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The fungal pathogen Candida albicans can form biofilms that protect it from drugs and the immune system. The biofilm cells release extracellular vesicles (EVs) that promote extracellular matrix formation and resistance to antifungal drugs. Here, we define functions for numerous EV cargo proteins in biofilm matrix assembly and drug resistance, as well as in fungal cell adhesion and dissemination. We use a machine-learning analysis of cargo proteomic data from mutants with EV production defects to identify 63 candidate gene products for which we construct mutant and complemented strains for study. Among these, 17 mutants display reduced biofilm matrix accumulation and antifungal drug resistance. An additional subset of 8 cargo mutants exhibit defects in adhesion and/or dispersion. Representative cargo proteins are shown to function as EV cargo through the ability of exogenous wild-type EVs to complement mutant phenotypic defects. Most functionally assigned cargo proteins have roles in two or more of the biofilm phases. Our results support that EVs provide community coordination throughout biofilm development in C. albicans., The fungal pathogen Candida albicans can release extracellular vesicles that promote biofilm formation and antifungal resistance. Here, Zarnowski et al. define functions for numerous vesicle cargo proteins in biofilm matrix assembly and drug resistance, as well as in fungal cell adhesion and dissemination.

Candida albicans is among the most significant human fungal pathogens. However, the vast majority of C. albicans studies have focused on a single clinical isolate and its marked derivatives. We investigated natural variation among clinical C. albicans isolates in gene regulatory control of biofilm formation, a process crucial to virulence. The transcription factor Efg1 is required for biofilm-associated gene expression and biofilm formation. Previously, we found extensive variation in Efg1-responsive gene expression among 5 diverse clinical isolates. However, chromatin immunoprecipitation sequencing analysis showed that Efg1 binding to genomic loci was uniform among the isolates. Functional dissection of strain differences identified three transcription factors, Brg1, Tec1, and Wor1, for which small changes in expression levels reshaped the Efg1 regulatory network. Brg1 and Tec1 are known biofilm activators, and their role in Efg1 network variation may be expected. However, Wor1 is a known repressor of

Extracellular vesicles mediate community interactions among cells ranging from unicellular microbes to complex vertebrates. Extracellular vesicles of the fungal pathogen Candida albicans are vital for biofilm communities to produce matrix, which confers environmental protection and modulates community dispersion. Infections are increasingly due to diverse Candida species, such as the emerging pathogen Candida auris , as well as mixed Candida communities. Here, we define the composition and function of biofilm-associated vesicles among five species across the Candida genus. We find similarities in vesicle size and release over the biofilm lifespan. Whereas overall cargo proteomes differ dramatically among species, a group of 36 common proteins is enriched for orthologs of C. albicans biofilm mediators. To understand the function of this set of proteins, we asked whether mutants in select components were important for key biofilm processes, including drug tolerance and dispersion. We found that the majority of these cargo components impact one or both biofilm processes across all five species. Exogenous delivery of wild-type vesicle cargo returned mutant phenotypes toward wild type. To assess the impact of vesicle cargo on interspecies interactions, we performed cross-species vesicle addition and observed functional complementation for both biofilm phenotypes. We explored the biologic relevance of this cross-species biofilm interaction in mixed species and mutant studies examining the drug-resistance phenotype. We found a majority of biofilm interactions among species restored the community’s wild-type behavior. Our studies indicate that vesicles influence the development of protective monomicrobial and mixed microbial biofilm communities.

Engineered conditional gene expression is used in appraisal of gene function and pathway relationships. For pathogens like the fungus Candida albicans, conditional expression systems are most useful if they are active in the infection environment and if they can be utilized in multiple clinical isolates. Here, we describe such a system. It employs the

Candida albicans is an important cause of human fungal infections. A widely studied virulence trait of C. albicans is its ability to undergo filamentation to hyphae and pseudohyphae. Although yeast, pseudohyphae, and hyphae are present in pathological samples of infected mammalian tissue, it has been challenging to characterize the role of regulatory networks and specific genes during in vivo filamentation. In addition, the phenotypic heterogeneity of C. albicans clinical isolates is becoming increasingly recognized, while correlating this heterogeneity with pathogenesis remains an important goal. Here, we describe the use of an intravital imaging approach to characterize C. albicans filamentation in a mammalian model of infection by taking advantage of the translucence of mouse pinna (ears). Using this model, we have found that the in vitro and in vivo filamentation phenotypes of different C. albicans isolates can vary significantly, particularly when in vivo filamentation is compared to solid agar-based assays. We also show that the well-characterized transcriptional regulators Efg1 and Brg1 appear to play important roles both in vivo and in vitro. In contrast, Ume6 is much more important in vitro than in vivo. Finally, strains that are dependent on Bcr1 for in vitro filamentation are able to form filaments in vivo in its absence. This intravital imaging approach provides a new approach to the systematic characterization of this important virulence trait during mammalian infection. Our initial studies provide support for the notion that the regulation and initiation of C. albicans filamentation in vivo is distinct from in vitro induction. IMPORTANCE Candida albicans is one of the most common causes of fungal infections in humans. C. albicans undergoes a transition from a round yeast form to a filamentous form during infection, which is critical for its ability to cause disease. Although this transition has been studied in the laboratory for years, methods to do so in an animal model of infection have been limited. We have developed a microscopy method to visualize fluorescently labeled C. albicans undergoing this transition in the subcutaneous tissue of mice. Our studies indicate that the regulation of C. albicans filamentation during infection is distinct from that observed in laboratory conditions.

The fungal pathogen Candida albicans is surrounded by a cell wall that is the target of caspofungin and other echinocandin antifungals. Candida albicans can grow in several morphological forms, notably budding yeast and hyphae. Yeast and hyphal forms differ in cell wall composition, leading us to hypothesize that there may be distinct genes required for yeast and hyphal responses to caspofungin. Mutants in 27 genes reported previously to be caspofungin hypersensitive under yeast growth conditions were all caspofungin hypersensitive under hyphal growth conditions as well. However, a screen of mutants defective in transcription factor genes revealed that Cup9 is required for normal caspofungin tolerance under hyphal and not yeast growth conditions. In a hyphal-defective efg1Δ/Δ background, Cup9 is still required for normal caspofungin tolerance. This result argues that Cup9 function is related to growth conditions rather than cell morphology. RNA-seq conducted under hyphal growth conditions indicated that 361 genes were up-regulated and 145 genes were down-regulated in response to caspofungin treatment. Both classes of caspofungin-responsive genes were enriched for cell wall-related proteins, as expected for a response to disruption of cell wall integrity and biosynthesis. The cup9Δ/Δ mutant, treated with caspofungin, had reduced RNA levels of 40 caspofungin up-regulated genes, and had increased RNA levels of 8 caspofungin down-regulated genes, an indication that Cup9 has a narrow rather than global role in the cell wall integrity response. Five Cup9-activated surface-protein genes have roles in cell wall integrity, based on mutant analysis published previously (PGA31 and IFF11) or shown here (ORF19.3499, ORF19.851, or PGA28), and therefore may explain the hypersensitivity of the cup9Δ/Δmutant to caspofungin. Our findings define Cup9 as a new determinant of caspofungin susceptibility.

Here we present a protocol of interest to those who want to look at the functional consequences of decreasing expression of essential genes constitutively as well as being able to study the regulatory pathways of that essential gene by also deleting one or two additional genes. This allows epistasis relationships to be determined.

Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality., Author summary All organisms tailor genetic programs to the available nutrients, such as sources of carbon. Here we define two key regulators of the genetic programs for carbon source utilization in the fungal pathogen Candida albicans. The two regulators have many shared roles, yet are partially specialized to control carbon acquisition and metabolism, respectively. In addition, the regulators together control traits associated with pathogenicity, an indication that carbon regulation is integrated into the pathogenicity program. Finally, the regulators help to explain a long-standing riddle—that the central carbon regulator Snf1 is essential for C. albicans viability.

The fungus Candida albicans can grow as either yeast or filaments, which include hyphae and pseudohyphae, depending on environmental conditions. Filamentous growth is of particular interest because it is required for biofilm formation and for pathogenesis. Environmentally induced filamentous growth is associated with expression of filamentation-associated genes, and both filamentous growth and associated gene expression depend upon several well characterized transcription factors. Surprisingly, strains with reduced expression of many essential genes display filamentous growth under non-inducing conditions – those in which the wild type grows as yeast. We found recently that diminished expression of several essential protein kinase genes leads to both filamentous cell morphology and filamentation-associated gene expression under non-inducing conditions. Reduced expression of the essential protein kinase gene CAK1 promoted filamentation-associated gene expression and biofilm formation in strains that lacked key transcriptional activators of these processes, thus indicating that CAK1 expression is critical for both environmental and genetic control of filamentation. In this study we extend our genetic interaction analysis to a second essential protein kinase gene, KIN28. Reduced expression of KIN28 also permits filamentation-associated gene expression, though not biofilm formation, in the absence of several key transcriptional activators. Our results argue that impairment of several essential cellular processes can alter the regulatory requirements for filamentation-associated gene expression. Our results also indicate that levels of filamentation-associated gene expression are not fully predictive of biofilm formation ability.

Cells from all kingdoms of life produce extracellular vesicles (EVs). Their cargo is protected from the environment by the surrounding lipid bilayer. EVs from many organisms have been shown to function in cell–cell communication, relaying signals that impact metazoan development, microbial quorum sensing, and pathogenic host–microbe interactions. Here, we have investigated the production and functional activities of EVs in a surface-associated microbial community or biofilm of the fungal pathogen Candida albicans. Crowded communities like biofilms are a context in which EVs are likely to function. Biofilms are noteworthy because they are encased in an extracellular polymeric matrix and because biofilm cells exhibit extreme tolerance to antimicrobial compounds. We found that biofilm EVs are distinct from those produced by free-living planktonic cells and display strong parallels in composition to biofilm matrix material. The functions of biofilm EVs were delineated with a panel of mutants defective in orthologs of endosomal sorting complexes required for transport (ESCRT) subunits, which are required for normal EV production in diverse eukaryotes. Most ESCRT-defective mutations caused reduced biofilm EV production, reduced matrix polysaccharide levels, and greatly increased sensitivity to the antifungal drug fluconazole. Matrix accumulation and drug hypersensitivity of ESCRT mutants were reversed by addition of wild-type (WT) biofilm EVs. Vesicle complementation showed that biofilm EV function derives from specific cargo proteins. Our studies indicate that C. albicans biofilm EVs have a pivotal role in matrix production and biofilm drug resistance. Biofilm matrix synthesis is a community enterprise; prior studies of mixed cell biofilms have demonstrated extracellular complementation. Therefore, EVs function not only in cell–cell communication but also in the sharing of microbial community resources., Author summary Candida albicans—the most common fungal pathogen in humans—often grows as a biofilm, resulting in an infection that is difficult to treat. These adherent communities tolerate extraordinarily high concentrations of antifungals due in large part to the protective extracellular matrix. The present study identifies extracellular vesicles (EVs) that are distinct to biofilms. These EVs deliver the functional extracellular matrix and are essential for resistance to antifungals. Our findings not only reveal a coordinated mechanism by which the defining trait of the biofilm lifestyle arises but also identify a number of potential therapeutic targets.

Our understanding of new genes is often built upon the knowledge of well-characterized genes. One avenue toward revealing such connections involves creation of strains with mutations in two or more defined genes to permit genetic interaction analysis. Strain manipulations can yield unexpected mutations at loci outside the defined targeted genes. In this report, we describe a method for rapid validation of multigene mutants, thus allowing an appraisal of the contribution of the defined targeted genes to the strain’s phenotype., The biological function of a gene is often probed through its interactions with other genes. This general approach has been especially useful to build knowledge about poorly understood genes upon the bedrock of well-characterized genes. Genetic interaction analysis requires the construction of strains with mutations in two or more genes. Single-gene mutants of microbial pathogens are generally validated through introduction of a wild-type copy of the affected gene to create a complemented or reconstituted strain, followed by testing for restoration of a wild-type phenotype. This practice, formalized as one of Falkow’s “molecular Koch’s postulates” ensures that the phenotype of the mutant depends upon the known mutation. However, multigene mutants are seldom validated because of the labor required to assemble multiple genomic segments into a vector that can be introduced into the mutant strain. We present here an approach, concatemer assembly for rescue of mutant abilities (CARMA), that circumvents this impediment through an in vivo recombinational assembly strategy that does not require cloning at all. Our results show that CARMA allows genetic rescue of two double-gene mutant strains of the fungal pathogen Candida albicans. IMPORTANCE Our understanding of new genes is often built upon the knowledge of well-characterized genes. One avenue toward revealing such connections involves creation of strains with mutations in two or more defined genes to permit genetic interaction analysis. Strain manipulations can yield unexpected mutations at loci outside the defined targeted genes. In this report, we describe a method for rapid validation of multigene mutants, thus allowing an appraisal of the contribution of the defined targeted genes to the strain’s phenotype.

The Rim101 protein is a conserved pH-responsive transcription factor that mediates important interactions between several fungal pathogens and the infected host. In the human fungal pathogen Cryptococcus neoformans, the Rim101 protein retains conserved functions to allow the microorganism to respond to changes in pH and other host stresses. This coordinated cellular response enables this fungus to effectively evade the host immune response. Preliminary studies suggest that this conserved transcription factor is uniquely regulated in C. neoformans both by the canonical pH-sensing pathway and by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Here we present comparative transcriptional data that demonstrate a strong concordance between the downstream effectors of PKA and Rim101. To define Rim101-dependent gene expression during a murine lung infection, we used nanoString profiling of lung tissue infected with a wild-type or rim101Δ mutant strain. In this setting, we demonstrated that Rim101 controls the expression of multiple cell wall-biosynthetic genes, likely explaining the enhanced immunogenicity of the rim101Δ mutant. Despite its divergent upstream regulation, the C. neoformans Rim101 protein recognizes a conserved DNA binding motif. Using these data, we identified direct targets of this transcription factor, including genes involved in cell wall regulation. Therefore, the Rim101 protein directly controls cell wall changes required for the adaptation of C. neoformans to its host environment. Moreover, we propose that integration of the cAMP/PKA and pH-sensing pathways allows C. neoformans to respond to a broad range of host-specific signals.

In Saccharomyces cerevisiae , the vacuolar protein sorting complexes Vps51/52/53/54 and Vps15/30/34/38 are essential for efficient endosome-to-Golgi complex retrograde transport. Here we investigated the function of Vps15 and Vps51, representative members of these complexes, in the stress resistance, host cell interactions, and virulence of Candida albicans . We found that C. albicans vps15 Δ/Δ and vps51 Δ/Δ mutants had abnormal vacuolar morphology, impaired retrograde protein trafficking, and dramatically increased susceptibility to a variety of stressors. These mutants also had reduced capacity to invade and damage oral epithelial cells in vitro and attenuated virulence in the mouse model of oropharyngeal candidiasis. Proteomic analysis of the cell wall of the vps51 Δ/Δ mutant revealed increased levels of the Crh11 and Utr2 transglycosylases, which are targets of the calcineurin signaling pathway. The transcript levels of the calcineurin pathway members CHR11 , UTR2 , CRZ1 , CNA1 , and CNA2 were elevated in the vps15 Δ/Δ and vps51 Δ/Δ mutants. Furthermore, these strains were highly sensitive to the calcineurin-specific inhibitor FK506. Also, deletion of CHR11 and UTR2 further increased the stress susceptibility of these mutants. In contrast, overexpression of CRH11 and UTR2 partially rescued their defects in stress resistance, but not host cell interactions. Therefore, intact retrograde trafficking in C. albicans is essential for stress resistance, host cell interactions, and virulence. Aberrant retrograde trafficking stimulates the calcineurin signaling pathway, leading to the increased expression of Chr11 and Utr2, which enables C. albicans to withstand environmental stress.

Biofilm formation on implanted medical devices is a major source of lethal invasive infection by Candida albicans. Filamentous growth of this fungus is tied to biofilm formation because many filamentation-associated genes are required for surface adherence. Cell cycle or cell growth defects can induce filamentation, but we have limited information about the coupling between filamentation and filamentation-associated gene expression after cell cycle/cell growth inhibition. Here we identified the CDK activating protein kinase Cak1 as a determinant of filamentation and filamentation-associated gene expression through a screen of mutations that diminish expression of protein kinase-related genes implicated in cell cycle/cell growth control. A cak1 diminished expression (DX) strain displays filamentous growth and expresses filamentation-associated genes in the absence of typical inducing signals. In a wild-type background, expression of filamentation-associated genes depends upon the transcription factors Bcr1, Brg1, Efg1, Tec1, and Ume6. In the cak1 DX background, the dependence of filamentation-associated gene expression on each transcription factor is substantially relieved. The unexpected bypass of filamentation-associated gene expression activators has the functional consequence of enabling biofilm formation in the absence of Bcr1, Brg1, Tec1, Ume6, or in the absence of both Brg1 and Ume6. It also enables filamentous cell morphogenesis, though not biofilm formation, in the absence of Efg1. Because these transcription factors are known to have shared target genes, we suggest that cell cycle/cell growth limitation leads to activation of several transcription factors, thus relieving dependence on any one., Author Summary The ability of the pathogen Candida albicans to grow on surfaces as biofilms is a determinant of infection ability, because biofilms on implanted medical devices seed infections. Biofilm formation by this organism requires growth in the form of filamentous cells and the expression of filamentation-associated genes. Inhibition of cell proliferation can induce filamentous cell formation, as we find here for strains that express greatly reduced levels of the cell cycle regulator Cak1. Surprisingly, biofilm formation occurs independently of many central biofilm regulatory genes when Cak1 levels are reduced. This response to proliferation inhibition may reflect the activation of numerous biofilm regulators, thus relieving the dependence on any one regulator. The stimulation of biofilm formation by proliferation inhibition, a property of many bacterial pathogens as well, may contribute to the limited effectiveness of antimicrobials against biofilms.

Background. The pathogenesis of intra-abdominal candidiasis is poorly understood. Methods. Mice were intraperitoneally infected with Candida albicans (1 × 106 colony-forming units) and sterile stool. nanoString assays were used to quantitate messenger RNA for 145 C. albicans genes within the peritoneal cavity at 48 hours. Results. Within 6 hours after infection, mice developed peritonitis, characterized by high yeast burdens, neutrophil influx, and a pH of 7.9 within peritoneal fluid. Organ invasion by hyphae and early abscess formation were evident 6 and 24 hours after infection, respectively; abscesses resolved by day 14. nanoString assays revealed adhesion and responses to alkaline pH, osmolarity, and stress as biologic processes activated in the peritoneal cavity. Disruption of the highly-expressed gene RIM101, which encodes an alkaline-regulated transcription factor, did not impact cellular morphology but reduced both C. albicans burden during early peritonitis and C. albicans persistence within abscesses. RIM101 influenced expression of 49 genes during intra-abdominal candidiasis, including previously unidentified Rim101 targets. Overexpression of the RIM101-dependent gene SAP5, which encodes a secreted protease, restored the ability of a rim101 mutant to persist within abscesses. Conclusions. A mouse model of intra-abdominal candidiasis is valuable for studying pathogenesis and C. albicans gene expression. RIM101 contributes to persistence within intra-abdominal abscesses, at least in part through activation of SAP5.

We previously showed that phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and septin regulation play major roles in maintaining Candida albicans cell wall integrity in response to caspofungin and other stressors. Here, we establish a link between PI(4,5)P2 signaling and septin localization and demonstrate that rapid redistribution of PI(4,5)P2 and septins is part of the natural response of C. albicans to caspofungin. First, we studied caspofungin-hypersusceptible C. albicans irs4 and inp51 mutants, which have elevated PI(4,5)P2 levels due to loss of PI(4,5)P2-specific 5′-phosphatase activity. PI(4,5)P2 accumulated in discrete patches, rather than uniformly, along surfaces of mutants in yeast and filamentous morphologies, as visualized with a green fluorescent protein (GFP)-pleckstrin homology domain. The patches also contained chitin (calcofluor white staining) and cell wall protein Rbt5 (Rbt5-GFP). By transmission electron microscopy, patches corresponded to plasma membrane invaginations that incorporated cell wall material. Fluorescently tagged septins Cdc10 and Sep7 colocalized to these sites, consistent with well-described PI(4,5)P2-septin physical interactions. Based on expression patterns of cell wall damage response genes, irs4 and inp51 mutants were firmly positioned within a group of caspofungin-hypersusceptible, septin-regulatory protein kinase mutants. irs4 and inp51 were linked most closely to the gin4 mutant by expression profiling, PI(4,5)P2-septin-chitin redistribution and other phenotypes. Finally, sublethal 5-min exposure of wild-type C. albicans to caspofungin resulted in redistribution of PI(4,5)P2 and septins in a manner similar to those of irs4 , inp51 , and gin4 mutants. Taken together, our data suggest that the C. albicans Irs4-Inp51 5′-phosphatase complex and Gin4 function upstream of PI(4,5)P2 and septins in a pathway that helps govern responses to caspofungin.

Candida albicans is a causative agent of oropharyngeal candidiasis (OPC), a biofilm-like infection of the oral mucosa. Biofilm formation depends upon the C. albicans transcription factor Bcr1, and previous studies indicate that Bcr1 is required for OPC in a mouse model of infection. Here we have used a nanoString gene expression measurement platform to elucidate the role of Bcr1 in OPC-related gene expression. We chose for assays a panel of 134 genes that represent a range of morphogenetic and cell cycle functions as well as environmental and stress response pathways. We assayed gene expression in whole infected tongue samples. The results sketch a portrait of C. albicans gene expression in which numerous stress response pathways are activated during OPC. This one set of experiments identifies 64 new genes with significantly altered RNA levels during OPC, thus increasing substantially the number of known genes in this expression class. The bcr1 Δ/Δ mutant had a much more limited gene expression defect during OPC infection than previously reported for in vitro growth conditions. Among major functional Bcr1 targets, we observed that ALS3 was Bcr1 dependent in vivo while HWP1 was not. We used null mutants and complemented strains to verify that Bcr1 and Hwp1 are required for OPC infection in this model. The role of Als3 is transient and mild, though significant. Our findings suggest that the versatility of C. albicans as a pathogen may reflect its ability to persist in the face of multiple stresses and underscore that transcriptional circuitry during infection may be distinct from that detailed during in vitro growth.

The cell wall of Candida albicans lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in SUN41 , Orf19.5412, Orf19.1277, MSB2 , Orf19.3869, and WSC1 caused hypersensitivity to the cell wall inhibitor caspofungin, while two different ecm33 insertions caused mild caspofungin resistance. Insertion mutations in SUN41 and Orf19.5412 caused biofilm defects. Through analysis of homozygous sun41 Δ/ sun41 Δ deletion mutants and sun41 Δ/ sun41 Δ+p SUN41 -complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The sun41 Δ/ sun41 Δ mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the sun41 Δ/ sun41 Δ cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target.

The ability of Candida albicans to invade mucosal tissues is a major virulence determinant of this organism; however, the mechanism of invasion is not understood in detail. Proteolytic breakdown of E-cadherin, the major protein in epithelial cell junctions, has been proposed as a mechanism of invasion of certain bacteria in the oral mucosa. The objectives of this study were (i) to assess whether C. albicans degrades E-cadherin expressed by oral epithelial cells in vitro; (ii) to compare the abilities of strains with different invasive potentials to degrade this protein; and (iii) to investigate fungal virulence factors responsible for E-cadherin degradation. We found that while E-cadherin gene expression was not altered, E-cadherin was proteolytically degraded during the interaction of oral epithelial cells with C. albicans . Moreover, C. albicans -mediated degradation of E-cadherin was completely inhibited in the presence of protease inhibitors. Using a three-dimensional model of the human oral mucosa, we found that E-cadherin was degraded in localized areas of tissue invasion by C. albicans . An invasion-deficient rim101 − / rim101 − strain was deficient in degradation of E-cadherin, and this finding suggested that proteases may depend on Rim101p for expression. Indeed, reverse transcription-PCR data indicated that expression of the SAP4 , SAP5 , and SAP6 genes is severely reduced in the rim101 − / rim101 − mutant. These SAP genes are functional Rim101p targets, because engineered expression of SAP5 in the rim101 − / rim101 − strain restored E-cadherin degradation and invasion in the mucosal model. Our data support the hypothesis that there is a mechanism by which C. albicans invades mucosal tissues by promoting the proteolytic degradation of E-cadherin in epithelial adherens junctions.

In Aspergillus nidulans , the AcuK and AcuM transcription factors form a complex that regulates gluconeogenesis. In Aspergillus fumigatus , AcuM governs gluconeogenesis and iron acquisition in vitro and virulence in immunosuppressed mice. However, the function of AcuK was previously unknown. Through in vitro studies, we found that A. fumigatus Δ acuK single and Δ acuK Δ acuM double mutants had impaired gluconeogenesis and iron acquisition, similar to the Δ acuM mutant. Also, the Δ acuK , Δ acuM , and Δ acuK Δ acuM mutants had similar virulence defects in mice. However, the Δ acuK mutant had a milder defect in extracellular siderophore activity and induction of epithelial cell damage in vitro than did the Δ acuM mutant. Moreover, overexpression of acuM in the Δ acuK mutant altered expression of 3 genes and partially restored growth under iron-limited conditions, suggesting that AcuM can govern some genes independently of AcuK. Although the Δ acuK and Δ acuM mutants had very similar transcriptional profiles in vitro , their transcriptional profiles during murine pulmonary infection differed both from their in vitro profiles and from each other. While AcuK and AcuM governed the expression of only a few iron-responsive genes in vivo , they influenced the expression of other virulence-related genes, such as hexA and dvrA . Therefore, in A. fumigatus , while AcuK and AcuM likely function as part of the same complex, they can also function independently of each other. Furthermore, AcuK and AcuM have different target genes in vivo than in vitro , suggesting that in vivo infection stimulates unique transcriptional regulatory pathways in A. fumigatus .

Gene expression dynamics have provided foundational insight into almost all biological processes. Here, we analyze expression of environmentally responsive genes and transcription factor genes to infer signals and pathways that drive pathogen gene regulation during invasive Candida albicans infection of a mammalian host. Environmentally responsive gene expression shows that there are early and late phases of infection. The early phase includes induction of zinc and iron limitation genes, genes that respond to transcription factor Rim101, and genes characteristic of invasive hyphal cells. The late phase includes responses related to phagocytosis by macrophages. Transcription factor gene expression also reflects early and late phases. Transcription factor genes that are required for virulence or proliferation in vivo are enriched among highly expressed transcription factor genes. Mutants defective in six transcription factor genes, three previously studied in detail (Rim101, Efg1, Zap1) and three less extensively studied (Rob1, Rpn4, Sut1), are profiled during infection. Most of these mutants have distinct gene expression profiles during infection as compared to in vitro growth. Infection profiles suggest that Sut1 acts in the same pathway as Zap1, and we verify that functional relationship with the finding that overexpression of either ZAP1 or the Zap1-dependent zinc transporter gene ZRT2 restores pathogenicity to a sut1 mutant. Perturbation with the cell wall inhibitor caspofungin also has distinct gene expression impact in vivo and in vitro. Unexpectedly, caspofungin induces many of the same genes that are repressed early during infection, a phenomenon that we suggest may contribute to drug efficacy. The pathogen response circuitry is tailored uniquely during infection, with many relevant regulatory relationships that are not evident during growth in vitro. Our findings support the principle that virulence is a property that is manifested only in the distinct environment in which host–pathogen interaction occurs., A study of the invasive infection of a mammalian host by the pathogenic fungus Candida albicans reveals characteristic gene regulation patterns in response to the host environment, distinct from those seen when growing in vitro., Author Summary We have a limited understanding of how the expression of pathogens’ genes changes during infection of humans or other animal hosts, in contrast to in vitro models of infection. Here we profile the alteration in gene expression over time as a predictor of functional consequences during invasive growth of Candida in the kidney; a situation in which the limited number of pathogen cells makes gene expression challenging to assay. Our findings reveal that there are distinct early and late phases of infection, and identify new genes that govern the early zinc acquisition response necessary for proliferation in vivo—and thus required for infection. We also find that the response to drug treatment that manifests during infection can be distinct from that detected in vitro. We show that a well-known gene expression response to the antifungal drug caspofungin is naturally down-regulated in infecting cells, suggesting that the efficacy of the drug may be enhanced by a susceptible state of the pathogen during invasive proliferation.

Candida albicansis a major opportunistic pathogen in immunocompromised patients. Production of proinflammatory cytokines by host cells in response toC. albicansplays a critical role in the activation of immune cells and final clearance of the organism. Invasion of host cells and tissues is considered one of the virulence attributes of this organism. The purpose of this study was to investigate whether the ability ofC. albicansto invade host cells and tissues affects the proinflammatory cytokine responses by epithelial and endothelial cells. In this study we used the invasion-deficientRIM101gene knockout strain DAY25, the highly invasive strain SC5314, and highly invasiveRIM101-complemented strain DAY44 to compare the proinflammatory cytokine responses by oral epithelial or endothelial cells. Using a high-throughput approach, we found both qualitative and quantitative differences in the overall inflammatory responses toC. albicansstrains with different invasive potentials. Overall, the highly invasive strains triggered higher levels of proinflammatory cytokines in host cells than the invasion-deficient mutant triggered. Significant differences compared to the attenuated mutant were noted in interleukin-1α (IL-1α), IL-6, IL-8, and tumor necrosis factor alpha in epithelial cells and in IL-6, growth-related oncogene, IL-8, monocyte chemoattractant protein 1 (MCP-1), MCP-2, and granulocyte colony-stimulating factor in endothelial cells. Our results indicate that invasion of host cells and tissues byC. albicansenhances the host proinflammatory response to infection.

The impact of many microorganisms on their environment depends upon their ability to form surface bound communities called biofilms [1]. Biofilm formation on implanted medical devices has severe consequences for human health by providing both a portal of entry and a sanctuary for invasive bacterial and fungal pathogens [1 and 2]. Biofilm regulators and adherence molecules are extensively defined for many bacterial pathogens [3, 4, and 5], but not for fungal pathogens such as Candida albicans. Elongated filaments called hyphae are a prominent feature of C. albicans biofilms, and known genes that promote biofilm formation are required for hyphal development [2, 6, 7 and 8]. From a new library of transcription-factor mutants, we identify Bcr1p, a zinc finger protein required for formation of biofilms but not hyphae. Expression analysis shows that Bcr1p activates cell-surface protein and adhesin genes, including several induced during hyphal development. BCR1 expression depends upon the hyphal regulator Tec1p. Thus, BCR1 is a downstream component of the hyphal regulatory network that couples expression of cell-surface genes to hyphal differentiation. Our results indicate that hyphal cells are specialized to present adherence molecules that support biofilm integrity.

Environmental pH changes have broad consequences for growth and differentiation. The best-understood eukaryotic pH response pathway acts through the zinc-finger transcription factor PacC of Aspergillus nidulans, which activates alkaline pH-induced genes directly. We show here that Saccharomyces cerevisiae Rim101p, the pH response regulator homologous to PacC, functions as a repressor in vivo. Chromatin immunoprecipitation assays show that Rim101p is associated in vivo with the promoters of seven Rim101p-repressed genes. A reporter gene containing deduced Rim101p binding sites is negatively regulated by Rim101p and is associated with Rim101p in vivo. Deletion mutations of the Rim101p repression targets NRG1 and SMP1 suppress rim101Delta mutant defects in ion tolerance, haploid invasive growth, and sporulation. Therefore, transcriptional repression is the main biological function of Rim101p. The Rim101p repression target Nrg1p is in turn required for repression of two alkaline pH-inducible genes, including the Na+ pump gene ENA1, which is required for ion tolerance. Thus, Nrg1p, a known transcriptional repressor, functions as an inhibitor of alkaline pH responses. Our findings stand in contrast to the well-characterized function of PacC as a direct activator of alkaline pH-induced genes yet explain many aspects of Rim101p and PacC function in other organisms.

SummaryTemperature affects diverse biological processes. In fungi such as the pathogen Candida albicans, temperature governs a morphogenetic switch between yeast and hyphal growth. A new report connects the thermosensor Hsp90 to a CDK–cyclin–transcription factor module that controls morphogenesis.

Septin proteins are conserved structural proteins that often demarcate regions of cell division. The essential nature of the septin ring, composed of several septin proteins, complicates investigation of the functions of the ring, although careful analysis in the model yeast Saccharomyces cerevisiae has elucidated the role that septins play in the cell cycle. Mutation analysis of nonessential septins in the pathogenic fungus Candida albicans has shown that septins also have vital roles in c ell w all r egulation (CWR), hyphal formation, and pathogenesis. While mutations in nonessential septins have been useful in establishing phenotypes, the septin defect is so slight that identifying causative associations between septins and downstream effectors has been difficult. In this work, we describe decreased abundance by mRNA perturbation (DAmP) alleles of essential septins, which display a septin defect more severe than the defect observed in deletions of nonessential septins. The septin DAmP alleles have allowed us to genetically separate the roles of septins in hyphal growth and CWR and to identify the cyclic AMP pathway as a pathway that likely acts in a parallel manner with septins in hyphal morphogenesis.

Virulence of Candida is linked with its ability to form biofilms. Once established, biofilm infections are nearly impossible to eradicate. Biofilm cells live immersed in a self-produced matrix, a blend of extracellular biopolymers, many of which are uncharacterized. In this study, we provide a comprehensive analysis of the matrix manufactured by Candida albicans both in vitro and in a clinical niche animal model. We further explore the function of matrix components, including the impact on drug resistance. We uncovered components from each of the macromolecular classes (55% protein, 25% carbohydrate, 15% lipid, and 5% nucleic acid) in the C. albicans biofilm matrix. Three individual polysaccharides were identified and were suggested to interact physically. Surprisingly, a previously identified polysaccharide of functional importance, β-1,3-glucan, comprised only a small portion of the total matrix carbohydrate. Newly described, more abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched β-1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx). Functional matrix proteomic analysis revealed 458 distinct activities. The matrix lipids consisted of neutral glycerolipids (89.1%), polar glycerolipids (10.4%), and sphingolipids (0.5%). Examination of matrix nucleic acid identified DNA, primarily noncoding sequences. Several of the in vitro matrix components, including proteins and each of the polysaccharides, were also present in the matrix of a clinically relevant in vivo biofilm. Nuclear magnetic resonance (NMR) analysis demonstrated interaction of aggregate matrix with the antifungal fluconazole, consistent with a role in drug impedance and contribution of multiple matrix components., IMPORTANCE This report is the first to decipher the complex and unique macromolecular composition of the Candida biofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs. The availability of these biochemical analyses provides a unique resource for further functional investigation of the biofilm matrix, a defining trait of this lifestyle.

The Saccharomyces cerevisiae zinc finger protein Rim101p is activated by cleavage of its C-terminal region, which resembles PEST regions that confer susceptibility to proteolysis. Here we report that Rim20p, a member of the broadly conserved PalA/AIP1/Alix family, is required for Rim101p cleavage. Two-hybrid and coimmunoprecipitation assays indicate that Rim20p binds to Rim101p, and a two-hybrid assay shows that the Rim101p PEST-like region is sufficient for Rim20p binding. Rim101p-Rim20p interaction is conserved in Candida albicans , supporting the idea that interaction is functionally significant. Analysis of Rim20p mutant proteins indicates that some of its broadly conserved regions are required for processing of Rim101p and for stability of Rim20p itself but are not required for interaction with Rim101p. A recent genome-wide two-hybrid study (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569–4574, 2000) indicates that Rim20p interacts with Snf7p and that Snf7p interacts with Rim13p, a cysteine protease required for Rim101p proteolysis. We suggest that Rim20p may serve as part of a scaffold that places Rim101p and Rim13p in close proximity.

The ability of Candida albicans to respond to diverse environments is critical for its success as a pathogen. The RIM101 pathway controls gene expression and the yeast-to-hyphal transition in C. albicans in response to changes in environmental pH in vitro. In this study, we found that the RIM101 pathway is necessary in vivo for pathogenesis. First, we show that rim101 − /rim101 − and rim8 − /rim8 − mutants have a significant reduction in virulence using the mouse model of hematogenously disseminated systemic candidiasis. Second, these mutants show a marked reduction in kidney pathology. Third, the rim101 − /rim101 − and rim8 − /rim8 − mutants show defects in the ability to damage endothelial cells in situ. Finally, we show that an activated allele of RIM101 , RIM101-405 , is a suppressor of the rim8 − mutation in vivo as it rescues the virulence, histological, and endothelial damage defects of the rim8 − /rim8 − mutant. These results demonstrate that the RIM101 pathway is required for C. albicans virulence in vivo and that the function of Rim8p in pathogenesis is to activate Rim101p.

The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression.

Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

In order to colonize the host and cause disease, Candida albicans must avoid being killed by host defense peptides. Previously, we determined that the regulatory protein Ssd1 governs antimicrobial peptide resistance in C. albicans . Here, we sought to identify additional genes whose products govern susceptibility to antimicrobial peptides. We discovered that a bcr1 Δ/Δ mutant, like the ssd1 Δ/Δ mutant, had increased susceptibility to the antimicrobial peptides, protamine, RP-1, and human β defensin-2. Homozygous deletion of BCR1 in the ssd1 Δ/Δ mutant did not result in a further increase in antimicrobial peptide susceptibility. Exposure of the bcr1 Δ/Δ and ssd1 Δ/Δ mutants to RP-1 induced greater loss of mitochondrial membrane potential and increased plasma membrane permeability than with the control strains. Therefore, Bcr1 and Ssd1 govern antimicrobial peptide susceptibility and likely function in the same pathway. Furthermore, BCR1 mRNA expression was downregulated in the ssd1 Δ/Δ mutant, and the forced expression of BCR1 in the ssd1 Δ/Δ mutant partially restored antimicrobial peptide resistance. These results suggest that Bcr1 functions downstream of Ssd1. Interestingly, overexpression of 11 known Bcr1 target genes in the bcr1 Δ/Δ mutant failed to restore antimicrobial peptide resistance, suggesting that other Bcr1 target genes are likely responsible for antimicrobial peptide resistance. Collectively, these results demonstrate that Bcr1 functions downstream of Ssd1 to govern antimicrobial peptide resistance by maintaining mitochondrial energetics and reducing membrane permeabilization.

Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall β-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis., Author Summary Invasive aspergillosis is the most common mold infection in humans, predominately affecting immunocompromised patients. The mechanisms by which the mold Aspergillus fumigatus adheres to host tissues and causes disease are poorly understood. In this report, we compared mutants of Aspergillus with different adhesive properties to identify fungal factors involved in adherence to host cells. This approach identified a cell wall associated polysaccharide, galactosaminogalactan, which is required for adherence to a wide variety of substrates. Galactosaminogalactan was also observed to suppress inflammation by concealing β-glucans, key pattern associated microbial pattern molecules in Aspergillus hyphae, from recognition by the innate immune system. Mutants that were deficient in galactosaminogalactan were less virulent in mouse models of invasive aspergillosis. These data identify a bifunctional role for galactosaminogalactan in the pathogenesis of invasive aspergillosis, and suggest that it may serve as a useful target for antifungal therapy.

Ime2p is a protein kinase that is expressed only during meiosis in Saccharomyces cerevisiae. Ime2p stimulates early, middle, and late meiotic gene expression and down-regulates expression of IME1, which specifies an activator of early meiotic genes that acts independently of Ime2p. We have identified a new gene, IDS2 (for IME2-dependent signaling), which has a functional relationship to Ime2p. An ids2 null mutation delays down-regulation of IME1 and expression of middle and late meiotic genes. In an ime1 null mutant that express IME2 from the GAL1 promoter (ime1 delta PGAL1-IME2 mutant), early meiotic gene expression depends only upon Ime2p. In such strains, Ids2p is dispensable for expression of the early genes HOP1 and SPO13 but is essential for expression of the middle and late genes SPS1, SPS2, and SPS100. Ids2p is also essential for the autoregulatory pathway through which Ime2p activates its own expression via the IME2 upstream activation sequences (UAS). An PGAL1-IME2 derivative that produces a truncated Ime2p (lacking its C-terminal 174 residues) permits IME2 UAS activation in the absence of Ids2p. This observation suggests that Ids2p acts upstream of Ime2p or that Ids2p and Ime2p act in independent, convergent pathways to stimulate IME2 UAS activity. Accumulation of epitope-tagged Ids2p derivatives is greatest in growing cells and declines during meiosis. We propose that Ids2p acts indirectly to modify Ime2p activity, thus permitting Ime2p to carry out later meiotic functions.

Extracellular polysaccharides are key constituents of the biofilm matrix of many microorganisms. One critical carbohydrate component of Candida albicans biofilms, β-1,3 glucan, has been linked to biofilm protection from antifungal agents. In this study, we identify three glucan modification enzymes that function to deliver glucan from the cell to the extracellular matrix. These enzymes include two predicted glucan transferases and an exo-glucanase, encoded by BGL2, PHR1, and XOG1, respectively. We show that the enzymes are crucial for both delivery of β-1,3 glucan to the biofilm matrix and for accumulation of mature matrix biomass. The enzymes do not appear to impact cell wall glucan content of biofilm cells, nor are they necessary for filamentation or biofilm formation. We demonstrate that mutants lacking these genes exhibit enhanced susceptibility to the commonly used antifungal, fluconazole, during biofilm growth only. Transcriptional analysis and biofilm phenotypes of strains with multiple mutations suggest that these enzymes act in a complementary fashion to distribute matrix downstream of the primary β-1,3 glucan synthase encoded by FKS1. Furthermore, our observations suggest that this matrix delivery pathway works independently from the C. albicans ZAP1 matrix formation regulatory pathway. These glucan modification enzymes appear to play a biofilm-specific role in mediating the delivery and organization of mature biofilm matrix. We propose that the discovery of inhibitors for these enzymes would provide promising anti-biofilm therapeutics., Author Summary Biofilms are a community of microbes that grow attached to each other and adherent to a surface. One distinguishing feature of this form of growth is the presence of a surrounding extracellular matrix which is proposed to provide a structural scaffold and protection for biofilm cells. This later function contributes to the extreme resistance to anti-infective therapies, another innate characteristic of biofilms. One carbohydrate component of the matrix of Candida albicans, β-1, 3 glucan, has been linked to overall accumulation of matrix material and the antifungal drug resistance phenotype. Although the glucan synthase pathway has been implicated in glucan production, the delivery and incorporation of these carbohydrates into the matrix remains a mystery. The current investigation describes three gene products that serve a matrix delivery role. The functions of these gene products include glucanase and glucanosyltransferase activities. Mutants unable to produce these enzymes demonstrate reduced matrix glucan, decreased total matrix biomass accumulation, and enhanced susceptibility to antifungal drug therapy. The observations here offer insight into a novel pathway that contributes to biofilm maintenance. Enzymes in this biofilm-specific process may provide useful anti-biofilm drug targets.

Cell-substrate adherence is a fundamental property of microorganisms that enables them to exist in biofilms. Our study focuses on adherence of the fungal pathogen Candida albicans to one substrate, silicone, that is relevant to device-associated infection. We conducted a mutant screen with a quantitative flow-cell assay to identify thirty transcription factors that are required for adherence. We then combined nanoString gene expression profiling with functional analysis to elucidate relationships among these transcription factors, with two major goals: to extend our understanding of transcription factors previously known to govern adherence or biofilm formation, and to gain insight into the many transcription factors we identified that were relatively uncharacterized, particularly in the context of adherence or cell surface biogenesis. With regard to the first goal, we have discovered a role for biofilm regulator Bcr1 in adherence, and found that biofilm regulator Ace2 is a major functional target of chromatin remodeling factor Snf5. In addition, Bcr1 and Ace2 share several target genes, pointing to a new connection between them. With regard to the second goal, our findings reveal existence of a large regulatory network that connects eleven adherence regulators, the zinc-response regulator Zap1, and approximately one quarter of the predicted cell surface protein genes in this organism. This limited yet sensitive glimpse of mutant gene expression changes had thus defined one of the broadest cell surface regulatory networks in C. albicans., Author Summary Most microorganisms adhere to surfaces in nature, leading to formation of complex communities called biofilms. Pathogen adherence to medical devices is the basis for device-associated infection. We have focused on the control of adherence in the fungal pathogen Candida albicans. We find that this process is under control of thirty transcriptional regulators. Our analysis of gene expression in regulatory mutants with altered adherence provides new understanding of the relationships among known regulators. In addition, we find evidence for a large regulatory network that connects one quarter of all cell surface protein genes.

Biofilms of Candida albicans include both yeast cells and hyphae. Prior studies indicated that a zap1 Δ/Δ mutant, defective in zinc regulator Zap1, has increased accumulation of yeast cells in biofilms. This altered yeast-hypha balance may arise from internal regulatory alterations or from an effect on the production of diffusible quorum-sensing (QS) molecules. Here, we develop biosensor reporter strains that express yeast-specific YWP1-RFP or hypha-specific HWP1-RFP , along with a constitutive TDH3-GFP normalization standard. Seeding these biosensor strains into biofilms allows a biological activity assay of the surrounding biofilm milieu. A zap1 Δ/Δ biofilm induces the yeast-specific YWP1-RFP reporter in a wild-type biosensor strain, as determined by both quantitative reverse transcription-PCR (qRT-PCR) gene expression measurements and confocal microscopy. Remediation of the zap1 Δ/Δ zinc uptake defect through zinc transporter gene ZRT2 overexpression reverses induction of the yeast-specific YWP1-RFP reporter. Gas chromatography-mass spectrometry (GC-MS) measurements of known organic QS molecules show that the zap1 Δ/Δ mutant accumulates significantly less farnesol than wild-type or complemented strains and that ZRT2 overexpression does not affect farnesol accumulation. Farnesol is a well-characterized inhibitor of hypha formation; hence, a reduction in farnesol levels in zap1 Δ/Δ biofilms is unexpected. Our findings argue that a Zap1- and zinc-dependent signal affects the yeast-hypha balance and that it is operative in the low-farnesol environment of the zap1 Δ/Δ biofilm. In addition, our results indicate that Zap1 is a positive regulator of farnesol accumulation.

Diploid a/alpha Saccharomyces cerevisiae cells cease mitotic growth and enter meiosis in response to starvation. Expression of meiotic genes depends on the IME1 gene product, which accumulates only in meiotic cells. We report here an analysis of the regulatory region of IME2, an IME1-dependent meiotic gene. Deletion and substitution studies identified a 48-bp IME1-dependent upstream activation sequence (UAS). Activity of the UAS also requires the RIM11, RIM15, and RIM16 gene products, which are required for expression of the chromosomal IME2 promoter and for meiosis. Through a selection for suppressors that permit UAS activity in an ime1 deletion mutant, we identified recessive mutations in three genes, SIN3 (also called RPD1, UME4, and SDI1), RPD3, and UME6 (also called CAR80), that were previously known as negative regulators of other early meiotic genes. Mutational analysis of the IME2 UAS reveals two critical sequence elements: a G+C-rich sequence (called URS1), previously identified at many meiotic genes, and a newly described element, the T4C site, that we found at a subset of meiotic genes. In agreement with prior studies, URS1 mutations lead to elevated IME2 UAS activity in the absence of IME1. However, the URS1 mutations prevent any further stimulation of UAS activity by IME1. Repression through URS1 has been shown to require the UME6 gene product. We find that activation of the IME2 UAS by IME1 also requires the UME6 gene product. Thus, UME6 and the URS1 site both have dual negative and positive roles at the IME2 UAS. We propose that IME1 modifies UME6 to convert it from a negulator to a positive Regulor.

IME1 is required in yeast for meiosis and for expression of IME2 and other early meiotic genes. IME1 is a 360-amino acid polypeptide with central and C-terminal tyrosine-rich regions. We report here that a fusion protein composed of the lexA DNA-binding domain and IME1 activates transcription in vivo of a reporter gene containing upstream lexA binding sites. Activation by the fusion protein shares several features with natural IME1 activity: both are dependent on the RIM11 gene product; both are impaired by the same ime1 missense mutations; both are restored by intragenic suppressors. The central tyrosine-rich region is sufficient to activate transcription when fused to lexA. Deletion of this putative activation domain results in a defective IME1 derivative. Function of the deletion derivative is restored by fusion to the acidic Herpesvirus VP16 activation domain. The C-terminal tyrosine-rich region is dispensable for transcriptional activation; rather it renders activation dependent upon starvation and RIM11. Immunofluorescence studies indicate that an IME1-lacZ fusion protein is concentrated in the nucleus. These observations are consistent with a model in which IME1 normally stimulates IME2 expression by providing a transcriptional activation domain at the IME2 5' regulatory region.

In the yeast Saccharomyces cerevisiae, genetic studies suggest that the RIM1 gene encodes a positive regulator of meiosis. rim1 mutations cause reduced expression of IME1, which is required for expression of many meiotic genes, and thus lead to a partial defect in meiosis and spore formation. We report the sequence of RIM1 and functional analysis of its coding region. The RIM1 gene product (RIM1) contains three regions similar to C2H2 zinc fingers. Serine substitutions for cysteine in each of the putative zinc fingers abolish RIM1 function. The carboxyl-terminus of RIM1 is enriched in acidic amino acids and is required for full RIM1 activity. RIM1 also contains two putative cAMP-dependent protein kinase (cAPK) phosphorylation sites. At one site, substitution of alanine for serine does not affect RIM1 activity; at the other site, this substitution impairs activity. This analysis of RIM1 suggests that the protein may function as a transcriptional activator. We have used the cloned RIM1 gene to create a complete rim1 deletion. This null allele, like previously isolated rim1 mutations, causes a partial meiotic defect. In addition to RIM1, maximum IME1 expression requires the MCK1 and IME4 gene products. Defects associated with rim1, mck1, and ime4 mutations in expression of a meiotic reporter gene (ime2-lacZ) and in sporulation are additive. These findings suggest that RIM1 acts independently of MCK1 and IME4 to stimulate IME1 expression.

Human β-defensins 2 and 3 are small cationic peptides with antimicrobial activity against the fungal pathogen Candida albicans . We found that hog1 and pbs2 mutants were hypersensitive to treatment with these peptides, pointing to a role of the high-osmolarity glycerol (HOG) pathway in the response to defensin-induced cell injury.

Candida albicans is the most common cause of invasive fungal infections in humans. It is unclear how C. albicans escapes from phagocytic attack and survives in the hostile blood environment during life-threatening systemic infections. Using a conditional overexpression or suppression genetic strategy, we discovered that HYR1 gene reduced phagocytic killing activity of C. albicans in vitro and increased tissue fungal burden in vivo. Concordant with its positive regulation by the transcription factor Bcr1p, autonomous expression of HYR1 complemented the hypersusceptibility to phagocyte-mediated killing of a bcr1 null mutant of C. albicans in vitro. As for C. albicans, heterologous expression of HYR1 in Candida glabrata rendered the organism more resistant to neutrophil killing activity. Vaccination with a recombinant Hyr1p significantly protected mice against hematogenously disseminated candidiasis (P = .001). Finally, anti-rHyr1p polyclonal antibodies enhanced mouse neutrophil killing activity by directly neutralizing rHyr1p effects in vitro. Thus, Hyr1 is an important virulence factor for C. albicans, mediating resistance to phagocyte killing. Hyr1p is a promising target for vaccine or other immunological or small molecule intervention to improve the outcomes of disseminated candidiasis.

Protein kinases play key roles in signaling and response to changes in the external environment. The ability of Candida albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. Our guiding hypothesis was that creating and screening a set of protein kinase mutant strains would reveal signaling pathways that mediate stress response in C. albicans. A library of protein kinase mutant strains was created and screened for sensitivity to a variety of stresses. For the majority of stresses tested, stress response was largely conserved between C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. However, we identified eight protein kinases whose roles in cell wall regulation (CWR) were not expected from functions of their orthologs in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. Analysis of the conserved roles of these protein kinases indicates that establishment of cell polarity is critical for CWR. In addition, we found that septins, crucial to budding, are both important for surviving and are mislocalized by cell wall stress. Our study shows an expanded role for protein kinase signaling in C. albicans cell wall integrity. Our studies suggest that in some cases, this expansion represents a greater importance for certain pathways in cell wall biogenesis. In other cases, it appears that signaling pathways have been rewired for a cell wall integrity response., Author Summary Candida albicans is the major fungal commensal and pathogen of humans. Like most microorganisms, C. albicans is surrounded and protected by a cell wall. This cell wall has two purposes: to act as a rigid “exoskeleton” to prevent cells from bursting, and to provide a surface where a cell can interact with the outside environment while protecting the cell itself from this environment. Maintenance of this structure has been well studied in the model fungus, Saccharomyces cerevisiae, but previous evidence suggested that C. albicans might have additional inputs to this process. By creating and testing a library of mutant strains for sensitivity to cell wall stress, we were able to identify a number of conserved genes with roles in this process not shared by their S. cerevisiae counterparts. Although some of these genes had previously been linked to cell wall integrity, it appears that they have increased impact on this process in C. albicans. For other genes, their role in cell wall integrity may represent a novel connection. Our findings provide insight into novel aspects of cell wall integrity in this pathogen and lay a foundation for its more detailed analysis.

The zinc-responsive transcription factor Zap1 has a striking role in fungal biofilm formation and is reported to regulate matrix formation., A biofilm is a surface-associated population of microorganisms embedded in a matrix of extracellular polymeric substances. Biofilms are a major natural growth form of microorganisms and the cause of pervasive device-associated infection. This report focuses on the biofilm matrix of Candida albicans, the major fungal pathogen of humans. We report here that the C. albicans zinc-response transcription factor Zap1 is a negative regulator of a major matrix component, soluble β-1,3 glucan, in both in vitro and in vivo biofilm models. To understand the mechanistic relationship between Zap1 and matrix, we identified Zap1 target genes through expression profiling and full genome chromatin immunoprecipitation. On the basis of these results, we designed additional experiments showing that two glucoamylases, Gca1 and Gca2, have positive roles in matrix production and may function through hydrolysis of insoluble β-1,3 glucan chains. We also show that a group of alcohol dehydrogenases Adh5, Csh1, and Ifd6 have roles in matrix production: Adh5 acts positively, and Csh1 and Ifd6, negatively. We propose that these alcohol dehydrogenases generate quorum-sensing aryl and acyl alcohols that in turn govern multiple events in biofilm maturation. Our findings define a novel regulatory circuit and its mechanism of control of a process central to infection., Author Summary A biofilm is a surface-associated population of microbes that is embedded in a cement of extracellular compounds. This cement is known as matrix. The two main functions of matrix are to protect cells from their surrounding environment, preventing drugs and other stresses from penetrating the biofilm, and to maintain the architectural stability of the biofilm, acting as a glue to hold the cells together. The presence of matrix is a contributing factor to the high degree of resistance to antimicrobial drugs observed in biofilms. Because biofilms have a major impact on human health, and because matrix is such a pivotal component of biofilms, it is important to understand how the production of matrix is regulated. We have begun to address this question in the major human fungal pathogen Candida albicans. We found that the zinc-responsive regulatory protein Zap1 controls the expression of several genes important for matrix formation in C. albicans. These target genes encode glucoamylases and alcohol dehydrogenases, enzymes that probably govern the synthesis of distinct matrix constituents. The findings here offer insight into the metabolic processes that contribute to biofilm formation and indicate that Zap1 functions broadly as a negative regulator of biofilm maturation.

The detection of protein–protein interactions through two-hybrid assays has revolutionized our understanding of biology. The remarkable impact of two-hybrid assay platforms derives from their speed, simplicity, and broad applicability. Yet for many organisms, the need to express test proteins in Saccharomyces cerevisiae or Escherichia coli presents a substantial barrier because variations in codon specificity or bias may result in aberrant protein expression. In particular, nonstandard genetic codes are characteristic of several eukaryotic pathogens, for which there are currently no genetically based systems for detection of protein–protein interactions. We have developed a protein–protein interaction assay that is carried out in native host cells by using GFP as the only foreign protein moiety, thus circumventing these problems. We show that interaction can be detected between two protein pairs in both the model yeast S. cerevisiae and the fungal pathogen Candida albicans. We use computational analysis of microscopic images to provide a quantitative and automated assessment of confidence.

In the yeast Saccharomyces cerevisiae, a/alpha cells can enter meiosis whereas a and alpha cells cannot. The a/alpha cell type is determined by presence of a repressor, a1-alpha 2. Previous studies indicate that a/alpha cells lack an inhibitor of meiosis, the RME1 gene product, and that a and alpha cells express RME1. We report here the sequence of RME1 and functional analysis of its regulatory and coding regions. The 5'-region of RME1 includes a sequence resembling a1-alpha 2 repression sites. Deletion of this site at RME1 relieves repression by a1-alpha 2, and insertion of the site into a heterologous regulatory region (CYC1) confers weak repression in a/alpha cells. These observations indicate that RME1 is directly repressed by a1-alpha 2. The RME1 product has three regions that resemble C2H2 zinc fingers, which are characteristic of a class of nucleic-acid-binding proteins. Substitution of serine for cysteine in each of the putative fingers abolishes RME1 function; serine substitutions in the second and third putative fingers do not affect RME1 stability. These findings indicate that at least two putative zinc fingers are critical for RME1 structure or activity. Therefore RME1, which is formally a negative regulator of the meiotic gene IME1, may act directly as a repressor.

pH-responsive transcription factors of the Rim101/PacC family govern virulence in many fungal pathogens. These family members control expression of target genes with diverse functions in growth, morphology and environmental adaptation, so the mechanistic relationship between Rim101/PacC and infection is unclear. We have focused on Rim101 from Candida albicans, which we find to be required for virulence in an oropharyngeal candidiasis model. Rim101 affects the yeast-hypha morphological transition, a major virulence requirement in disseminated infection models. However, virulence in the oropharyngeal candidiasis model is independent of the yeast-hypha transition because it is unaffected by an nrg1 mutation, which prevents formation of yeast cells. Here we have identified Rim101 target genes in an nrg1Delta/Delta mutant background and surveyed function using an overexpression-rescue approach. Increased expression of Rim101 target genes ALS3, CHT2, PGA7/RBT6, SKN1 or ZRT1 can partially restore pathogenic interaction of a rim101Delta/Delta mutant with oral epithelial cells. Four of these five genes govern cell wall structure. Our results indicate that Rim101-dependent cell wall alteration contributes to C. albicans pathogenic interactions with oral epithelial cells, independently of cell morphology.

Summary Background Biofilms are surface-associated microbial communities with significant environmental and medical impact. Here, we focus on an adherence mechanism that permits biofilm formation by Candida albicans , the major invasive fungal pathogen of humans. Results The Als surface-protein family has been implicated in biofilm formation, and we show that Als1 and Als3 have critical but redundant roles. Overexpression of several other Als proteins permits biofilm formation in a biofilm-defective als1/als1 als3/als3 strain, thus arguing that the function of Als proteins in this process is governed by their respective expression levels. The surface protein Hwp1 is also required for biofilm formation, and we find that a mixture of biofilm-defective hwp1/hwp1 and als1/als1 als3/als3 strains can form a hybrid biofilm both in vitro and in vivo in a catheter infection model. Complementary function of Hwp1 and Als1 and 3 seems to reflect their interaction because expression of Hwp1 in the heterologous host S. cerevisiae permits adherence to wild-type C. albicans , but not to an als1/als1 als3/als3 strain. Conclusions The complementary roles of Hwp1 and Als1 and Als3 in biofilm formation are analogous to the roles of sexual agglutinins in mating reactions. This analogy suggests that biofilm-adhesin complementarity may promote formation of monospecies biofilms.

Hwp1 is a well-characterized Candida albicans cell surface protein, expressed only on hyphae, that mediates tight binding to oral epithelial cells. Prior studies indicate that HWP1 expression is dependent upon Bcr1, a key regulator of biofilm formation. Here we test the hypothesis that Hwp1 is required for biofilm formation. In an in vitro model, the hwp1 / hwp1 mutant produces a thin biofilm that lacks much of the hyphal mass found in the hwp1 / HWP1 reconstituted strain. In a biofilm cell retention assay, we find that the hwp1 / hwp1 mutant is defective in retention of nonadherent bcr1 / bcr1 mutant cells. In an in vivo rat venous catheter model, the hwp1 / hwp1 mutant has a severe biofilm defect, yielding only yeast microcolonies in the catheter lumen. These properties of the hwp1 / hwp1 mutant are consistent with its role as a hypha-specific adhesin and indicate that it is required for normal biofilm formation. Overexpression of HWP1 in a bcr1 / bcr1 mutant background improves adherence in the in vivo catheter model. This finding provides additional support for the model that Hwp1 is critical for biofilm adhesion. Hwp1 is the first cell surface protein known to be required for C. albicans biofilm formation in vivo and is thus an excellent therapeutic target.

To identify Candida albicans genes whose proteins are necessary for host cell interactions and virulence, a collection of C. albicans insertion mutants was screened for strains with reduced capacity to damage endothelial cells in vitro. This screen identified CKA2. CKA2 and its homologue CKA1 encode the catalytic subunits of the protein kinase CK2. cka2delta/cka2delta strains of C. albicans were constructed and found to have significantly reduced capacity to damage both endothelial cells and an oral epithelial cell line in vitro. Although these strains invaded endothelial cells similarly to the wild-type strain, they were defective in oral epithelial cell invasion. They were also hypersusceptible to hydrogen peroxide, but not to high salt or to cell wall damaging agents. A cka1delta/cka1delta mutant caused normal damage to both endothelial cells and oral epithelial cells, and it was not hypersusceptible to hydrogen peroxide. However, overexpression of CKA1 in a cka2delta/cka2delta strain restored wild-type phenotype. Although the cka2delta/cka2delta mutant had normal virulence in the mouse model of haematogenously disseminated candidiasis, it had significantly attenuated virulence in the mouse model of oropharyngeal candidiasis. Therefore, Cka2p governs the interactions of C. albicans with endothelial and oral epithelial cells in vitro and virulence during oropharyngeal candidiasis.