligate parasites. Some Pfam families of transcription factors have undergone significant reduction during their evolution in the Pneumocystis genus, and other Pfam families have been lost or appear to be in the process of being lost. Meanwhile, other transcription factor families have been retained in Pneumocystis species, and some even appear to have undergone expansion. On this basis, Pneumocystis species seem to have retained transcriptional regulators that control chromosome maintenance, ribosomal gene regulation, RNA processing and modification, and respiration. Meanwhile, regulators that promote the assimilation of alternative carbon sources, amino acid, lipid, and sterol biosynthesis, and iron sensing and homeostasis appear to have been lost. Our analyses of transcription factor retention, loss, and gain provide important insights into the biology and lifestyle of Pneumocystis. IMPORTANCE Pneumocystis jirovecii is a major fungal pathogen of humans that infects healthy individuals, colonizing the lungs of infants. In immunocompromised and transplant patients, this fungus causes life-threatening pneumonia, and these Pneumocystis infections remain among the most common and serious infections in HIV/AIDS patients. Yet we remain remarkably ignorant about the biology and epidemiology of Pneumocystis due to the inability to culture this fungus in vitro. Our analyses of transcription factor retentions, losses, and gains in sequenced Pneumocystis species provide valuable new views of their specialized biology, suggesting the retention of many metabolic and stress regulators and the loss of others that are essential in free-living fungi. Given the lack of in vitro culture methods for Pneumocystis, this powerful bioinformatic approach has advanced our understanding of the lifestyle of P. jirovecii and the nature of its dependence on the host for survival.

The publisher regrets that the Ethical Statement for sensory evaluation was missed during the publication process. The authors and Publisher wished to bring more information to light regarding human and animal welfare. The Publisher apologizes for any inconvenience caused. Work involving animals (mice) to generate peritoneal macrophages was carried out in accordance with UK Home Office regulations under an existing Home Office project licence (PPL 60/4135). The use of animals in experiments and testing was regulated under the Animals (Scientific Procedures) Act 1986 (ASPA) and European Directive 2010/63/EU.

We are constantly exposed to the threat of fungal infection. The outcome-clearance, commensalism or infection-depends largely on the ability of our innate immune defences to clear infecting fungal cells versus the success of the fungus in mounting compensatory adaptive responses. As each seeks to gain advantage during these skirmishes, the interactions between host and fungal pathogen are complex and dynamic. Nevertheless, simply compromising the physiological robustness of fungal pathogens reduces their ability to evade antifungal immunity, their virulence, and their tolerance against antifungal therapy. In this article I argue that this physiological robustness is based on a 'Resilience Network' which mechanistically links and controls fungal growth, metabolism, stress resistance and drug tolerance. The elasticity of this network probably underlies the phenotypic variability of fungal isolates and the heterogeneity of individual cells within clonal populations. Consequently, I suggest that the definition of the fungal Resilience Network represents an important goal for the future which offers the clear potential to reveal drug targets that compromise drug tolerance and synergise with current antifungal therapies.

Candida auris is an emerging, multidrug-resistant fungal pathogen that causes refractory colonization and life-threatening, invasive nosocomial infections. The high proportion of C. auris isolates that display antifungal resistance severely limits treatment options. Combination therapies provide a possible strategy by which to enhance antifungal efficacy and prevent the emergence of further resistance. Therefore, we examined drug combinations using antifungals that are already in clinical use or are undergoing clinical trials. Using checkerboard assays, we screened combinations of 5-flucytosine and manogepix (the active form of the novel antifungal drug fosmanogepix) with anidulafungin, amphotericin B, or voriconazole against drug resistant and susceptible C. auris isolates from clades I and III. Fractional inhibitory concentration indices (FICI values) of 0.28 to 0.75 and 0.36 to 1.02 were observed for combinations of anidulafungin with manogepix or 5-flucytosine, respectively, indicating synergistic activity. The high potency of these anidulafungin combinations was confirmed using live-cell microfluidics-assisted imaging of the fungal growth. In summary, combinations of anidulafungin with manogepix or 5-flucytosine show great potential against both resistant and susceptible C. auris isolates.

The uptake of cholesterol from the host is closely linked to the proliferation of pathogenic fungi and protozoa during infection. For some pathogenic fungi, cholesterol uptake is an important strategy for decreasing susceptibility to antifungals that inhibit ergosterol biosynthesis. In this study, we show that Candida glabrata ERG25, which encodes an enzyme that demethylates 4,4-dimethylzymosterol, is required for cholesterol uptake from host serum. Based on the screening of C. glabrata conditional knockdown mutants for each gene involved in ergosterol biosynthesis, ERG25 knockdown was found to decrease lethality of infected mice. ERG25 knockdown impairs the plasma membrane localization of the sterol importer Aus1p, suggesting that the accumulated 4,4-dimethylzymosterol destabilizes the lipid domain with which Aus1p functionally associates. ERG25 knockdown further influences the structure of the membrane compartment of Can1p (MCC)/eisosomes (ergosterol-rich lipid domains), but not the localization of the membrane proteins Pma1p and Hxt1p, which localize to sterol-poor domains. In the sterol-rich lipid domain, Aus1p-contining domain was mostly independent of MCC/eisosomes, and the nature of these domains was also different: Ausp1-contining domain was a dynamic network-like domain, whereas the MCC/eisosomes was a static dot-like domain. However, deletion of MCC/eisosomes was observed to influence the localization of Aus1p after Aus1p was transported from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane. These findings suggest that ERG25 plays a key role in stabilizing sterol-rich lipid domains, constituting a promising candidate target for antifungal therapy.

The human gut microbiota protects the host from invading pathogens and the overgrowth of indigenous opportunistic species via a process called colonization resistance. Here, we investigated the antagonistic activity of human gut bacteria towards Candida albicans, an opportunistic fungal pathogen that can cause severe infections in susceptible individuals. Coculture batch incubations of C. albicans in the presence of faecal microbiota from six healthy individuals revealed varying levels of inhibitory activity against C. albicans. 16S rRNA gene amplicon profiling of these faecal coculture bacterial communities showed that the Bifidobacteriaceae family, and Bifidobacterium adolescentis in particular, were most correlated with antagonistic activity against C. albicans. Follow-up mechanistic studies performed under anaerobic conditions confirmed that culture supernatants of Bifidobacterium species, particularly B. adolescentis, inhibited C. albicans in vitro. Fermentation acids (FA), including acetate and lactate, present in the bifidobacterial supernatants were important contributors to inhibitory activity. However, increasing the pH of both bacterial supernatants and mixtures of FA reduced their anti-Candida effects, indicating a combinatorial effect of prevailing pH and FA. This work, therefore, demonstrates potential mechanisms underpinning gut microbiome-mediated colonization resistance against C. albicans, and identifies particularly inhibitory components such as bifidobacteria and FA as targets for further study.

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.

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.

Aspergillus fumigatus is the leading cause of the fungal invasive disease called aspergillosis, which is associated with a high mortality rate that can reach 50% in some groups of immunocompromised individuals. The increasing prevalence of azole-resistant A. fumigatus isolates, both in clinical settings and the environment, highlights the importance of discovering new fungal virulence factors that can potentially become targets for novel antifungals. Nitronate monooxygenases (Nmos) represent potential targets for antifungal compounds as no orthologs of those enzymes are present in humans. Nmos catalyse the denitrification of nitroalkanes, thereby detoxifying these mediators of nitro-oxidative stress, and therefore we tested whether Nmos provide protection for A. fumigatus against host-imposed stresses at sites of infection. The results of inhibition zone assays indicated that Nmo2 and Nmo5 are not essential for the oxidative stress resistance of A. fumigatus in vitro. In addition, the resazurin-based metabolic activity assay revealed that the growth of mutants lacking the nmo2 or nmo5 genes was only slightly reduced in the presence of 0.05 mM peroxynitrite. Nevertheless, both Nmo2 and Nmo5 were shown to contribute to defense against murine bone marrow-derived macrophages, and this was no longer observed when NADPH oxidase, the main generator of reactive oxygen species during infection, was inhibited in macrophages. Furthermore, we revealed that Nnmos promote the virulence of the fungus in the Galleria mellonella model of infection. Both nmo2 and nmo5 knock-out strains were less virulent than the wild-type control as recorded 72 h post-infection. Our results indicate that Nmos play a role in the virulence of A. fumigatus.

In certain niches, microbes encounter environmental challenges that are temporally linked. In such cases, microbial fitness is enhanced by the evolution of anticipatory responses where the initial challenge simultaneously activates pre-emptive protection against the second impending challenge. The accumulation of anticipatory responses in domesticated yeasts, which have been termed 'adaptive prediction', has led to the emergence of 'core stress responses' that provide stress cross-protection. Protective anticipatory responses also seem to be common in fungal pathogens of humans. These responses reflect the selective pressures that these fungi have faced relatively recently in their evolutionary history. Consequently, some pathogens have evolved 'core environmental responses' which exploit host signals to trigger immune evasion strategies that protect them against imminent immune attack.

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.

Reactive Oxygen Species (ROS) are highly reactive molecules that can induce oxidative stress. For instance, the oxidative burst of immune cells is well known for its ability to inhibit the growth of invading pathogens. However, ROS also mediate redox signalling, which is important for the regulation of antimicrobial immunity. Here, we report a crucial role of mitochondrial ROS (mitoROS) in antifungal responses of macrophages. We show that mitoROS production rises in murine macrophages exposed to swollen conidia of the fungal pathogen Aspergillus fumigatus compared to untreated macrophages, or those treated with resting conidia. Furthermore, the exposure of macrophages to swollen conidia increases the activity of complex II of the respiratory chain and raises mitochondrial membrane potential. These alterations in mitochondria of infected macrophages suggest that mitoROS are produced via reverse electron transport (RET). Significantly, preventing mitoROS generation via RET by treatment with rotenone, or a suppressor of site IQ electron leak, S1QEL1.1, lowers the production of pro-inflammatory cytokines TNF-α and IL-1β in macrophages exposed to swollen conidia of A. fumigatus. Rotenone and S1QEL1.1 also reduces the fungicidal activity of macrophages against swollen conidia. Moreover, we have established that elevated recruitment of NADPH oxidase 2 (NOX2, also called gp91phox) to the phagosomal membrane occurs prior to the increase in mitoROS generation. Using macrophages from gp91phox-/- mice, we have further demonstrated that NOX2 is required to regulate cytokine secretion by RET-associated mitoROS in response to infection with swollen conidia. Taken together, these observations demonstrate the importance of RET-mediated mitoROS production in macrophages infected with A. fumigatus.

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Background: Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection. Methods: in this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches. Results: Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages. Conclusion: Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.

Antimicrobial peptides and proteins (AMPs) provide an important line of defence against invading microorganisms. However, the activity of AMPs against the human fungal pathogen Aspergillus fumigatus remains poorly understood. Therefore, the aim of this study was to characterise the anti-Aspergillus activity of specific human AMPs, and to determine whether A. fumigatus can possess resistance to specific AMPs, as a result of in-host adaptation. AMPs were tested against a wide range of clinical isolates of various origins (including cystic fibrosis patients, as well as patients with chronic and acute aspergillosis). We also tested a series of isogenic A. fumigatus isolates obtained from a single patient over a period of 2 years. A range of environmental isolates, obtained from soil in Scotland, was also included. Firstly, the activity of specific peptides was assessed against hyphae using a measure of fungal metabolic activity. Secondly, the activity of specific peptides was assessed against germinating conidia, using imaging flow cytometry as a measure of hyphal growth. We showed that lysozyme and histones inhibited hyphal metabolic activity in all the A. fumigatus isolates tested in a dose-dependent fashion. In addition, imaging flow cytometry revealed that histones, β-defensin-1 and lactoferrin inhibited the germination of A. fumigatus conidia.

Candida albicans is a normal member of the human microbiome. It is also an opportunistic pathogen, which can cause life-threatening systemic infections in severely immunocompromized individuals. Despite the availability of antifungal drugs, mortality rates of systemic infections are high and new drugs are needed to overcome therapeutic challenges including the emergence of drug resistance. Targeting known disease pathways has been suggested as a promising avenue for the development of new antifungals. However,

The fungal cell wall is an essential organelle that maintains cellular morphology and protects the fungus from environmental insults. For fungal pathogens such as Candida albicans, it provides a degree of protection against attack by host immune defences. However, the cell wall also presents key epitopes that trigger host immunity and attractive targets for antifungal drugs. Rather than being a rigid shield, it has become clear that the fungal cell wall is an elastic organelle that permits rapid changes in cell volume and the transit of large liposomal particles such as extracellular vesicles. The fungal cell wall is also flexible in that it adapts to local environmental inputs, thereby enhancing the fitness of the fungus in these microenvironments. Recent evidence indicates that this cell wall adaptation affects host-fungus interactions by altering the exposure of major cell wall epitopes that are recognised by innate immune cells. Therefore, we discuss the impact of environmental adaptation upon fungal cell wall structure, and how this affects immune recognition, focussing on C. albicans and drawing parallels with other fungal pathogens.

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.

© 2020, the Author(s). Candida glabrata is an important human fungal pathogen known to trigger serious infections in immune-compromised individuals. Its ability to form biofilms, which exhibit high tolerance to antifungal treatments, has been considered as an important virulence factor. However, the mechanisms involving antifungal resistance in biofilms and the impact of host niche environments on these processes are still poorly defined. In this study, we performed a whole-transcriptome analysis of C. glabrata biofilm cells exposed to different environmental conditions and constraints in order to identify the molecular pathways involved in fluconazole resistance and understand how acidic pH niches, associated with the presence of acetic acid, are able to modulate these responses. We show that fluconazole treatment induces gene expression reprogramming in a carbon source and pH-dependent manner. This is particularly relevant for a set of genes involved in DNA replication, ergosterol, and ubiquinone biosynthesis. We also provide additional evidence that the loss of mitochondrial function is associated with fluconazole resistance, independently of the growth condition. Lastly, we propose that C. glabrata Mge1, a cochaperone involved in iron metabolism and protein import into the mitochondria, is a key regulator of fluconazole susceptibility during carbon and pH adaptation by reducing the metabolic flux towards toxic sterol formation. These new findings suggest that different host microenvironments influence directly the physiology of C. glabrata, with implications on how this pathogen responds to antifungal treatment. Our analyses identify several pathways that can be targeted and will potentially prove to be useful for developing new antifungals to treat biofilm-based infections.

Candida glabrata is an important human fungal pathogen known to trigger serious infections in immune-compromised individuals. Its ability to form biofilms, which exhibit high tolerance to antifungal treatments, has been considered as an important virulence factor. However, the mechanisms involving antifungal resistance in biofilms and the impact of host niche environments on these processes are still poorly defined. In this study, we performed a whole-transcriptome analysis of C. glabrata biofilm cells exposed to different environmental conditions and constraints in order to identify the molecular pathways involved in fluconazole resistance and understand how acidic pH niches, associated with the presence of acetic acid, are able to modulate these responses. We show that fluconazole treatment induces gene expression reprogramming in a carbon source and pH-dependent manner. This is particularly relevant for a set of genes involved in DNA replication, ergosterol, and ubiquinone biosynthesis. We also provide additional evidence that the loss of mitochondrial function is associated with fluconazole resistance, independently of the growth condition. Lastly, we propose that C. glabrata Mge1, a cochaperone involved in iron metabolism and protein import into the mitochondria, is a key regulator of fluconazole susceptibility during carbon and pH adaptation by reducing the metabolic flux towards toxic sterol formation. These new findings suggest that different host microenvironments influence directly the physiology of C. glabrata, with implications on how this pathogen responds to antifungal treatment. Our analyses identify several pathways that can be targeted and will potentially prove to be useful for developing new antifungals to treat biofilm-based infections.

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.

Azole-resistance in Aspergillus fumigatus is an emerging worldwide threat as it precludes the use of one of the 3 major classes of antifungal drugs to treat chronic and invasive aspergillosis [1]. In addition to the well-known environmental emergence of azole-resistant A. fumigatus strains, associated with the use of fungicides in agriculture [2], [3], the development of in-host resistance, facilitated by medical antifungal use, has been described [4]. Investigations involving linked sets of (isogenic) clinical isolates of A. fumigatus sequentially recovered from individual patients, are extremely important in order to improve our understanding of how azole resistance develops in-host. Here we present the whole genome sequences of 13 clinical isogenic A. fumigatus isolates. These isolates were cultured from a single patient suffering from invasive aspergillosis over a period of 2 years. This patient underwent a wide range of antifungal therapies and the resultant isolates acquired multiple azole resistance in-host during the course of infection. The data presented here is related to our research paper titled "In-host microevolution of Aspergillus fumigatus: a phenotypic and genotypic analysis" which describes the phenotypic characterisation of these clinical isolates [5]. The raw sequence data was deposited in the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra), under BioProject ID number PRJNA528395.

The human host comprises a range of specific niche environments. In order to successfully persist, pathogens such as Aspergillus fumigatus must adapt to these environments. One key example of in-host adaptation is the development of resistance to azole antifungals. Azole resistance in A. fumigatus is increasingly reported worldwide and the most commonly reported mechanisms are cyp51A mediated. Using a unique series of A. fumigatus isolates, obtained from a patient suffering from persistent and recurrent invasive aspergillosis over 2 years, this study aimed to gain insight into the genetic basis of in-host adaptation. Single nucleotide polymorphisms (SNPs) unique to a single isolate in this series, which had developed multi-azole resistance in-host, were identified. Two nonsense SNPs were recreated using CRISPR-Cas9; these were 213. in svf1 and 167. in uncharacterised gene AFUA_7G01960. Phenotypic analyses including antifungal susceptibility testing, mycelial growth rate assessment, lipidomics analysis and statin susceptibility testing were performed to associate genotypes to phenotypes. This revealed a role for svf1 in A. fumigatus oxidative stress sensitivity. In contrast, recapitulation of 167. in AFUA_7G01960 resulted in increased itraconazole resistance. Comprehensive lipidomics analysis revealed decreased ergosterol levels in strains containing this SNP, providing insight to the observed itraconazole resistance. Decreases in ergosterol levels were reflected in increased resistance to lovastatin and nystatin. Importantly, this study has identified a SNP in an uncharacterised gene playing a role in azole resistance via a non-cyp51A mediated resistance mechanism. This mechanism is of clinical importance, as this SNP was identified in a clinical isolate, which acquired azole resistance in-host.

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.

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.

In order to survive, Aspergillus fumigatus must adapt to specific niche environments. Adaptation to the human host includes modifications facilitating persistent colonisation and the development of azole resistance. The aim of this study is to advance understanding of the genetic and physiological adaptation of A. fumigatus in patients during infection and treatment. Thirteen A. fumigatus strains were isolated from a single chronic granulomatous disease patient suffering from persistent and recurrent invasive aspergillosis over a period of 2 years. All strains had identical microsatellite genotypes and were considered isogenic. Whole genome comparisons identified 248 non-synonymous single nucleotide polymorphisms. These non-synonymous mutations have potential to play a role in in-host adaptation. The first 2 strains isolated were azole susceptible, whereas later isolates were itraconazole, voriconazole and/or posaconazole resistant. Growth assays in the presence and absence of various antifungal stressors highlighted minor changes in growth rate and stress resistance, with exception of one isolate showing a significant growth defect. Poor conidiation was observed in later isolates. In certain drug resistant isolates conidiation was restored in the presence of itraconazole. Differences in virulence were observed as demonstrated in a Galleria mellonella infection model. We conclude that the microevolution of A. fumigatus in this patient has driven the emergence of both Cyp51A-independent and Cyp51A-dependent, azole resistance mechanisms, and additional phenotypes that are likely to have promoted fungal persistence.

In all eukaryotic kingdoms, mitogen-activated protein kinases (MAPKs) play critical roles in cellular responses to environmental cues. These MAPKs are activated by phosphorylation at highly conserved threonine and tyrosine residues in response to specific inputs, leading to their accumulation in the nucleus and the activation of their downstream targets. A specific MAP kinase can regulate different downstream targets depending on the nature of the input signal, thereby raising a key question: what defines the stress-specific outputs of MAP kinases? We find that the Hog1 MAPK contributes to nitrosative-stress resistance in Candida albicans even though it displays minimal stress-induced phosphorylation under these conditions. We show that Hog1 becomes oxidized in response to nitrosative stress, accumulates in the nucleus, and regulates the nitrosative stress-induced transcriptome. Mutation of specific cysteine residues revealed that C156 and C161 function together to promote stress resistance, Hog1-mediated nitrosative-stress-induced gene expression, resistance to phagocytic killing, and C. albicans virulence. We propose that the oxidation of Hog1, rather than its phosphorylation, contributes to the nitrosative-stress-specific responses of this MAP kinase.

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 Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the ‘p38-related’ Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation.

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.

Stress-activated protein kinase (SAPK) pathways are evolutionarily conserved eukaryotic signalling modules that are essential for the virulence of human pathogenic fungi. The Hog1 SAPK in Candida albicans is robustly phosphorylated in response to a number of host-imposed stresses, and is essential for virulence. The current dogma is that stress-induced phosphorylation activates the SAPK, and promotes its nuclear accumulation that is necessary for the expression of SAPK-dependent stress-protective genes. Here we challenge this dogma. C. albicans strains were constructed in which Hog1 was either tethered to the plasma membrane or constitutively nuclear. Strikingly, tethering Hog1 to the plasma membrane did not abrogate stress resistance or stress-induced gene expression. Furthermore, preventing the nuclear accumulation of Hog1 had no impact on C. albicans virulence in two distinct models of systemic infection. However, tethering Hog1 to the plasma membrane did impact on signal fidelity, and on the magnitude and kinetics of the stress-induced phosphorylation of this SAPK. Taken together, these findings challenge the dogma that nuclear accumulation of SAPKs is a pre-requisite for SAPK-dependent gene expression, and reveal that stress-induced nuclear accumulation of Hog1 is dispensable for the virulence of a major human fungal pathogen.

Fever is a universal response to infection, and opportunistic pathogens such as Candida albicans have evolved complex circuitry to sense and respond to heat. Here we harness RNA-seq and ChIP-seq to discover that the heat shock transcription factor, Hsf1, binds distinct motifs in nucleosome-depleted promoter regions to regulate heat shock genes and genes involved in virulence in C. albicans. Consequently, heat shock increases C. albicans host cell adhesion, damage and virulence. Hsf1 activation depends upon the molecular chaperone Hsp90 under basal and heat shock conditions, but the effects are opposite and in part controlled at the level of Hsf1 expression and DNA binding. Finally, we demonstrate that Hsp90 regulates global transcription programs by modulating nucleosome levels at promoters of stress-responsive genes. Thus, we describe a mechanism by which C. albicans responds to temperature via Hsf1 and Hsp90 to orchestrate gene expression and chromatin architecture, thereby enabling thermal adaptation and virulence.

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.

Following phagocytosis, microbes are exposed to an array of antimicrobial weapons that include reactive oxygen species (ROS) and cationic fluxes. This is significant as combinations of oxidative and cationic stresses are much more potent than the corresponding single stresses, triggering the synergistic killing of the fungal pathogen Candida albicans by “stress pathway interference.” Previously we demonstrated that combinatorial oxidative plus cationic stress triggers a dramatic increase in intracellular ROS levels compared to oxidative stress alone. Here we show that activation of Cap1, the major regulator of antioxidant gene expression in C. albicans, is significantly delayed in response to combinatorial stress treatments and to high levels of H2O2. Cap1 is normally oxidized in response to H2O2; this masks the nuclear export sequence, resulting in the rapid nuclear accumulation of Cap1 and the induction of Cap1-dependent genes. Here we demonstrate that following exposure of cells to combinatorial stress or to high levels of H2O2, Cap1 becomes trapped in a partially oxidized form, Cap1OX-1. Notably, Cap1-dependent gene expression is not induced when Cap1 is in this partially oxidized form. However, while Cap1OX-1 readily accumulates in the nucleus and binds to target genes following high-H2O2stress, the nuclear accumulation of Cap1OX-1 following combinatorial H2O2and NaCl stress is delayed due to a cationic stress-enhanced interaction with the Crm1 nuclear export factor. These findings define novel mechanisms that delay activation of the Cap1 transcription factor, thus preventing the rapid activation of the stress responses vital for the survival of C. albicans within the host.

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.

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.

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.

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.

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.

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.

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.

Candida albicans is a major fungal pathogen of humans. This yeast is carried by many individuals as a harmless commensal, but when immune defences are perturbed it causes mucosal infections (thrush). Additionally, when the immune system becomes severely compromised, C. albicans often causes life-threatening systemic infections. A battery of virulence factors and fitness attributes promote the pathogenicity of C. albicans. Fitness attributes include robust responses to local environmental stresses, the inactivation of which attenuates virulence. Stress signalling pathways in C. albicans include evolutionarily conserved modules. However, there has been rewiring of some stress regulatory circuitry such that the roles of a number of regulators in C. albicans have diverged relative to the benign model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This reflects the specific evolution of C. albicans as an opportunistic pathogen obligately associated with warm-blooded animals, compared with other yeasts that are found across diverse environmental niches. Our understanding of C. albicans stress signalling is based primarily on the in vitro responses of glucose-grown cells to individual stresses. However, in vivo this pathogen occupies complex and dynamic host niches characterised by alternative carbon sources and simultaneous exposure to combinations of stresses (rather than individual stresses). It has become apparent that changes in carbon source strongly influence stress resistance, and that some combinatorial stresses exert non-additive effects upon C. albicans. These effects, which are relevant to fungus-host interactions during disease progression, are mediated by multiple mechanisms that include signalling and chemical crosstalk, stress pathway interference and a biological transistor.

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 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.

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.

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.

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.

Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21Δ/Δ mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity. Hsp21 therefore represents the first reported example of a small heat shock protein functioning as a virulence factor in a eukaryotic pathogen.

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.

Background the yeast Saccharomyces cerevisiae responds to amino acid starvation by inducing the transcription factor Gcn4. This is mainly mediated via a translational control mechanism dependent upon the translation initiation eIF2·GTP·Met-tRNAiMet ternary complex, and the four short upstream open reading frames (uORFs) in its 5’ mRNA leader. These uORFs act to attenuate GCN4 mRNA translation under normal conditions. During amino acid starvation, levels of ternary complex are reduced. This overcomes the GCN4 translation attenuation effect via a scanning/reinitiation control mechanism dependent upon uORF spacing. Results Using published experimental data, we have developed and validated a probabilistic formulation of GCN4 translation using the Chemical Master Equation (Model 1). Model 1 explains GCN4 translation’s nonlinear dependency upon uORF placements, and predicts that an as yet unidentified factor, which was proposed to regulate GCN4 translation under some conditions, only has pronounced effects upon GCN4 translation when intercistronic distances are unnaturally short. A simpler Model 2 that does not include this unidentified factor could well represent the regulation of a natural GCN4 mRNA. Using parameter values optimised for this algebraic Model 2, we performed stochastic simulations by Gillespie algorithm to investigate the distribution of ribosomes in different sections of GCN4 mRNA under distinct conditions. Our simulations demonstrated that ribosomal loading in the 5’-untranslated region is mainly determined by the ratio between the rates of 5’-initiation and ribosome scanning, but was not significantly affected by rate of ternary complex binding. Importantly, the translation rate for codons starved of cognate tRNAs is predicted to be the most significant contributor to the changes in ribosomal loading in the coding region under repressing and derepressing conditions. Conclusions Our integrated probabilistic Models 1 and 2 explained GCN4 translation and helped to elucidate the role of a yet unidentified factor. The ensuing stochastic simulations evaluated different factors that may impact on the translation of GCN4 mRNA, and integrated translation status with ribosomal density.

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.

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.

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.

Candida glabrata is a major fungal pathogen of humans, and the virulence of C. glabrata is increased by inactivation of the transcription factor, Ace2. Our previous examination of the effects of Ace2 inactivation upon the intracellular proteome suggested that the hypervirulence of C. glabrata ace2 mutants might be caused by differences in the secretome. Therefore in this study we have characterised the C. glabrata secretome and examined the effects of Ace2 inactivation upon this extracellular proteome. We have identified 31 distinct proteins in the secretome of wild-type C. glabrata cells by MS/MS of proteins that were precipitated from the growth medium and enriched by affinity chromatography on concanavalin A. Most of these proteins are predicted to be cell wall proteins, cell wall modifying enzymes and aspartyl proteinases. The endochitinase Cts1 and the endoglucanase Egt2 were not detected in the C. glabrata secretome following Ace2 inactivation. This can account for the cell separation defect of C. glabrata ace2 cells. Ace2 inactivation also resulted in the detection of new proteins in the C. glabrata secretome. The release of such proteins might contribute to the hypervirulence of ace2 cells.

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.

Stress responses are important for the virulence of the major fungal pathogen of humans, Candida albicans. In this study we employed a 2-DE approach to examine the impact of exposure to peroxide (5 mM H(2)O(2)), salt (300 mM NaCl) or cadmium stress (0.5 mM Cd(2+)) upon the C. albicans proteome. Highly reproducible changes in the C. albicans proteome were observed in response to each stress condition. Significantly more proteins were up-regulated in response to cadmium (77) than to the salt (35) or peroxide stresses (35). These proteomic changes displayed minimal overlap with those observed in the transcriptome under equivalent conditions and, importantly, revealed functional categories that respond to stress at the protein level but not the transcript level. Six proteins were up-regulated by all three conditions: Adh1, Atp2, Cip1, Eft2, Ssa1 and Ssb1, which is consistent with the concept that a core stress response exists in C. albicans. This is the first time that a fungal core stress response has been defined at the proteomic level. We have also shown that the Hog1 stress-activated mitogen-activated protein kinase, which is activated in response to the stresses examined in this study, makes a major contribution to the C. albicans stress proteome.

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.

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.

Fungal pathogenicity has arisen in polyphyletic manner during evolution, yielding fungal pathogens with diverse infection strategies and with differing degrees of evolutionary adaptation to their human host. Not surprisingly, these fungal pathogens display differing degrees of resistance to the reactive oxygen and nitrogen species used by human cells to counteract infection. Furthermore, whilst evolutionarily conserved regulators, such as Hog1, are central to such stress responses in many fungal pathogens, species-specific differences in their roles and regulation abound. In contrast, there is a high degree of commonality in the cellular responses to reactive oxygen and nitrogen species evoked in evolutionarily divergent fungal pathogens.

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.

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.

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 unfolded protein response (UPR) regulates the expression of genes involved in the protein secretory pathway and in endoplasmic reticulum (ER) stress in yeasts and filamentous fungi. We have characterized the global transcriptional response of Candida albicans to ER stresses (dithiothreitol and tunicamycin) and established the impact of the transcription factor Hac1 upon this response. Expression of C. albicans Hac1, which is the functional homologue of Saccharomyces cerevisiae Hac1p, is predicted to be translationally regulated via an atypical mRNA splicing event during ER stress. C. albicans genes involved in secretion, vesicle trafficking, stress responses and cell wall biogenesis are up-regulated in response to ER stress, and translation and ribosome biogenesis genes are down-regulated. Hac1 is not essential for C. albicans viability, but plays a major role in this stress-related transcriptional response and is required for resistance to ER stress. In addition, we show that Hac1 plays an important role in regulating the morphology of C. albicans and in the expression of genes encoding cell surface proteins during ER stress, factors that are important in virulence of this fungal pathogen.

MNL1, the Candida albicans homologue of an orphan Msn2-like gene (YER130c in Saccharomyces cerevisiae) has no known function. Here we report that MNL1 regulates weak acid stress responses. Deletion of MNL1 prevents the long-term adaptation of C. albicans cells to weak acid stresses and compromises their global transcriptional response under these conditions. The promoters of Mnl1-dependent genes contain a novel STRE-like element (SLE) that imposes Mnl1-dependent, weak acid stress-induced transcription upon a lacZ reporter in C. albicans. The SLE (HHYYCCCCTTYTY) is related to the Nrg1 response element (NRE) element recognized by the transcriptional repressor Nrg1. Deletion of NRG1 partially restores the ability of C. albicans mnl1 cells to adapt to weak acid stress, indicating that Mnl1 and Nrg1 act antagonistically to regulate this response. Molecular, microarray, and proteomic analyses revealed that Mnl1-dependent adaptation does not occur in cells exposed to proapoptotic or pronecrotic doses of weak acid, suggesting that Ras-pathway activation might suppress the Mnl1-dependent weak acid response in dying cells. Our work defines a role for this YER130c orthologue in stress adaptation and cell death.

Micro-organisms must adapt to environmental change to survive, and this is particularly true for fungal pathogens such as Candida glabrata. C. glabrata is found both in the environment and in diverse niches in its human host. The ambient pH of these niches varies considerably, and therefore we have examined the response of C. glabrata to changes in ambient pH using a proteomic approach. Proteins expressed in C. glabrata cells growing at pH 4.0, 7.4 or 8.0 were compared by 2-DE, and 174 spots displaying reproducible and statistically significant changes in expression level were identified by peptide mass fingerprinting, thereby extending our 2-DE map of the C. glabrata proteome to a total of 272 identified spots. Proteins involved in glucose metabolism, the TCA cycle, respiration and protein synthesis were expressed at lower levels during growth at pH 7.4 and/or 8.0, whereas proteins involved in stress responses and protein catabolism were expressed at higher levels under these alkaline conditions. Our data suggest that C. glabrata perceives low pH as less stressful than higher pH. This contrasts with another opportunistic fungal pathogen of humans, Candida albicans.

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.

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.

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.

The central metabolic enzyme fructose-1,6-bisphosphate aldolase (Fba1p) catalyzes a reversible reaction required for both glycolysis and gluconeogenesis. Fba1p is a potential antifungal target because it is essential in yeast and because fungal and human aldolases differ significantly. To test the validity of Fba1p as an antifungal target, we have examined the effects of depleting this enzyme in the major fungal pathogen Candida albicans. Using a methionine/cysteine-conditional mutant (MET3-FBA1/fba1), we have shown that Fba1p is required for the growth of C albicans. However, Fba1p must be depleted to below 5% of wild-type levels before growth is blocked. Furthermore, Fba1p depletion exerts static rather than cidal effects upon C albicans. Fba1p is a relatively abundant and stable protein in C albicans, and hence, Fba1p levels decay relatively slowly following MET3-FBA1 shutoff. Taken together, our observations can account for our observation that the virulence of MET3-FBA1/fba1 cells is only partially attenuated in the mouse model of systemic candidiasis. We conclude that an antifungal drug directed against Fba1p would have to be potent to be effective.

We outline a framework for managing information quality (IQ) in eScience, using ontologies, semantic annotation of resources, and data bindings. Scientists define the quality characteristics that are of importance in their particular domain by extending an OWL DL IQ ontology, which classifies and organises these domain-specific quality characteristics within an overall quality management framework. RDF is used to annotate data resources, with reference to IQ indicators defined in the ontology. Data bindings - again defined in RDF - are used to represent mappings between data elements (e.g. defined in XML Schemas) and the IQ ontology. As a practical illustration of our approach, we present a case study from the domain of proteomics.

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.

The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.

CandidaDB is a database dedicated to the genome of the most prevalent systemic fungal pathogen of humans, Candida albicans. CandidaDB is based on an annotation of the Stanford Genome Technology Center C.albicans genome sequence data by the European Galar Fungail Consortium. CandidaDB Release 2.0 (June 2004) contains information pertaining to Assembly 19 of the genome of C.albicans strain SC5314. The current release contains 6244 annotated entries corresponding to 130 tRNA genes and 5917 protein-coding genes. For these, it provides tentative functional assignments along with numerous pre-run analyses that can assist the researcher in the evaluation of gene function for the purpose of specific or large-scale analysis. CandidaDB is based on GenoList, a generic relational data schema and a World Wide Web interface that has been adapted to the handling of eukaryotic genomes. The interface allows users to browse easily through genome data and retrieve information. CandidaDB also provides more elaborate tools, such as pattern searching, that are tightly connected to the overall browsing system. As the C.albicans genome is diploid and still incompletely assembled, CandidaDB provides tools to browse the genome by individual supercontigs and to examine information about allelic sequences obtained from complementary contigs. CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.

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 development of the molecular toolbox for the fungal pathogen Candida albicans has been hampered by its lack of an exploitable sexual cycle, its diploid nature, and its non-canonical genetic code. We describe the adaptation of the Cre-loxP site-specific recombination system as a tool for the efficient and controlled disruption of C. albicans genes. We have validated this system by disrupting two C. albicans loci: ADE2 and MET15. Ade2 and met15 null mutants were made using loxP-flanked ARG4- and HIS1-based disruption cassettes. These markers were then resolved from the C. albicans genome using a synthetic codon-optimised cre recombinase gene, with near 100% efficiency. Finally, CIp plasmids containing the URA3, HIS1, and ARG4 markers were generated for the reintegration of markers and target genes in control strains. This system allows multiple and sequential genetic manipulations, which will facilitate the functional analysis of multigene families in C. albicans.

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.

Inactivation of the gene encoding the transcriptional activator Ace2 in the fungal pathogen Candida glabrata results in an almost 200-fold increase in virulence characterised by acute mortality and a massive over-stimulation of the pro-inflammatory arm of the innate immune system. In this study we have adopted a proteomics approach to identify cellular functions regulated by C. glabrata Ace2 that might contribute to this increase in virulence. A two-dimensional polyacrylamide gel electrophoresis map of the C. glabrata proteome was constructed. We identified a total of 123 proteins, 61 of which displayed reproducible and statistically significant alterations in their levels following inactivation of ACE2. of these, the levels of 32 proteins were elevated, and 29 were reduced in ace2 cells. These data show that Ace2 influences metabolism, protein synthesis, folding and targeting, and aspects of cell growth and polarisation. Some of these functions are likely to contribute to the effects of Ace2 upon the virulence of C. glabrata.

The ALS (agglutinin-like sequence) gene family of Candida albicans encodes eight cell-surface glycoproteins, some of which are involved in adherence to host surfaces. A mutational analysis of each ALS gene is currently being performed to deduce the functions of the encoded proteins and to better understand the role of these proteins in C. albicans biology and pathogenesis. This paper describes construction of an als3/als3 mutant and comparison of its phenotype to an als1/als1 strain. Efforts to disrupt ALS3 indicated that the gene could be deleted in two transformation steps, suggesting that the gene is encoded by a single locus and that the ALS3-like locus, ALS8, does not exist. Strains lacking ALS3 or ALS1 did not exhibit a defect in germ tube formation when grown in RPMI 1640 medium, but the als1/als1 mutant formed significantly fewer germ tubes in Lee medium. Analysis of ALS3 and ALS1 promoter activity using green fluorescent protein (GFP) reporter strains and flow cytometry showed that when cells are placed into medium that promotes germ tube formation, ALS1 is transcribed prior to ALS3. Comparison of the mutant strains in adhesion assays showed that the als3/als3 strain was defective in adhesion to both human umbilical vein endothelial cells (HUVEC) and buccal epithelial cells (BEC), but not to fibronectin-coated plastic plates. In contrast, the als1/als1 strain showed decreased adherence to HUVEC, but adherence to BEC and fibronectin were the same as wild-type controls. Inoculation of the buccal reconstituted human epithelium (RHE) model of oral candidiasis with the mutant strains showed nearly a total lack of adhesion and epithelial destruction by the als3/als3 mutant while the als1/als1 strain showed only a slightly reduced degree of epithelial destruction compared to the wild-type control. Adhesion data presented here suggest that, in the assays performed, loss of Als3p affects C. albicans adhesion more than loss of Als1p. Collectively, these results demonstrate functional similarities and differences between Als1p and Als3p, and suggest the potential for more complex interrelationships between the ALS genes and their encoded proteins.

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.

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.

BACKGROUND: Proteomics is rapidly evolving into a high-throughput technology, in which substantial and systematic studies are conducted on samples from a wide range of physiological, developmental, or pathological conditions. Reference maps from 2D gels are widely circulated. However, there is, as yet, no formally accepted standard representation to support the sharing of proteomics data, and little systematic dissemination of comprehensive proteomic data sets. RESULTS: This paper describes the design, implementation and use of a Proteome Experimental Data Repository (PEDRo), which makes comprehensive proteomics data sets available for browsing, searching and downloading. It is also serves to extend the debate on the level of detail at which proteomics data should be captured, the sorts of facilities that should be provided by proteome data management systems, and the techniques by which such facilities can be made available. CONCLUSIONS: the PEDRo database provides access to a collection of comprehensive descriptions of experimental data sets in proteomics. Not only are these data sets interesting in and of themselves, they also provide a useful early validation of the PEDRo data model, which has served as a starting point for the ongoing standardisation activity through the Proteome Standards Initiative of the Human Proteome Organisation.

Saccharomyces cerevisiae activates general amino acid control (GCN) in response to amino acid starvation. Some aspects of this response are known to be conserved in other fungi including Candida albicans, the major systemic fungal pathogen of humans. Here, we describe a proteomic comparison of the GCN responses in S. cerevisiae and C. albicans. We have used high-resolution two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting to develop a 2-D protein map of C. albicans. A total of 391 protein spots, representing 316 open reading frames, were identified. Fifty-five C. albicans and 65 S. cerevisiae proteins were identified that responded reproducibly to 3-aminotriazole (3AT) in a Gcn4p-dependent fashion. The changes in the S. cerevisiae proteome correlated with the response in the S. cerevisiae transcript profile to 3AT treatment (rank correlation coefficient = 0.59; Natarajan et al. Molec. Cell. Biol. 2001, 21, 4347-4368). Significant aspects of the GCN response were conserved in C. albicans and S. cerevisiae. In both fungi, amino acid biosynthetic enzymes on multiple metabolic pathways were induced by 3AT in a Gcn4p-dependent fashion. Carbon metabolism functions were also induced. However, subtle differences were observed between these fungi. For example, purine biosynthetic enzymes were induced in S. cerevisiae, but were not significantly induced in C. albicans. These differences presumably reflect the contrasting niches of these relatively benign and pathogenic yeasts, respectively.

Both the generation and the analysis of proteome data are becoming increasingly widespread, and the field of proteomics is moving incrementally toward high-throughput approaches. Techniques are also increasing in complexity as the relevant technologies evolve. A standard representation of both the methods used and the data generated in proteomics experiments, analogous to that of the MIAME (minimum information about a microarray experiment) guidelines for transcriptomics, and the associated MAGE (microarray gene expression) object model and XML (extensible markup language) implementation, has yet to emerge. This hinders the handling, exchange, and dissemination of proteomics data. Here, we present a UML (unified modeling language) approach to proteomics experimental data, describe XML and SQL (structured query language) implementations of that model, and discuss capture, storage, and dissemination strategies. These make explicit what data might be most usefully captured about proteomics experiments and provide complementary routes toward the implementation of a proteome repository.

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.

Glucose exerts profound effects upon yeast physiology. In general, the effects of high glucose concentrations (>1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of> 0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post-transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile. In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient.

Protein A-tagging has become an important tool in characterization of protein-protein interactions in many systems, allowing purification of multicomponent complexes under native conditions. Here we provide a set of vectors that allow protein A-tagging in Candida albicans, through addition of the tag to open reading frames. These vectors were successfully used to generate stably tagged proteins that were functional, shown to be localized appropriately or assembled into complexes. These new vectors comprise a useful addition to the C. albicans molecular toolbox. Copyright (C) 2003 John Wiley Sons, Ltd.

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.

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.

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.

The mechanisms by which the weak acid preservative benzoic acid inhibits the growth of Saccharomyces cerevisiae have been investigated, a reduction in the pyruvate kinase level, which decreases glycolytic flux, did not increase the sensitivity of yeast to benzoic acid, However, a decrease in 6-phosphofructo-1-kinase (PF1K), which does not affect glycolytic flux, did increase sensitivity to benzoic acid. Also, resistance was increased by elevating PF1K levels. Hence, resistance to benzoic acid was not dependent upon optimum glycolytic flux, but upon an adequate PF1K activity. Benzoic acid was shown to depress fructose 2,6-bisphosphate levels in YKC14 a mutant with low PF1K levels, This effect was partially suppressed by overexpressing constitutively active 6-phosphofructo-2-kinase (pfk26(Asp644)) or by inactivating fructose-2.6-bisphosphatase (in a Delta fbp26 mutant). The inactivation of PF2K (in a Delta pfk26 Delta pfk27 mutant) increased benzoic acid sensitivity, Therefore, the antimicrobial effects of benzoic acid can be relieved, at least in part, by the genetic manipulation of PF1K or fructose 2,6-bisphosphate levels.

We have characterized CaNrg1 from Candida albicans the major fungal pathogen in humans. CaNrg1 contains a zinc finger domain that is conserved in transcriptional regulators from fungi to humans. It is most closely related to ScNrg1, which represses transcription in a Tup1-dependent fashion in Saccharomyces cerevisiae. Inactivation of CaNrg1 in C. albicans causes filamentous and invasive growth, derepresses hypha-specific genes, increases sensitivity to some stresses and attenuates virulence. A tup1 mutant displays similar phenotypes. However, unlike tupl cells, nrg1 cells can form normal hyphae, generate chlamydospores at normal rates and grow at 42°C. Transcript profiling of 2002 C. albicans genes reveals that CaNrgl represses a subset of CaTupl-regulated genes, which includes known hypha-specific genes and other virulence factors. Most of these genes contain an Nrg1 response element (NRE) in their promoter. CaNrg1 interacts specifically with an NRE in vitro. Also, deletion of two NREs from the ALS8 promoter releases it from Nrg1-mediated repression. Hence, CaNrg1 is a transcriptional repressor that appears to target CaTup1 to a distinct set of virulence-related functions, including yeast-hypha morphogenesis.

Yeast phosphofructo-1-kinase (Pf1k) and pyruvate kinase (Pyk1) are allosterically regulated enzymes that catalyse essentially irreversible reactions in glycolysis. Both the synthesis and activity of these enzymes are tightly regulated, to separate experimentally the control of Pf1k and Pyk1 synthesis from their allosteric regulation, a congenic set of PFK1, PFK2 and PYK1 mutants was constructed in which these wild-type coding regions were driven by alternative promoters, Mutants carrying PGK1 promoter fusions displayed normal rates of growth, glucose consumption and ethanol production, indicating that the relatively tight regulation of Pyk1 and Pf1k synthesis is not essential for glycolytic control under fermentative growth conditions, Mutants carrying fusions to an enhancer-less version of the PGK1 promoter (PGK1(Delta 767)) expressed Pyk1 and Pf1k at about 2.5-fold lower levels than normal, Physiological and metabolic analysis of the PFK1 PFK2 double mutant indicated that decreased Pf1k had no significant effect on growth, apparently due to compensatory increases in its positive effector, fructose 2,6-bisphosphate. In contrast, growth rate and glycolytic flux were reduced in the PGK1(Delta 767)-PYK1 mutant, which had decreased Pyk1 levels, Unexpectedly, the reduced Pyk1 levels caused the flow of carbon to the TCA cycle to increase, even under fermentative growth conditions. Therefore, Pyk1 exerts a significant level of control over both the rate and direction of carbon flux in yeast.

The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S, cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not, These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

The pathogenic fungus, Candida albicans contains homologues of the transcriptional repressors ScTup1, ScMig1 and ScNrg1 found in budding yeast. In Saccharomyces cerevisiae, ScMig1 targets the ScTup1/ScSsn6 complex to the promoters of glucose repressed genes to repress their transcription. ScNrg1 is thought to act in a similar manner at other promoters. We have examined the roles of their homologues in C. albicans by transcript profiling with an array containing 2002 genes, representing about one quarter of the predicted number of open reading frames (ORFs) in C. albicans. The data revealed that CaNrg1 and CaTup1 regulate a different set of C. albicans genes from CaMig1 and CaTup1. This is consistent with the idea that CaMig1 and CaNrg1 target the CaTup1 repressor to specific subsets of C. albicans genes. However, CaMig1 and CaNrg1 repress other C. albicans genes in a CaTup1-independent fashion. The targets of CaMig1 and CaNrg1 repression, and phenotypic analyses of nrg1/nrg1 and mig1/mig1 mutants, indicate that these factors play differential roles in the regulation of metabolism, cellular morphogenesis and stress responses. Hence, the data provide important information both about the modes of action of these transcriptional regulators and their cellular roles. The transcript profiling data are available at http://www.pasteur.fr/recherche/unites/RIF/transcriptdata/.

Glucose regulates yeast gene expression at both transcriptional and post-transcriptional levels. Glucose strongly represses the transcription of the gluconeogenic genes, FBP1 and PCK1, and accelerates the degradation of their mRNAs. Together these mechanisms are responsible for the rapid decrease in gluconeogenic enzyme synthesis when yeast cells switch to glycolytic metabolism. In this study, we show that accelerated gluconeogenic mRNA degradation can be triggered by low concentrations of glucose (1%). We also show that accelerated gluconeogenic mRNA degradation is co-ordinated with transcriptional repression by common signalling components that include sugar kinases and Ras-cAMP signalling. Furthermore, the ability of the low glucose signal to trigger accelerated gluconeogenic mRNA degradation depends upon the low glucose sensor, Snf3p, but not on the high glucose sensor, Rgt2p. Also, this response is influenced by reg1 and ume5 mutations, but not by grr1 or rgt1 mutations. Our data suggest that several signalling pathways co-ordinate differential post-transcriptional and transcriptional responses in yeast, depending upon the amount of glucose available in the medium.

Rad6p plays important roles in post-replication DNA repair, chromatin organization, gene silencing and meiosis. In this study, we show that Rad6p also regulates yeast-hypha morphogenesis in the human pathogen Candida albicans. CaRAD6 gene and cDNAs were isolated and characterized revealing that the gene carries two 5’-proximal introns. CaRad6p shows a high degree of sequence similarity to Rad6 proteins from fungi to man (60-83% identity), and it suppresses the UV sensitivity and lack of induced mutagenesis displayed by a Saccharomyces cerevisiae rad6 mutant. In C. albicans, CaRAD6 expression is induced in response to UV, and CaRad6p depletion confers UV sensitivity, confirming that Rad6p serves a role in protecting this fungus against UV damage. CaRAD6 overexpression inhibits hyphal development, whereas CaRad6p depletion enhances hyphal growth. Also, CaRAD6 mRNA levels decrease during the yeast-hypha transition. These effects are dependent on Efg1p, but not Cph1p, indicating that CaRad6p acts specifically through the Efg1p morphogenetic signalling pathway to repress yeast-hypha morphogenesis.

The Saccharomyces cerevisiae gene YNL234w encodes a 426-amino acid-long protein that shares significant similarities with the globin family. Compared with known globins from unicellular organisms, the Ynl234wp polypeptide is characterized by an unusual structure. In this protein, a central putative heme-binding domain of about 140 amino acids is flanked by two sequences of about 160 and 120 amino acids, respectively, which share no similarity with known polypeptides. Northern analysis indicates that YNL234w transcription is very low in cells grown under normal aerobic conditions but is induced by oxygen-limited growth conditions and by other stress conditions such as glucose repression, heat shock, osmotic stress, and nitrogen starvation. However the deletion of the gene had no detectable effect on yeast growth. The Ynl234wp polypeptide has been expressed in Escherichia coli, and the hemoprotein nature of the recombinant protein was demonstrated by heme staining after SDS/polyacrylamide gel electrophoresis and spectroscopic analysis. Our data indicate that purified recombinant Ynl234wp possesses a noncovalently bound heme molecule that is predominantly found in a low spin form.

Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase was purified to apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells efficiently overexpressing the GFA1 gene. The pure enzyme exhibited K-m(Gln) = 1.56 mM and Km(Fru-6-P) = 1.41 mM and catalyzed GlcN-6-P formation with k(cat) = 1150 min(-1). The isoelectric point of 4.6 +/- 0.05 was estimated from isoelectric chromatofocusing, Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330-340 kDa, Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Flu-6-P-binding site. GlcN-6-P synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC50 = 0.67 mM). Purification of the enzyme markedly decreased the sensitivity to the inhibitor, but this could be restored by addition of another effector, glucose g-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with RN = 0.54, whereas in the absence of this sugar phosphate no cooperative effect was observed. Pure enzyme was a substrate for cAMP-dependent protein kinase, the action of which led to the substantial increase of GlcN-6-P synthase activity, correlated with an extent of protein phosphorylation, the maximal level of activity was observed for the enzyme molecules containing 1.21 +/- 0.08 mol of phosphate/mol of GlcN-6-P synthase, Monitoring of GlcN-6-P synthase activity and its sensitivity to UDP-GlcNAc during yeast –> mycelia transformation of C, albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter.

A Candida albicans mitochondrial ribosomal protein S9 (MRPS9) cDNA was identified in a screen for sequences whose expression induce galactose lethality in Saccharomyces cerevisiae. MRPS9 appears to encode a protein of 346 amino acids with an N-terminal mitochondrial targeting sequence and an internal S9 signature that is conserved amongst eukaryotic mitochondrial and prokaryotic ribosomal protein S9 sequences. Expression of a GAL1-CaMRPS9 fusion in S. cerevisiae caused the slow development of a galactose-negative phenotype upon repeated subculturing, and this correlated with an increased frequency of petite mutant formation. Therefore, over-expression of CaMRPS9 interferes with S. cerevisiae mitochondrial function, which accounts for the inhibition of growth on galactose. The sequence has been entered in the EMBL data library under Accession Number AF067962. Copyright (C) 1999 John Wiley & Sons, Ltd.

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.

The yeast LPD1 gene encoding lipoamide dehydrogenase is subject to the general control of amino acid biosynthesis mediated by the GCN4 transcription factor. This is striking in that it demonstrates that CCN4-mediated regulation extends much farther upstream than simply to the direct pathways for amino acid and purine biosynthesis. In yeast, lipoamide dehydrogenase functions in at least three multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (which function in the entry of pyruvate into, and metabolism via, the citric acid cycle) and glycine decarboxylase. When wild-type cells were shifted from growth on amino acid-rich to amino acid-deficient medium, the expression of lipoamide dehydrogenase was induced approx. 2-fold. In a similar experiment no such induction was observed in isogenic gcn4 mutant cells. Northern analysis indicated that amino acid starvation affected levels of the LPD1 transcript. In the upstream region of LPD1 are three matches to the consensus for control mediated by GCN4. Directed mutagenesis of each site, and of all combinations of sites, suggests that only one site might be important for the general control response under the conditions tested. Gel-retardation analysis with GCN4 protein synthesized in vitro has indicated that GCN4 can bind in vitro to at least two of the consensus motifs.

The yeast LPD1 gene encoding lipoamide dehydrogenase is subject to the general control of amino acid biosynthesis mediated by the GCN4 transcription factor. This is striking in that it demonstrates that GCN4-mediated regulation extends much farther upstream than simply to the direct pathways for amino acid and purine biosynthesis. In yeast, lipoamide dehydrogenase functions in at least three multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (which function in the entry of pyruvate into, and metabolism via, the citric acid cycle) and glycine decarboxylase. When wild-type cells were shifted from growth on amino acid-rich to amino acid-deficient medium, the expression of lipoamide dehydrogenase was induced approx. 2-fold. In a similar experiment no such induction was observed in isogenic gcn4 mutant cells. Northern analysis indicated that amino acid starvation affected levels of the LPD1 transcript. In the upstream region of LPD1 are three matches to the consensus for control mediated by GCN4. Directed mutagenesis of each site, and of all combinations of sites, suggests that only one site might be important for the general control response under the conditions tested. Gel-retardation analysis with GCN4 protein synthesized in vitro has indicated that GCN4 can bind in vitro to at least two of the consensus motifs.

A Candida albicans mitochondrial ribosomal protein S9 (MRPS9) cDNA was identified in a screen for sequences whose expression induce galactose lethality in Saccharomyces cerevisiae. MRPS9 appears to encode a protein of 346 amino acids with an N-terminal mitochondrial targeting sequence and an internal S9 signature that is conserved amongst eukaryotic mitochondrial and prokaryotic ribosomal protein S9 sequences. Expression of a GAL1-CaMRPS9 fusion in S. cerevisiae caused the slow development of a galactose-negative phenotype upon repeated subculturing, and this correlated with an increased frequency of petite mutant formation. Therefore, over-expression of CaMRPS9 interferes with S. cerevisiae mitochondrial function, which accounts for the inhibition of growth on galactose. The sequence has been entered in the EMBL data library under Accession Number AF067962.

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.

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.

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), 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 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.

The transcription of the yeast FBP1 and PCK1 genes, which encode the gluconeogenic enzymes fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, is repressed by glucose. Here, we show that this repression is both very strong and exceptionally sensitive to glucose, being triggered by glucose at concentrations less than 0.005% (0.27 mM). This repression remains operative in yeast mutants carrying any one of the three hexose kinases, but is lost in a triple hxk1, hxk2, glk1 mutant. In addition, 2-deoxyglucose can trigger the repression, but 6-deoxyglucose cannot, suggesting that internalization and phosphorylation of the glucose is essential for repression to occur. While gluconeogenic gene transcription is subject to the Mig 1p-dependent pathway of glucose repression, the exquisite response to glucose is maintained in hxk2 and mig1 mutants, suggesting that this pathway is not essential for the response. The response can also be triggered by the addition of exogenous cAMP, suggesting that the Ras/cAMP pathway can mediate repression of the FPB1 and PCK1 mRNAs. However, the response is not dependent upon this pathway because it remains intact in Ras, adenyl cyclase and protein kinase a mutants. The data show that yeast cells can detect very low glucose concentrations in the environment, and suggest that several distinct signalling pathways operate to repress FPB1 and PCK1 transcription in the presence of glucose.

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.

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.

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.

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.

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

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.

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.

A new modular in vivo/in vitro expression system was constructed which facilitates studies of the control and regulation of gene expression in the yeast Saccharomyces cerevisiae. We studied the influence of stem-loop structures inserted into the non-translated leader region upon the steady- state levels and translation of mRNAs bearing the cat gene from the bacterial transposon Tn9. mRNA abundance changed relatively little in response to alterations in the leader sequence and structure, whereas stem-loop structures clearly inhibited translation to a degree that was dependent upon the predicted stability as well as the position of the inserted secondary structure. A stem-loop structure with a predicted stability greater than -28 kcal mol-1 and with a stem comprising at least 15 (mainly G/C) base pairs inhibited translation in vivo by at least 98%. A stem-loop structure with a predicted stability of approximately -14 kcal mol-1, whose stem comprised at least six G/C base pairs, inhibited translation in vivo by at least 66%. The hairpins were more inhibitory when placed close to the start codon than when positioned near the 5' end of the leader. An mRNA showing extensive complementarity between the leader and trailer regions was not only poorly translated but also had a steady-state level at least three times higher than the average for all the cat constructs examined. Translation of the various mRNAs in a yeast cell-free system followed qualitatively the same pattern as the results obtained in vivo. The stem-loop structures were far less inhibitory in a reticulocyte lysate system. Overall, the data are likely to reflect the full spectrum of translational activities of yeast mRNAs in vivo determined by secondary structure and emphasize the importance of translation as a control step in gene expression.

A well‐defined set of isogenic yeast strains has been constructed whereby each strain contains a different LPD::lacZ gene fusion integrated at the ura3 locus. These LPD::lacZ fusions differ in the amount of the LPD1 gene (encoding lipoamide dehydrogenase) that is fused to the lacZ reporter. Comparison of the β‐galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the LPD1 coding region between +13 and +700 is involved in activating gene expression in a carbon source‐dependent manner. This activation occurs at the mRNA level, and is not mediated by changes in mRNA stability. Therefore, the LPD1 gene appears to contain a transcriptional enhancer that lies 3′ to the transcriptional start site, and which responds to carbon source. Copyright © 1992, Wiley Blackwell. All rights reserved

Previous studies have proposed that specific translational pauses have evolved to promote protein folding inside the cell by temporally separating the folding of specific regions of some polypeptide chains during their synthesis. Here we show that this is the case for a bifunctional protein in Saccharomyces cerevisiae. The yeast TRP3 gene contains a translational pause comprising ten contiguous non-preferred codons within its second functional domain (indoleglycerol phosphate synthase). Site-directed mutagenesis was used to remove this translational pause by increasing the codon bias of the region without changing the amino acid sequence of the protein (to create the gene TRP3pr: pause replaced). The TRP3pr gene was able to complement a trp3 :: URA3 null mutation in yeast. No significant differences in the doubling times of TRP3 or TRP3pr yeast transformants were observed during growth at 25 °C, 30 °C or 37 °C, or in the presence of sublethal concentrations of the analogue, 5-methyltryptophan. However, further analysis of TRP3 and TRP3pr transformants revealed that the removal of the translational pause causes a 1.5-fold decrease in indoleglycerol phosphate synthase activity per TRP3 mRNA. This observation which is statistically significant (P < 0.05) and reproducible, suggests that translational pausing promotes the correct intracellular folding of the TRP3 protein. © 1992.

The Saccharomyces cerevisiae kinase-encoding gene (PYK1) has been transformed back into yeast using a derivative of the multicopy vector, pJDB207. High levels of PYK1 expression in these transformants are limited by at least two separate mechanisms. Pyruvate kinase assays and polysome analyses demonstrate that the translation of the PYK1 mRNA is inhibited as its abundance increases. The abundance of the PYK1 mRNA per gene copy also decreases as the copy number of the PYKI gene increases. This is the first report which demonstrates that a eukaryotic glycolytic gene is subject to dosage limitation at the translational level. © 1990.

The effects of poor codon bias and secondary structure formation upon the translation of the pyruvate kinase (PYK1) mRNA have been investigated in Saccharomyces cerevisiae. Following insertion mutagenesis at the 5′‐end of the PYK1 coding region, the gene was transformed into yeast, and translation assessed directly in vivo by determining the distribution of the modified PYK1 mRNAs across polysomes fractionated by sucrose density gradient centrifugation. The chromosomally‐encoded (wild‐type) PYK1 mRNA, and the actin, ribosomal protein L3 and glyceraldehyde‐3‐phosphate dehydrogenase mRNAs were used to control for minor differences between polysome preparations. An insertion containing 13 non‐preferred codons at the 5′‐end of the coding region was found to have no significant effect upon PYK1 mRNA translation. In contrast, translation was inhibited by an insertion which increased the formation of secondary structures at the 5′‐end of the mRNA (overall ΔG = −36·6 kcal/mol). Control insertions were also analysed to exclude the possibility that alterations to the amino acid sequence of pyruvate kinase affect the translation of its mRNA. These insertions, which introduced preferred codons or restored wild‐type levels of secondary structure formation, did not significantly influence PYK1 mRNA translation. Copyright © 1989 John Wiley & Sons Ltd.

The sequence of the gene encoding pyruvate kinase from Saccharomyces cerevisiae was re-determined because of failures with oligonucleotide-directed mutagenesis experiments involving a region thought to contain a string of five contiguous non-preferred codons. This region was found to be difficult to sequence and was shown to have three extra bases when compared with the published sequence [(1983) J. Biol. Chem. 258, 2193-2201]. The revised sequence demonstrates that the yeast pyruvate kinase gene does not have a cluster of non-preferred codons, and that it therefore is not an example of the class of genes which possibly exhibit translational control by the presence of non-preferred codons. © 1989.

The analysis of 17 functional mRNAs and two recombinant mRNAs in the yeast Saccharomyces cerevisiae suggests that the length of an mRNA influences its half-life in this organism. The mRNAs are clearly divisible into two populations when their lengths and half-lives are compared. Differences in ribosome loading amongst the mRNAs cannot account for this division into relatively stable and unstable populations. Also, specific mRNAs seem to be destabilized to differing extents when their translation is disrupted by N-tenninus-proximal stop codons. The analysis of a mutant mRNA, generated by the fusion of the yeast PYK1 and URA3 genes, suggests that a destabilizing element exists within the URA3 sequence. The presence of such elements within relatively unstable mRNAs might account for the division between the yeast mRNA populations. On the basis of these, and other previously published observations, a model is proposed for a general pathway of mRNA degradation in yeast. This model may be relevant to other eukaryotic systems. Also, only a minor extension to the model is required to explain how the stability of some eukaryotic mRNAs might be regulated. © 1988.

A comparison between the half-lives of 10 specific yeast mRNA3 and their distribution within polysones (fractionated on sucrose density gradients) was used to test the relationship between mRNA translation and degradation in the eukaryote Saccharomvces cerevisiae. Although the mRNAs vary in their distribution across the same polysorae gradients, there is no obvious correlation between the stability of an mRNA and the number of ribosoraes it carries in vivo. This suggests that ribosomal protection against nucleolytic attack is not a major factor in determining the stability of an mRNA in yeast. The relative lengths of the poly(A) tails of 9 yeast mRNAs were analysed using thermal elution from poly(U)-Sepharose. No dramatic differences in poly(A) tail length were observed amongst the mRNAs which could account for their wide ranging half-lives. Minor differences were consistent with shortening of the poly(A) tail as an mRNA ages. © 1987 IRL Press Limited.

A 53 basepair deletion was constructed within the 3' untranslated region (3' UTR) of the yeast pyruvate kinase (PYK) gene borne upon a centromeric plasmid. Various modular assemblies of the pUC13 polylinker DNA (single unit = 44 bp) were used to replace the deleted region, and the effects of these modifications upon both transcript stability and translation ascertained in yeast. The use of a differential probing stratagem, based on the hybridisation of specific oligonucleotides to either pUC13 polylinker or unaltered PYK 3' UTR sequences, allowed for discrimination between mutant (plasmid borne) and wild-type (chromosomal) PYK transcripts. In no construct was there any significant alteration in mRNA stability, but translation of the PYK mRNA was severely curtailed by truncation of the 3' UTR or the presence of a strong hairpin-loop structure in the 3' UTR. A specific mutation in the N-terminal coding sequences, which created a premature termination codon in both a 3' 'tagged' PYK plasmid and a PYK/LacZ fusion gene, aborted the translation of a majority of their transcripts but left their chemical half-lives unaltered. This observation is at variance with some previously published data (Losson & Lacroute (1979) Proc Natl Acad Sci USA 76, 5134; Pelsey & Lacroute (1984) Curr Genet 8, 277), but is consistent with our own earlier observation that there is no obvious link between ribosome loading and mRNA stability in yeast (Santiago et al. (1986) Nucleic Acids Res 14, 8347). Possible reasons for this disparity are discussed. © 1987 IRL Press Limited, Oxford, England.

We propose that the way in which some proteins fold is affected by the rates at which regions of their polypeptide chains are translated in vivo. Furthermore, we suggest that their gene sequences have evolved to control the rate of translational elongation such that the synthesis of defined portions of their polypeptide chains is separated temporally. We stress that many proteins are capable of folding efficiently into their native conformations without the help of differential translation rates. For these proteins the amino acid sequence does indeed contain all the information needed for the polypeptide chain to fold correctly (even in vitro, after denaturation). However, other proteins clearly do not fold efficiently into their native conformation in vitro. We argue that the efficiency of folding of these problematic proteins in vivo may be improved by controlled synthesis of the nascent polypeptide. © 1987.

Plasmids were assembled in which the coding region of the pyruvate kinase (PYK) gene of Saccharomvces cerevisiae was replaced by that of the B-galactosidase (LacZ) gene from Escherichia coli. Analysis of the resultant, chimaeric transcripts from low copy number, centromeric plasmids indicated that this substitution caused a dramatic reduction in the steady-state level of the messenger RNA (mRNA). This fluctuation cannot be wholly accounted for by the 2-fold decrease in mRNA stability observed. This is consistent with the existence of a transcriptional Downstream Activation Site (DAS) within the PYK coding region, analogous to the DAS reported within the yeast phosphoglycerate kinase gene (PGK; Kingsman, S M et al. (1985) Biotech. Gen, Eng. Rev. 3. 377). At these low levels of heterologous gene expression, comparison of the distribution of PYK and PYK/LacZ transcripts across polysome gradients revealed no significant effect mediated by their striking disparity in codon usage. Nevertheless, upon increasing B-galactosidase mRNA levels, via manipulation of plasmid copy number, a distinct decline in ribosome loading was observed for the heterologous PYK/LacZ transcript which was not mirrored by either endogenous PYK transcripts or other yeast mRNAs of high (Ribosomal protein 1) or moderate (Actin) codon bias. However, high levels of the PYK/LacZ mRNA did affect the translation of an endogenous mRNA with poor codon bias (TRP2). The possible basis for this phenomenon is discussed. © 1987 IRL Press Limited, Oxford, England.

A rapid and convenient procedure has been developed for the measurement of mRNA half-life in S.cerevisiae using the transcriptional inhibitor, 1,10-phenanthroline. A range of half-lives from 6.6 ± 0.67 minutes to over 100 minutes, relative to the stability of the 18S rRNA control, has been obtained for fifteen mRNAs. They include the pyruvate kinase and actin mRNAs, as well as 13 randomly picked mRNAs of unknown function. The mRNAs clearly fall into two populations when their lengths and half-lives are analysed; one population is considerably more stable than the other when mRNAs of similar length are compared. Also, within each population, there is an inverse relationship between mRNA length and half-life. These results suggest that mRNA length and at least one additional factor strongly influence mRNA stability in yeast. © 1986 IRL Press Limited.

Ferritin, an iron-storage protein found in all life forms examined, is composed of varying proportions of two subunits of different molecular weight, heavy (H) and light (L). Using cDNA clones, we have determined the nucleotide sequence corresponding to the mRNA of the L-subunit of rat liver ferritin. The coding region of 546 nucleotides (182 amino acids) is flanked by 5'- and 3'-untranslated regions of approximately 130 and 150 nucleotides, respectively. The rat liver L-subunit amino acid sequence derived from the reading frame of the cDNA showed 88% and 82% homology, respectively, with the amino acid sequences of horse spleen ferritin (Heusterspreute, M. and Crichton, R.R. (1981) FEBS Lett. 129, 322-327), and human spleen ferritin (Wustefeld, C. and Crichton, R. R. (1982) FEBS Lett. 150, 43-48), thus demonstrating evolutionary conservation of the L-subunit sequence. However, a major difference between the rat and the horse and human sequences is the insertion of an octopeptide near the COOH-terminus of the rat protein resulting in a slightly longer peptide chain in this species. The reading frame and parts of the derived amino acid sequence including the octopeptide seuence were confirmed by direct amino acid sequencing of cyanogen bromide peptides from rat liver ferritin. Minor fragments of rat liver ferritin, presumably derived from the H-subunit, were also isolated after cyanogen bromide treatment. On sequencing, these H-peptides showed limited homology with regions of the L-sequence but extensive homology with published H-sequences from human liver and spleen. The H-subunit sequence did not contain the octopeptide found as part of the L-subunit sequence.

The shell of the iron-storage protein ferritin consists of two types of subunit (heavy, Mr = 21,000; light, Mr = 19,000). To study the structure and expression of the ferritin subunit genes, recombinant plasmids containing ferritin cDNA have been isolated. A cDNA library was constructed in the vector pBR322 from rat liver polysomal mRNA and screened by using I125-labeled antibody to rat liver ferritin. Six positive clones were identified and were shown to contain cDNA inserts ranging in length from 800 to 950 base pairs. When these cDNA clones were used for hybrid selection of rat liver mRNA and the selected mRNAs were translated in vitro, the products from each clone migrated on denaturing gels in a position similar to that of the light subunit of ferritin. No evidence of translation of the heavy subunit was obtained, indicating that the two subunits are encoded by separate mRNAs. RNA blot analysis gave a length of 1,100 nucleotides for the light-subunit mRNA. One of the cDNA inserts was fractionated into four fragments by using the restriction enzyme Sau3A. When the fragments were hybridized with Southern blots of rat spleen DNA, each fragment yielded similar patterns of hybridization to that obtained with the intact cDNA. Therefore, all regions of the cDNA sequence contain homologous sequences to similar genomic restriction fragments. This is consistent with the existence of a family of genes that encode the light subunit of rat ferritin.