Professor Ken Haynes

Candida glabrata biofilms are recognized to have high resistance to antifungals. In order to understand the effect of mannans in the resistance profile of C. glabrata mature biofilms, C. glabrata Δmnn2 was evaluated. Biofilm cell walls were analysed by confocal laser scanning microscopy (CLSM) and their susceptibility was assessed for fluconazole, amphotericin B, caspofungin, and micafungin. Crystal violet and Alcian Blue methods were performed to quantify the biomass and the mannans concentration in the biofilm cells and matrices, respectively. The concentration of β-1,3 glucans was also measured. No visible differences were detected among cell walls of the strains, but the mutant had a high biomass reduction, after a drug stress. When compared with the reference strain, it was detected a decrease in the susceptibility of the biofilm cells and an increase of β-1,3 glucans in the C. glabrata Δmnn2. The deletion of the MNN2 gene in C. glabrata induces biofilm matrix and cell wall variabilities that increase the resistance to the antifungal drug treatments. The rise of β-1,3 glucans appears to have a role in this effect.

© 2015 Elsevier Ltd. Fungal pathogens pose serious threats to human, plant, and ecosystem health. Improved diagnostics and antifungal strategies are therefore urgently required. Here, we review recent developments in online bioinformatic tools and associated interactive data archives, which enable sophisticated comparative genomics and functional analysis of fungal pathogens in silico. Additionally, we highlight cutting-edge experimental techniques, including conditional expression systems, recyclable markers, RNA interference, genome editing, compound screens, infection models, and robotic automation, which are promising to revolutionize the study of both human and plant pathogenic fungi. These novel techniques will allow vital knowledge gaps to be addressed with regard to the evolution of virulence, host-pathogen interactions and antifungal drug therapies in both the clinic and agriculture. This, in turn, will enable delivery of improved diagnosis and durable disease-control strategies.

Gene overexpression is a widely used functional genomics approach in fungal biology. However, to date it has not been established in Zymoseptoria tritici which is an important pathogen of wheat (Triticum species). Here we report a suite of Gateway® recombination compatible ternary expression vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici. The suite of 32 vectors is based on a combination of four resistance markers for positive selection against glufosinate ammonium, geneticin, hygromycin and sulfonylurea; three constitutive Z. tritici promoters (pZtATUB, pZtGAPDH and pZtTEF) and a nitrogen responsive promoter (pZtNIA1) for controlled expression of the open reading frames. Half of the vectors facilitate expression of proteins tagged with C-terminal EGFP. All 32 vectors allow high frequency targeting of the overexpression cassette into the Ku70 locus and complement the Ku70 gene when transformed into a Z. tritici ku70 null strain, thus circumventing additional phenotypes that can arise from random integration. This suite of ternary expression vectors will be a useful tool for functional analysis through gene overexpression in Z. tritici.

In recent years, there has been a noticeable rise in fungal infections related to non-albicans Candida species, including Candida glabrata which has both intrinsic resistance to and commonly acquired resistance to azole antifungals. Phylogenetically, C. glabrata is more closely related to the mostly non-pathogenic model organism Saccharomyces cerevisiae than to other Candida species. Despite C. glabrata's designation as a pathogen by Wickham in 1957, relatively little is known about its mechanism of virulence. Over the past few years, technology to analyse the molecular basis of infection has developed rapidly, and here we briefly review the major advances in tools and technologies available to explore and investigate the virulence of C. glabrata that have occurred over the past decade.

Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a β-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets.

Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen.

The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.

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

The trend for large-scale genetic and phenotypic screens has revealed a wealth of information on biological systems. A major challenge is understanding how genes function and putative roles in networks. The majority of current gene knowledge is garnered from studies utilising the model yeast Saccharomyces cerevisiae. We demonstrate that synthetic dosage lethal genetic array methodologies can be used to study genetic networks in other yeasts, namely the fungal pathogen Candida glabrata, which has limited forward genetic tools, due to the lack of 'natural' mating. We performed two SDL screens in S. cerevisiae, overexpressing the transcriptional regulator UME6 as bait in the first screen and its C. glabrata ortholog CAGL0F05357g in the second. Analysis revealed that SDL maps share 204 common interactors, with 10 genetic interactions unique to C. glabrata indicating a level of genetic rewiring, indicative of linking genotype to phenotype in fungal pathogens. This was further validated by incorporating our results into the global genetic landscape map of the cell from Costanzo et al. to identify common and novel gene attributes. This data demonstrated the utility large data sets and more robust analysis made possible by interrogating exogenous genes in the context of the eukaryotic global genetic landscape.

Candida parapsilosis and Candida albicans are human fungal pathogens that belong to the CTG clade in the Saccharomycotina. In contrast to C. albicans, relatively little is known about the virulence properties of C. parapsilosis, a pathogen particularly associated with infections of premature neonates. We describe here the construction of C. parapsilosis strains carrying double allele deletions of 100 transcription factors, protein kinases and species-specific genes. Two independent deletions were constructed for each target gene. Growth in >40 conditions was tested, including carbon source, temperature, and the presence of antifungal drugs. The phenotypes were compared to C. albicans strains with deletions of orthologous transcription factors. We found that many phenotypes are shared between the two species, such as the role of Upc2 as a regulator of azole resistance, and of CAP1 in the oxidative stress response. Others are unique to one species. For example, Cph2 plays a role in the hypoxic response in C. parapsilosis but not in C. albicans. We found extensive divergence between the biofilm regulators of the two species. We identified seven transcription factors and one protein kinase that are required for biofilm development in C. parapsilosis. Only three (Efg1, Bcr1 and Ace2) have similar effects on C. albicans biofilms, whereas Cph2, Czf1, Gzf3 and Ume6 have major roles in C. parapsilosis only. Two transcription factors (Brg1 and Tec1) with well-characterized roles in biofilm formation in C. albicans do not have the same function in C. parapsilosis. We also compared the transcription profile of C. parapsilosis and C. albicans biofilms. Our analysis suggests the processes shared between the two species are predominantly metabolic, and that Cph2 and Bcr1 are major biofilm regulators in C. parapsilosis.

Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.

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.

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes.

Yeast cells are able to tolerate and adapt to a variety of environmental stresses. An essential aspect of stress adaptation is the regulation of monovalent ion concentrations. Ion regulation determines many fundamental physiological parameters, such as cell volume, membrane potential, and intracellular pH. It is achieved through the concerted activities of multiple cellular components, including ion transporters and signaling molecules, on both short and long time scales. Although each component has been studied in detail previously, it remains unclear how the physiological parameters are maintained and regulated by the concerted action of all components under a diverse range of stress conditions. In this study, we have constructed an integrated mathematical model of ion regulation in Saccharomyces cerevisiae to understand this coordinated adaptation process. Using this model, we first predict that the interaction between phosphorylated Hog1p and Tok1p at the plasma membrane inhibits Tok1p activity and consequently reduces Na(+) influx under NaCl stress. We further characterize the impacts of NaCl, sorbitol, KCl and alkaline pH stresses on the cellular physiology and the differences between the cellular responses to these stresses. We predict that the calcineurin pathway is essential for maintaining a non-toxic level of intracellular Na(+) in the long-term adaptation to NaCl stress, but that its activation is not required for maintaining a low level of Na(+) under other stresses investigated. We provide evidence that, in addition to extrusion of toxic ions, Ena1p plays an important role, in some cases alongside Nha1p, in re-establishing membrane potential after stress perturbation. To conclude, this model serves as a powerful tool for both understanding the complex system-level properties of the highly coordinated adaptation process and generating further hypotheses for experimental investigation.

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

Alkaline pH adaptation represents an important environmental stress response in Aspergillus nidulans. It is mediated by the pal signalling pathway and the PacC transcription factor. Although studied extensively experimentally, the activation mechanism of PacC has not been quantified, and it is not clear how this activation is regulated. Here, by constructing mathematical models, we first show that the pattern of PacC activation observed in previously published experiments cannot be explained based on existing knowledge about PacC activation. Extending the model with a negative feedback loop is necessary to produce simulation results that are consistent with the data, suggesting the existence of a negative feedback loop in the PacC activation process. This extended model is then validated against published measurements for cells with drug treatment and mutant cells. Furthermore, we investigate the role of an intermediate form of PacC in the PacC activation process, and propose experiments that can be used to test our predictions. Our work illustrates how mathematical models can be used to uncover regulatory mechanisms in the transcription regulation, and generate hypotheses that guide further laboratory investigations.

Understanding how populations and communities respond to competition is a central concern of ecology. A seminal theoretical solution first formalised by Levins (and re-derived in multiple fields) showed that, in theory, the form of a trade-off should determine the outcome of competition. While this has become a central postulate in ecology it has evaded experimental verification, not least because of substantial technical obstacles. We here solve the experimental problems by employing synthetic ecology. We engineer strains of Escherichia coli with fixed resource allocations enabling accurate measurement of trade-off shapes between bacterial survival and multiplication in multiple environments. A mathematical chemostat model predicts different, and experimentally verified, trajectories of gene frequency changes as a function of condition-specific trade-offs. The results support Levins' postulate and demonstrates that otherwise paradoxical alternative outcomes witnessed in subtly different conditions are predictable. © 2013 John Wiley & Sons Ltd/CNRS.

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.

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.

β-Glucan is a (1→3)-β-linked glucose polymer with (1→6)-β-linked side chains and a major component of fungal cell walls. β-Glucans provide structural integrity to the fungal cell wall. The nature of the (1-6)-β-linked side chain structure of fungal (1→3,1→6)-β-D-glucans has been very difficult to elucidate. Herein, we report the first detailed structural characterization of the (1→6)-β-linked side chains of Candida glabrata using high-field NMR. The (1→6)-β-linked side chains have an average length of 4 to 5 repeat units spaced every 21 repeat units along the (1→3)-linked polymer backbone. Computer modeling suggests that the side chains have a bent curve structure that allows for a flexible interconnection with parallel (1→3)-β-D-glucan polymers, and/or as a point of attachment for proteins. Based on these observations we propose new approaches to how (1→6)-β-linked side chains interconnect with neighboring glucan polymers in a manner that maximizes fungal cell wall strength, while also allowing for flexibility, or plasticity.

The osmotic stress response signalling pathway of the model yeast Saccharomyces cerevisae is crucial for the survival of cells under osmotic stress, and is preserved to varying degrees in other related fungal species. We apply a method for inference of ancestral states of characteristics over a phylogeny to 17 fungal species to infer the maximum likelihood estimate of presence or absence in ancestral genomes of genes involved in osmotic stress response. The same method allows us furthermore to perform a statistical test for correlated evolution between genes. Where such correlations exist within the osmotic stress response pathway of S. cerevisae, we have used this in order to predict and subsequently test for the presence of physical protein-protein interactions in an attempt to detect novel interactions. Finally we assess the relevance of observed evolutionary correlations in predicting protein interactions in light of the experimental results. We do find that correlated evolution provides some useful information for the prediction of protein-protein interactions, but that these alone are not sufficient to explain detectable patterns of correlated evolution.

Background: in biological systems, redox reactions are central to most cellular processes and the redox potential of the intracellular compartment dictates whether a particular reaction can or cannot occur. Indeed the widespread use of redox reactions in biological systems makes their detailed description outside the scope of one review. Scope of the Review: Here we will focus on how system-wide redox changes can alter the reaction and transcriptional landscape of Saccharomyces cerevisiae. To understand this we explore the major determinants of cellular redox potential, how these are sensed by the cell and the dynamic responses elicited. Major Conclusions: Redox regulation is a large and complex system that has the potential to rapidly and globally alter both the reaction and transcription landscapes. Although we have a basic understanding of many of the sub-systems and a partial understanding of the transcriptional control, we are far from understanding how these systems integrate to produce coherent responses. We argue that this non-linear system self-organises, and that the output in many cases is temperature-compensated oscillations that may temporally partition incompatible reactions in vivo. General Significance: Redox biochemistry impinges on most of cellular processes and has been shown to underpin ageing and many human diseases. Integrating the complexity of redox signalling and regulation is perhaps one of the most challenging areas of biology. This article is part of a Special Issue entitled Systems Biology of Microorganisms. © 2011 Elsevier B.V. All rights reserved.

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.

BACKGROUND: Invasive aspergillosis (IA) is the most common cause of death associated with fungal infection in the developed world. Historically, susceptibility to IA has been associated with prolonged neutropenia; however, IA has now become a major problem in patients on calcineurin inhibitors and allogenic hematopoietic stem cell transplant patients following engraftment. These observations suggest complex cellular mechanisms govern immunity to IA. METHODS: to characterize the key early events that govern outcome from infection with Aspergillus fumigatus, we performed a comparative immunochip microarray analysis of the pulmonary transcriptional response to IA between cyclophosphamide-treated mice and immunocompetent mice at 24 h after infection. RESULTS: We demonstrate that death due to infection is associated with a failure to generate an incremental interferon-gamma response, increased levels of interleukin-5 and interleukin-17a transcript, coordinated expression of a network of tumor necrosis factor-alpha-related genes, and increased levels of tumor necrosis factor-alpha. In contrast, clearance of infection is associated with increased expression of a number genes encoding proteins involved in innate pathogen clearance, as well as apoptosis and control of inflammation. CONCLUSION: This first organ-level immune response transcriptional analysis for IA has enabled us to gain new insights into the mechanisms that govern fungal immunity in the lung.

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.

Aspergillosis is a disease determined by various factors that influence fungal growth and fitness. A conserved signal transduction cascade linking environmental stress to amino acid homeostasis is the Cross-Pathway Control (CPC) system that acts via phosphorylation of the translation initiation factor eIF2 by a sensor kinase to elevate expression of a transcription factor. Ingestion of Aspergillus fumigatus conidia by macrophages does not trigger this stress response, suggesting that their phagosomal microenvironment is not deficient in amino acids. The cpcC gene encodes the CPC eIF2alpha kinase, and deletion mutants show increased sensitivity towards amino acid starvation. CpcC is specifically required for the CPC response but has limited influence on the amount of phosphorylated eIF2alpha. Strains deleted for the cpcC locus are not impaired in virulence in a murine model of pulmonary aspergillosis. Accordingly, basal expression of the Cross-Pathway Control transcriptional activator appears sufficient to support aspergillosis in this disease model.

The DNA binding protein, Ace2, plays a major role in the control of cell-cycle progression in Saccharomyces cerevisiae, being a principal regulator of the M/G1 transition and cell separation in particular. Ace2 also plays a significant role in effecting virulence, though in completely different ways, in the fungal pathogens Candida albicans and Candida glabrata. Here we will briefly review Ace2 regulation and function in terms of cell-cycle progression, cell separation and fungal virulence. © 2008 Elsevier B.V. All rights reserved.

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.

© 2007 Springer. All Rights Reserved. This chapter deals with the yeast Candida,one of the increasingly important human pathogens, and addresses multiple factors that result in either immunity to or infection with this micro-organism. Factors contributing to the pathogenicity, antigenicity and type of infections caused by the genus Candidaand its' species are discussed. An overview of immunity to Candidaincludes previous and recent data on innate and adaptive immune responses, focusing on recognition and sensing of Candidavia pattern recognition receptors (Toll-like receptors, Dectin-1) and their role in directing the ensuing cascade of cytokine production, that leads to protective or non-protective immunity mediated by cellular and humoral adaptive immune responses. The disease Chronic Mucocutaneous Candidiasis (CMC), which includes a subgroup of patients with the APECED syndrome (Autoimmune PolyEndocrinopathy Candidasis Ectodermal Dystrophy), where patients show a selective susceptibility to infections with Candida,is discussed in detail. The role of cell-mediated and humoral immunity in impaired protection against Candidais presented, as well as recent data demonstrating dysregulated cytokine production in response to Candida,which is suggested could be the underlying immune defect in these patients. The importance of CMC as a non-conventional primary immune deficiency, manifesting as narrow susceptibility to infection with weakly pathogenic microbes is stressed, given that these unique human models have the potential of hugely increasing our understanding of basic immune mechanisms involved in protection against Candidaand other fungi

Beta-glucan is one of the most abundant polysaccharides in fungal pathogens, yet its importance in antifungal immunity is unclear. Here we show that deficiency of dectin-1, the myeloid receptor for beta-glucan, rendered mice susceptible to infection with Candida albicans. Dectin-1-deficient leukocytes demonstrated significantly impaired responses to fungi even in the presence of opsonins. Impaired leukocyte responses were manifested in vivo by reduced inflammatory cell recruitment after fungal infection, resulting in substantially increased fungal burdens and enhanced fungal dissemination. Our results establish a fundamental function for beta-glucan recognition by dectin-1 in antifungal immunity and demonstrate a signaling non-Toll-like pattern-recognition receptor required for the induction of protective immune responses.

Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase a activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant Delta sidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection.

Transglucosidases play a significant role in fungal cell wall biosynthesis. We identified three as yet undescribed genes encoding beta-glucan transglucosidases, homologues of the pH-regulated PHR1 and PHR2, in the genome of the pathogenic yeast Candida albicans. Transcript levels of the gene PGA4 encoding a putative GPI-anchored protein were elevated in C. albicans wild-type cells during infection of reconstituted human epithelial and mouse liver tissue, and transiently increased after induction of hyphal formation with serum. The serum-specific increase in PGA4 transcript was found to be dependent on the transcription factors Ras1p, Cyr1p, and Tec1p. The remaining C. albicans Phr homologues, PHR3 and PGA5, showed low expression levels. Unlike PHR1 and PHR2, the expression of PHR3, PGA4, and PGA5 was not dependent on the pH of the growth medium. Neither PHR3 deletion nor PGA4 disruption resulted in a distinct growth or morphology phenotype. A PGA4 disruption strain was found to have wild-type capacity of infecting reconstituted oral epithelial tissue. Our data suggest that PGA4, and potentially PHR3 and PGA5, are expressed under distinct conditions, which differ from those of PHR1 and PHR2.

Mutants of Candida albicans, Candida glabrata, and Saccharomyces cerevisiae with disruptions in the ACE2 gene and C. glabrata and S. cerevisiae swi5 disruption mutants were tested for virulence in a murine challenge model of disseminated yeast infection. All mutants showed a clumping phenotype, but clumping was minimized in challenge inocula by inclusion of chitinase in the growth medium. In animals rendered temporarily neutropenic by cyclophosphamide treatment, the C. glabrata ace2 null mutant was confirmed as hypervirulent: it led to early terminal illness and kidney, brain, and lung fungal burdens substantially and significantly larger than those in controls. The C. glabrata swi5 null mutant did not lead to terminal illness but generated significantly larger brain and lung burdens than those in controls. The C. albicans ace2 null mutant was very slightly attenuated and the S. cerevisiae ace2 and swi5 null mutants were substantially attenuated relative to their parental control strains. The phenotype of aggressive hypervirulence, unique to disruption of the C. glabrata ACE2 gene among the strains tested, was not seen when the C. glabrata ace2 strain was tested in immunologically intact mice. The different effects seen with these mutants rule out the clumping phenotype as the explanation for hypervirulence in the C. glabrata ace2 mutant. The absence of C. glabrata ace2 hypervirulence in healthy mice may be a tool for definitive future study of host-parasite cross talk in microbial opportunism.

The conserved family of fungal Ste11 mitogen activated protein kinase/kinases play important roles in several signalling cascades. We have cloned the STE11 homologue from the fungal pathogen Candida glabrata. The C. glabrata gene is present in a single copy in the genome, contains a well-conserved catalytic domain typical of the serine-threonine protein kinases and a sterile alpha motif widespread in signalling and nuclear proteins. Hypothetical translation of C. glabrata STE11 suggests that the protein has 64% identity and 77% similarity at the amino acid level to Saccharomyces cerevisiae Ste11. We have shown that C. glabrata STE11 can complement the mating defect and partially rescue the reduced nitrogen starvation induced filamentation of S. cerevisiae ste11 mutants. Functional analysis of a C. glabrata ste11 null mutant demonstrates that Ste11 is required for adaptation to hypertonic stress but is largely dispensable for maintenance of cell wall integrity. It also plays a role in C. glabrata nitrogen starvation induced filamentation. Survival analysis revealed that C. glabrata ste11 mutants, while still able to cause disease, are attenuated for virulence compared to reconstituted, STE11 cells. These data suggest that C. glabrata Ste11, in a similar fashion to the S. cerevisiae protein, functions in a number of different signalling modules.

The ascomycete Candida albicans is the most common fungal pathogen in immunocompromised patients. Its ability to change morphology, from yeast to filamentous forms, in response to host environmental cues is important for virulence. Filamentation is mediated by second messengers such as cyclic adenosine 3',5'-monophosphate (cAMP) synthesized by adenylyl cyclase. The distantly related basidiomycete Cryptococcus neoformans is an encapsulated yeast that predominantly infects the central nervous system in immunocompromised patients. Similar to the morphological change in C. albicans, capsule biosynthesis in C. neoformans, a major virulence attribute, is also dependent upon adenylyl cyclase activity. Here we demonstrate that physiological concentrations of CO2/HCO3- induce filamentation in C. albicans by direct stimulation of cyclase activity. Furthermore, we show that CO2/HCO3- equilibration by carbonic anhydrase is essential for pathogenesis of C. albicans in niches where the available CO2 is limited. We also demonstrate that adenylyl cyclase from C. neoformans is sensitive to physiological concentrations of CO2/HCO3-. These data demonstrate that the link between cAMP signaling and CO2/HCO3- sensing is conserved in fungi and reveal CO2 sensing to be an important mediator of fungal pathogenesis. Novel therapeutic agents could target this pathway at several levels to control fungal infections.

In this study, we describe a simple method for the identification of Candida albicans in clinical samples. A total of 383 clinical isolates of Candida species were streaked onto chocolate agar and incubated for 48 h at 37 degrees C in the presence of an atmosphere of 6% CO2. All 208 of the C. albicans isolates tested, developed an easy to identify filamentous colony morphology. of 175 other Candida species tested, 172 (98.3%) were distinguishable from C. albicans by their smooth colony morphology. Three isolates (1.7%) exhibited weak filamentation after prolonged incubation. Although not a routine medium in medical mycology a significant advantage of using chocolate agar lies in its use in clinical bacteriology laboratories for the isolation of fastidious bacteria. Implementation of the proposed method is applicable across a range of specimen types, thus allowing the direct identification of C. albicans in clinical samples. This simple method may allow a quicker entry into directed treatment.

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 ability of a pathogen to adapt to the host environment is usually required for the initiation of disease. Here we have investigated the importance of the Aspergillus nidulans PacC-mediated pH response in the pathogenesis of pulmonary aspergillosis. Using mutational analysis, we demonstrate that, in neutropenic mice, elimination of the A. nidulans pH-responsive transcription factor PacC, blocking the ambient pH signal transduction pathway or prevention of PacC proteolytic processing acutely attenuates virulence. Infections caused by these alkali-sensitive mutants are characterized by limited growth in vivo and a reduction of inflammatory cell infiltration. In stark contrast, constitutive activation of PacC causes increased mortality marked by extensive fungal invasive growth. PacC action is therefore required for, and able to enhance virulence, demonstrating that the A. nidulans pH-responsive transcription factor PacC plays a pivotal role in pulmonary pathogenesis.

While investigating the requirement for phagosomal alkalinization in the host defense against pulmonary aspergillosis, we observed high morbidity of p47(phox)(-/-) mice infected with pH-insensitive Aspergillus nidulans mutants despite a paucity of fungal growth. Fatal infection also resulted from a normally avirulent p-aminobenzoate auxotroph. This demonstrates that p47(phox)(-/-) murine immunity contributes significantly to A. nidulans lethality. These data have wider implications for microbial virulence studies with p47(phox)(-/-) mice.

The conserved family of fungal Ste20 p21-activated serine-threonine protein kinases regulate several signalling cascades. In Saccharomyces cerevisiae Ste20 is involved in pheromone signalling, invasive growth, the hypertonic stress response, cell wall integrity and binds Cdc42, a Rho-like small GTP-binding protein required for polarized morphogenesis. We have cloned the STE20 homologue from the fungal pathogen Candida glabrata and have shown that it is present in a single copy in the genome. Translation of the nucleotide sequence predicts that C. glabrata Ste20 contains a highly conserved p21-activated serine-threonine protein kinase domain, a binding site for G-protein beta subunits and a regulatory Rho-binding domain that enables the kinase to interact with Cdc42 and/or Rho-like small GTPases. C. glabrata Ste20 has 53% identity and 58% predicted amino acid similarity to S. cerevisiae Ste20 and can complement both the nitrogen starvation-induced filamentation and mating defects of S. cerevisiae ste20 mutants. Analysis of ste20 null and disrupted strains suggest that in C. glabrata Ste20 is required for a fully functional hypertonic stress response and intact cell wall integrity pathway. C. glabrata Ste20 is not required for nitrogen starvation-induced filamentation. Survival analysis revealed that C. glabrata ste20 mutants, while still able to cause disease, are mildly attenuated for virulence compared to reconstituted STE20 cells.

During an infection, the coordinated orchestration of many factors by the invading organism is required for disease to be initiated and to progress. The elucidation of the processes involved is critical to the development of a clear understanding of host-pathogen interactions. For Candida species, the inactivation of many fungal attributes has been shown to result in attenuation. Here we demonstrate that the Candida glabrata homolog of the Saccharomyces cerevisiae transcription factor gene ACE2 encodes a function that mediates virulence in a novel way. Inactivation of C. glabrata ACE2 does not result in attenuation but, conversely, in a strain that is hypervirulent in a murine model of invasive candidiasis. C. glabrata ace2 null mutants cause systemic infections characterized by fungal escape from the vasculature, tissue penetration, proliferation in vivo, and considerable overstimulation of the proinflammatory arm of the innate immune response. Compared to the case with wild-type fungi, mortality occurs much earlier in mice infected with C. glabrata ace2 cells, and furthermore, 200-fold lower doses are required to induce uniformly fatal infections. These data demonstrate that C. glabrata ACE2 encodes a function that plays a critical role in mediating the host-Candida interaction. It is the first virulence-moderating gene to be described for a Candida species.

The ability to acquire iron in vivo is essential for most microbial pathogens. Here we show that Aspergillus fumigatus does not have specific mechanisms for the utilization of host iron sources. However, it does have functional siderophore-assisted iron mobilization and reductive iron assimilation systems, both of which are induced upon iron deprivation. Abrogation of reductive iron assimilation, by inactivation of the high affinity iron permease (FtrA), has no effect on virulence in a murine model of invasive aspergillosis. In striking contrast, A. fumigatus L-ornithine-N5-monooxygenase (SidA), which catalyses the first committed step of hydroxamate-type siderophore biosynthesis, is absolutely essential for virulence. Thus, A. fumigatus SidA is an essential virulence attribute. Combined with the absence of a sidA ortholog-and the fungal siderophore system in general-in mammals, these data demonstrate that the siderophore biosynthetic pathway represents a promising new target for the development of antifungal therapies.

We have cloned and characterized the Aspergillus fumigatus cpcA gene encoding the transcriptional activator of the cross-pathway control system of amino acid biosynthesis. cpcA encodes a functional orthologue of Saccharomyces cerevisiae Gcn4p. The coding sequence of the 2.2 kb transcript is preceded by two short upstream open reading frames, the larger one being well conserved among Aspergilli. Deletion strains in which either the coding sequence or the entire locus are replaced by a bifunctional dominant marker are impaired in their cross-pathway control response upon amino acid starvation, as demonstrated by analyses of selected reporter genes and specific enzymatic activities. In a murine model of pulmonary aspergillosis, cpcAdelta strains display attenuated virulence. Pathogenicity is restored to wild-type levels in strains with reconstitution of the genomic locus. Competitive mixed infection experiments additionally demonstrate that cpcAdelta strains are less able to survive in vivo than their wild-type progenitor. Our data suggest that specific stress conditions are encountered by A. fumigatus within the mammalian host and that the fungal cross-pathway control system plays a significant role in pulmonary aspergillosis.

The highly conserved fungal Ste12 transcription factor family of proteins play critical roles in the regulation of many cellular processes including mating, cell wall biosynthesis, filamentation and invasive growth. They are also important mediators of fungal virulence. The Candida glabrata STE12 homologue was cloned. The encoded protein has a single DNA binding homeodomain but lacks both a C2H2 zinc finger DNA binding domain and an apparent Dig1/Dig2 regulatory motif. Candida glabrata STE12 can functionally complement the nitrogen starvation induced filamentation and mating defects of Saccharomyces cerevisiae ste12 mutants. We also show that C. glabrata STE12 is required for nitrogen starvation-induced filamentation as ste12 mutants rarely produce pseudohyphae on nitrogen depleted media. Finally we describe a novel murine model of C. glabrata systemic disease and use this to demonstrate that C. glabrata ste12 mutants, although still able to cause disease, are attenuated for virulence compared with STE12 reconstituted strains. Candida glabrata STE12 is therefore the first virulence factor encoding gene to be described in this increasingly important fungal pathogen.

In fungi, the cell wall plays a major role in host-pathogen interactions. Despite this, little is known about the molecular basis of cell wall assembly in Candida glabrata, which has emerged as the second most common cause of systemic candidosis. A C. glabrata gene family, CgGAS1-3, that shares significant homologies with both the GAS1 gene of Saccharomyces cerevisiae, which is necessary for cell wall assembly, and the pH-regulated genes PHR1 and PHR2 of Candida albicans, which are involved in cell wall assembly and required for virulence, has been cloned. Among the members of this family, CgGAS1-3 display a unique expression pattern. Both CgGAS1 and CgGAS2 are constitutively expressed. In contrast, CgGAS3 transcript was not detectable under any of the assayed conditions. The C. glabrata actin gene, CgACT1, has also been cloned to be used as a meaningful loading control in Northern blots. CgGAS1 and CgGAS2 were deleted by two different methodological approaches. A rapid PCR-based strategy by which gene disruption was achieved with short regions of homology (50 bp) was applied successfully to C. glabrata. DeltaCggas1 or DeltaCggas2 cells demonstrated similar aberrant morphologies, displaying an altered bud morphology and forming floccose aggregates. These phenotypes suggest a role for CgGAS1 and CgGAS2 in cell wall biosynthesis. Further evidence for this hypothesis was obtained by successful functional complementation of a gas1 null mutation in S. cerevisiae with the C. glabrata CgGAS1 or CgGAS2 gene.

Candida species other than Candida albicans now account for up to 50% of deep candidiasis cases, yet little attention has been paid to the virulence attributes of these fungi. Adherence to host tissues, response to environmental changes and the secretion of hydrolases are all thought to be important in Candida virulence. The identification of virulence attributes unique to a particular Candida species could provide powerful insights into the pathogenic process but will require the use of genome-wide approaches such as transcript profiling, signature-tagged mutagenesis and in vivo expression technology.

The ability to undergo morphological change has been reported as an advantageous trait in fungal pathogenesis. Here we demonstrate that Candida glabrata ATCC2001, like diploid Saccharomyces cerevisiae strains, forms elongated chains of pseudohyphal cells on solid nitrogen starvation media (SLAD). Constrictions were apparent between adjoining cells; no parallel-sided hyphae were seen and pseudohyphae invaded the agar. When SLAD was supplemented with ammonium sulfate both C. glabrata and diploid S. cerevisiae strains lost their ability to undergo pseudohyphal growth. However, on this media C. glabrata yeast cells invaded the agar in a similar fashion to the invasive growth mode exhibited by haploid strains of S. cerevisiae cultured on rich media such as YPD. C. glabrata was not capable of invading YPD demonstrating that the process of filamentation is distinct in these two fungi. To our knowledge this is the first report to demonstrate that C. glabrata can undergo morphological change and grow as an invasive filamentous organism.

The functionality of beta-galactosidase encoded by the E. coli lacZ gene as a reporter of gene expression in C. glabrata was investigated. C. glabrata/E. coli shuttle vectors were constructed, containing both a C. glabrata CEN-ARS cassette, to allow regular segregation and episomal replication of the plasmids, and the lacZ coding sequence of E. coli. The functionality of beta-galactosidase in C. glabrata was verified by inserting the promoter and the 5' coding region of the HIS3 gene from C. glabrata directionally upstream of the lacZ gene. By fusing the promoter of the copper-controlled MTII gene to the lacZ reporter, we showed that beta-galactosidase activity can be differentially induced in C. glabrata. beta-galactosidase reporter activities were detected qualitatively by an indirect filter assay and quantitatively from permeabilized cells.

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

Candida species other than C. albicans have become a significant cause of infection in humans. Several of the more commonly isolated of these species are less susceptible to commonly used azole antifungal drugs, a factor that poses significant difficulties for effective treatment. The modern mycology laboratory has an important role to play in several aspects relating to these organisms, including therapy, detection, identification and epidemiological analysis. The application of molecular techniques and phylogenetic analysis has led to the identification of a new species of Candida associated with mucosal candidiasis in HIV-infected individuals named Candida dubliniensis, the clinical significance of which is currently under investigation. Molecular techniques are also being applied to the analysis of determinants involved in pathogenicity of species such as Candida glabratta. These approaches should lead to a better understanding of these organisms and there ability to cause disease and should also provide more effective treatment.

Candida species other than C. albicans have become a significant cause of infection in humans. Several of the more commonly isolated of these species are less susceptible to commonly used azole antifungal drugs, a factor that poses significant difficulties for effective treatment. The modern mycology laboratory has an important role to play in several aspects relating to these organisms, including therapy, detection, identification and epidemiological analysis. The application of molecular techniques and phylogenetic analysis has led to the identification of a new species of Candida associated with mucosal candidiasis in HIV-infected individuals named Candida dubliniensis, the clinical significance of which is currently under investigation. Molecular techniques are also being applied to the analysis of determinants involved in pathogenicity of species such as Candida glabratta. These approaches should lead to a better understanding of these organisms and there ability to cause disease and should also provide more effective treatment.

The discovery and characterisation of a novel species of Candida, termed Candida dubliniensis, associated with oral candidosis in HIV-infected individuals is described. These organisms share several phenotypic characteristics in common with Candida albicans and Candida stellatoidea, including the ability to produce germ tubes and chlamydospores. However, in contrast to these latter two species, C. dubliniensis isolates produce abundant chlamydospores, which are often arranged in contiguous pairs, triplets and other multiples suspended from a single suspensor cell. They belong to C. albicans serotype a and exhibit atypical substrate assimilation profiles. Genomic DNA fingerprinting analysis with the C. albicans-specific probe 27A and five different oligonucleotide probes consisting of short repeat sequence-containing motifs, demonstrated that C. dubliniensis has a distinct genomic organisation relative to C. albicans and C. stellatoidea. This was confirmed by karyotype analysis and random amplified polymorphic DNA (RAPD) analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from 14 separate C. dubliniensis isolates and the corresponding sequences from C. albicans, C. stellatoidea, C. tropicalis, C. glabrata, C. parapsilosis, C. kefyr and C. krusei demonstrated that the C. dubliniensis isolates formed a homogenous cluster (100% similarity), representing a discrete taxon within the genus Candida that was significantly different from the other species analysed.

Candida dubliniensis is a recently identified chlamydospore-positive yeast species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected (HIV+) patients and is closely related to Candida albicans. Several recent reports have described atypical oral Candida isolates with phenotypic and genetic properties similar to those of C. dubliniensis. In this study 10 atypical chlamydospore-positive oral isolates from HIV+ patients in Switzerland, the United Kingdom, and Argentina and 1 isolate from an HIV-negative Irish subject were compared to reference strains of C. albicans and Candida stellatoidea and reference strains of C. dubliniensis recovered from Irish and Australian HIV+ individuals. All 11 isolates were phenotypically and genetically similar to and phylogenetically identical to C. dubliniensis. These findings demonstrate that the geographical distribution of C. dubliniensis is widespread, and it is likely that it is a significant constituent of the normal oral flora with the potential to cause oral candidiasis, particularly in immunocompromised patients.

Invasive aspergillosis is a significant and increasingly common life threatening fungal infection in susceptible patient groups. This review briefly describes the risk factors, symptoms and therapy of the disease. The problem of serological diagnosis is outlined and both previously described and novel approaches, aimed at improving current practice and its impact on the treatment of this difficult infection, are discussed. The potential of molecular techniques, in particular the polymerase chain reaction, for diagnosis is highlighted.

Six separate human immunodeficiency virus-positive patients with cryptococcal meningitis were each found to have been infected with a unique strain of Cryptococcus neoformans on the basis of genomic DNA finger-printing analysis with the microsatellite sequence-containing oligonucleotide probe (GGAT)4 and by random amplification of polymorphic DNA. Two patients (A and B) experienced a recurrent episode of infection. Between 12 and 16 single-colony isolates recovered from primary isolation media (> 50% of C. neoformans colonies recovered) from cerebrospinal fluid specimens were fingerprinted from both patients during each episode. The fingerprints of both isolate collections from patient B were very similar, although minor polymorphisms were evident in both sets of profiles. The fingerprints of the isolate collection from the initial episode of infection in patient a were also identical to each other, apart from minor polymorphisms, but they were clearly different from the corresponding profiles of the isolate collection from the recurrent episode, the latter of which were completely identical, apart from minor polymorphisms in a single isolate. Furthermore, prolonged storage and in vitro subculture of the isolates did not alter the fingerprint profiles. These results provided convincing evidence that patients a and B were each infected with a single C. neoformans strain during each episode of infection and that in patient B, the same strain persisted and caused both episodes, while in patient A, a different strain was responsible for each episode. The prevalence of polymorphisms in multiple single-colony isolates from both patients also suggested that C. neoformans populations may undergo microevolution.

A glycoprotein with an apparent molecular weight of 93 kDa was purified from a water-soluble extract of Aspergillus fumigatus NCPF 2109 by single step affinity chromatography using the mannose-specific snowdrop (Galanthus nivalis) lectin coupled to agarose. The carbohydrate moiety contained only mannose and galactose. Partial sequencing of cyanogen bromide fragments of the antigen yielded two sequences, KQNKP and GEIPMKF?PQL, with no homology to any reported proteins. In a preliminary evaluation of its diagnostic potential the 93 kDa antigen was recognized by the sera of four patients with allergic bronchopulmonary aspergillosis, in addition to a monoclonal antibody raised against a partially purified fraction of the A. fumigatus water-soluble extract.

A glycoprotein with an apparent molecular weight of 93 kDa was purified from a water-soluble extract of Aspergillus fumigatus NCPF 2109 by single step affinity chromatography using the mannose-specific snowdrop (Galanthus nivalis) lectin coupled to agarose. The carbohydrate moiety contained only mannose and galactose. Partial sequencing of cyanogen bromide fragments of the antigen yielded two sequences, KQNKP and GEIPMKF?PQL, with no homology to any reported proteins. In a preliminary evaluation of its diagnostic potential the 93 kDa antigen was recognized by the sera of four patients with allergic bronchopulmonary aspergillosis, in addition to a monoclonal antibody raised against a partially purified fraction of the A. fumigatus water-soluble extract.

A rapid PCR-based method for the identification of four Candida species is described. Primers to conserved sequences in the V3 region of large subunit rDNA were used to amplify DNA from C. albicans, C. glabrata, C. parapsilosis and C. krusei. The sequences were aligned and areas of non-concordance were used to design four species-specific forward primers. In a blind study of 82 yeast strains, four PCRs based on these primers correctly identified nine C. albicans, 18 C. glabrata, 13 C. parapsilosis and 18 C. krusei strains after gel electrophoresis of amplified DNA. Furthermore, of 17 other Candida strains (10 species) and seven strains from other yeast genera (Saccharomyces, Cryptococcus and Trichosporon) only one false positive amplification product (with C. dubliniensis) was seen. These PCRs offer a rapid alternative to conventional techniques for the identification of C. albicans, C. glabrata, C. parapsilosis and C. krusei.

Atypical oral Candida isolates were recovered from 60 HIV-infected and three HIV-negative individuals. These organisms were germ-tube-positive and produced abundant chlamydospores which were frequently arranged in triplets or in contiguous pairs. They belonged to C. albicans serotype a and had atypical carbohydrate assimilation profiles. Fingerprinting the genomic DNA of a selection of these organisms with the C. albicans-specific probe 27A and five separate oligonucleotides, homologous to eukaryotic microsatellite repeat sequences, demonstrated that they had a very distinct genomic organization compared to C. albicans and C. stellatoidea. This was further established by random amplified polymorphic DNA (RAPD) and karyotype analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from nine atypical isolates and the corresponding sequences determined from C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei showed that they atypical organisms formed a homogeneous cluster (100% similarity) that was significantly different from the other Candida species analysed, but was most closely related to C. albicans and C. stellatoidea. These genetic data combined with the phenotypic characteristics of these atypical organisms strongly suggest that they constitute a novel species within the genus Candida for which the name Candida dubliniensis is proposed.

We compared the abilities of random amplification of polymorphic DNA and DNA fingerprinting, with oligonucleotide probes, to type five pairs of Cryptococcus neoformans clinical isolates recovered from five separate human immunodeficiency virus-positive patients in London, England. The two techniques had comparable discriminatory abilities when applied to these isolates. A total of eight different isolate types were demonstrated in these patients. No isolate type was observed in more than one patient. Two of the isolate pairs recovered from single episodes of cryptococcosis within 1 day of each other were genotypically indistinguishable by both methods. The other three pairs of isolates were all distinguishable. One of these isolate pairs was obtained from a single episode of cryptococcosis, while the other two were obtained from recurrent infections. These results indicate that multiple strains of C. neoformans may be responsible for a single episode of cryptococcosis and that recurrent infection may occur as a result of reinfection with a novel strain.

We describe a polymerase chain reaction (PCR) based approach to the detection and identification of pathogenic fungi which has potential for the diagnosis of systemic mycoses. Primers to sequences of the large subunit ribosomal DNA genes, which are universally conserved within the fungal kingdom, were capable of amplifying DNA from 43 strains representing 20 species (12 genera) of medically important fungi. Sequence analysis of the products obtained from Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans allowed us to design species-specific primers which only amplified homologous DNA. The use of these two PCRs in tandem allows the detection (universal PCR) and identification (species-specific PCR) of a fungal pathogen within 8 h from simulated clinical specimens. © 1995 Informa UK Ltd all rights reserved: reproduction in whole or part not permitted.

We describe a polymerase chain reaction (PCR) based approach to the detection and identification of pathogenic fungi which has potential for the diagnosis of systemic mycoses. Primers to sequences of the large subunit ribosomal DNA genes, which are universally conserved within the fungal kingdom, were capable of amplifying DNA from 43 strains representing 20 species (12 genera) of medically important fungi. Sequence analysis of the products obtained from Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans allowed us to design species-specific primers which only amplified homologous DNA. The use of these two PCRs in tandem allows the detection (universal PCR) and identification (species-specific PCR) of a fungal pathogen within 8 h from simulated clinical specimens.

A commercial latex agglutination (LA) test for the detection of circulating Aspergillus galactomannan was evaluated in sera obtained from 121 immunocompromised patients, including 19 with proven invasive pulmonary aspergillosis. Patient urine (the specimen of choice for detection of galactomannan) was not tested with the LA test as 34 of 81 specimens from controls gave false-positive results. The sensitivity (95%), specificity (90%) and negative predictive value (99%) of the LA test were similar to previously published results obtained with two EIAs. However, the positive predictive value was only 67% compared to > or = 95% obtained with the EIAs. In addition, the LA test was also of less value than the EIAs in predicting the onset of invasive pulmonary aspergillosis. It was the earliest indicator of infection in only 1 of 19 proven cases.

Rhizopus oryzae was the causative organism in a fatal case of rhinocerebral and then pulmonary mucormycosis in a patient cured of her underlying leukaemia by bone marrow transplantation. We discuss the risk factors involved and the need for maintaining a high index of suspicion of fungal infection in the late post-transplant period.

One of the major antigens secreted in vitro by Aspergillus fumigatus is an 18-kDa basic protein which has been purified by cation-exchange chromatography. It is recognized by sera from aspergilloma patients. It is also the major circulating antigen found in urine of patients with invasive aspergillosis. Our results indicated that this antigen has potential for the diagnosis of both aspergilloma and invasive aspergillosis.

Serial urine samples were collected from 33 neutropenic patients, 10 of whom developed invasive aspergillosis (IA) while undergoing bone marrow transplantation or remission induction therapy for leukemia. Concentrated urine samples from the infected patients were subjected to polyacrylamide gel electrophoresis, blotted, and then incubated with antiserum raised to a cell wall extract of Aspergillus fumigatus (anti-CW) or an immunoglobulin G monoclonal antibody to A. fumigatus galactomannan (EBA1). When IA patient urine blots were probed with anti-CW, major bands at 11 and 18 kilodaltons (kDa); intermediate bands at 13, 14, and 29 kDa; and minor bands at 38 and 44 kDa were seen. In contrast, EBA1 showed diffuse staining at molecular masses larger than 45 kDa and a single weak band at 21 kDa. Urine samples from the 23 patients with no evidence of IA were unreactive with both anti-CW and EBA1. These antigen bands are likely to represent immunodominant antigens which are excreted during IA and should play a valuable role in the development of rapid diagnostic tests for aspergillosis.

Two ELISAs were used to detect serum and urinary aspergillus antigen in 121 patients who were profoundly neutropenic after leukaemia therapy or bone marrow transplantation. The presence of antigen correctly predicted development of invasive pulmonary aspergillosis (IPA) in 16 patients. In 2 other cases antigen appeared after the clinical diagnosis had been made, while in only 1 case was antigen not detected. In 11 of 13 episodes of clinically suspected fungal infection antigen was detected before clinical diagnosis was made. By contrast, antigen was detected in only 1 of 90 patients who had no evidence of IPA. Both ELISAs gave positive and negative predictive values for IPA of greater than 95%, demonstrating the value of antigen detection in early diagnosis of aspergillus infection and the assay's ability to predict subsequent development of IPA. We conclude that neutropenic patients should be screened for aspergillus antigen, and propose that initial detection of fungal antigen justifies commencement of empirical antifungal therapy. Such an approach should improve the survival of patients who are at risk of developing this usually fatal infection.