Albicans Gene Research Articles

Temporal dynamics of Candida albicans morphogenesis and gene expression reveals distinctions between in vitro and in vivo filamentation.

Candida albicans is a common human fungal pathogen that is also a commensal of the oral cavity and gastrointestinal tract. C. albicans pathogenesis is linked to its transition from budding yeast to filamentous morphologies including hyphae and pseudohyphae. The centrality of this virulence trait to C. albicans pathobiology has resulted in extensive characterization of a wide range of factors associated with filamentation with a strong focus on transcriptional regulation. The vast majority of these experiments have used in vitro conditions to induce the yeast-to-filament transition. Taking advantage of in vivo approaches to quantitatively characterize both morphology and gene expression during filamentation during mammalian infection, we have investigated the dynamics of these two aspects of filamentation in vivo and compared them to in vitro filament induction with "host-like" tissue culture media supplemented with serum at mammalian body temperature. Although filamentation shares many common features in the two conditions, we have found two significant differences. First, alternative carbon metabolism genes are expressed early during in vitro filamentation and late in vivo, suggesting significant differences in glucose availability. Second, C. albicans begins a hyphae-to-yeast transition after 4-h incubation while we find little evidence of hyphae-to-yeast transition in vivo up to 24 h post-infection. We show that the low rate of in vivo hyphae-to-yeast transition is likely due to the very low expression of PES1, a key driver of lateral yeast in vitro and that heterologous expression of PES1 is sufficient to trigger lateral yeast formation in vivo.IMPORTANCECandida albicans filamentation is correlated with virulence and is an intensively studied aspect of C. albicans biology. The vast majority of studies on C. albicans filamentation are based on in vitro induction of hyphae and pseudohyphae. Here we used an in vivo filamentation assay and in vivo expression profiling to compare the tempo of morphogenesis and gene expression between in vitro and in vivo filamentation. Although the hyphal gene expression profile is induced rapidly in both conditions, it remains stably expressed over a 12-h time course in vivo while it peaks after 4 h in vitro and is reduced. This reduced hyphal gene expression in vitro correlates with reduced hyphae and increased hyphae-to-yeast transition. By contrast, there is little evidence of hyphae-to-yeast transition in vivo.

The \u03b4 subunit of F1Fo-ATP synthase is required for pathogenicity of Candida albicans

Fungal infections, especially candidiasis and aspergillosis, claim a high fatality rate. Fungal cell growth and function requires ATP, which is synthesized mainly through oxidative phosphorylation, with the key enzyme being F1Fo-ATP synthase. Here, we show that deletion of the Candida albicans gene encoding the δ subunit of the F1Fo-ATP synthase (ATP16) abrogates lethal infection in a mouse model of systemic candidiasis. The deletion does not substantially affect in vitro fungal growth or intracellular ATP concentrations, because the decrease in oxidative phosphorylation-derived ATP synthesis is compensated by enhanced glycolysis. However, the ATP16-deleted mutant displays decreased phosphofructokinase activity, leading to low fructose 1,6-bisphosphate levels, reduced activity of Ras1-dependent and -independent cAMP-PKA pathways, downregulation of virulence factors, and reduced pathogenicity. A structure-based virtual screening of small molecules leads to identification of a compound potentially targeting the δ subunit of fungal F1Fo-ATP synthases. The compound induces in vitro phenotypes similar to those observed in the ATP16-deleted mutant, and protects mice from succumbing to invasive candidiasis. Our findings indicate that F1Fo-ATP synthase δ subunit is required for C. albicans lethal infection and represents a potential therapeutic target.

Identifying Candida albicans Gene Networks Involved in Pathogenicity.

Candida albicans is a normal member of the human microbiome. It is also an opportunistic pathogen, which can cause life-threatening systemic infections in severely immunocompromized individuals. Despite the availability of antifungal drugs, mortality rates of systemic infections are high and new drugs are needed to overcome therapeutic challenges including the emergence of drug resistance. Targeting known disease pathways has been suggested as a promising avenue for the development of new antifungals. However, <30% of C. albicans genes are verified with experimental evidence of a gene product, and the full complement of genes involved in important disease processes is currently unknown. Tools to predict the function of partially or uncharacterized genes and generate testable hypotheses will, therefore, help to identify potential targets for new antifungal development. Here, we employ a network-extracted ontology to leverage publicly available transcriptomics data and identify potential candidate genes involved in disease processes. A subset of these genes has been phenotypically screened using available deletion strains and we present preliminary data that one candidate, PEP8, is involved in hyphal development and immune evasion. This work demonstrates the utility of network-extracted ontologies in predicting gene function to generate testable hypotheses that can be applied to pathogenic systems. This could represent a novel first step to identifying targets for new antifungal therapies.

Transcriptional Profiling of Candida albicans in the Host.

Gene expression profiling has become an important tool for determining gene functions, performing phenotypic analysis, and quantifying the differential expression of individual transcripts under various conditions. Candida albicans gene expression is highly responsive to environmental conditions and rapidly adapts to various niches within the host. Here, we describe a mouse model of gastrointestinal colonization with C. albicans, the measurement of colonization in fecal pellets, and the collection of samples for transcriptional profiling. We describe how to extract and purify RNA suitable for analysis via RT-qPCR or microarray.

The expression of inflammatory cytokines, TAM tyrosine kinase receptors and their ligands is upregulated in venous leg ulcer patients: a novel insight into chronic wound immunity.

The systemic host defence mechanisms, especially innate immunity, in venous leg ulcer patients are poorly investigated. The aim of the current study was to measure Candida albicans killing activity and gene expressions of pro- and anti-inflammatory cytokines and innate immune response regulators, TAM receptors and ligands of peripheral blood mononuclear cells separated from 69 venous leg ulcer patients and 42 control probands. Leg ulcer patients were stratified into responder and non-responder groups on the basis of wound healing properties. No statistical differences were found in Candida killing among controls, responders and non-responders. Circulating blood mononuclear cells of patients overexpress pro-inflammatory (IL-1α, TNFα, CXCL-8) and anti-inflammatory (IL-10) cytokines as well as TAM receptors (Tyro, Axl, MerTK) and their ligands Gas6 and Protein S compared with those of control individuals. IL-1α is notably overexpressed in venous leg ulcer treatment non-responders; in contrast, Axl gene expression is robustly stronger among responders. These markers may be considered as candidates for the prediction of treatment response among venous leg ulcer patients.

The expression of the Candida albicans gene SAP4 during hyphal formation in human serum and in adhesion to monolayer cell culture of colorectal carcinoma Caco-2 (ATCC)

Abstract Candida albicans SAP4 gene encodes secretory aspartyl protease Sap4 which is involved in hyphae formation and virulence. Transcriptional factors Cph1 and Efg1 govern the expression of several C. albicans genes and contribute to morphogenesis. We investigated the expression of SAP4 in C. albicans clinical isolate and mutants lacking Efg1 or/ and Cph1 grown in human serum and during contact with Caco-2 cell line. mRNA was analyzed with the use of RT-PCR; relative quantification was normalized against an ACT1 in cells after 18-h growth either in serum or on monolayer as well as in their counterparts in YEPD medium. We assessed the role of Sap4, Efg1 and Cph1 in adhesion of C. albicans to epithelial cells. Additionally, adherence assay was performed with sap4/sap4. Adhesion was expressed as a percent of adherent cells to monolayer at 90 min vs. total cells added (100%). No differences were observed in adhesion of efg1/efg1 and sap4/sap4 compared with SC5314 (P≥0.05 statisitically insignificant). SAP4 expression indicated that it is not involved in adapting to the tested conditions. SAP4 expression can be strainspecific and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. Neither Efg1 nor Sap4 can influence adhesion.

Profiling of Candida albicans Gene Expression During Intra-abdominal Candidiasis Identifies Biologic Processes Involved in Pathogenesis

The pathogenesis of intra-abdominal candidiasis is poorly understood. Mice were intraperitoneally infected with Candida albicans (1 × 10(6) 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. 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. 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.

Pga26 mediates filamentation and biofilm formation and is required for virulence in Candida albicans

The Candida albicans gene PGA26 encodes a small cell wall protein and is upregulated during de novo wall synthesis in protoplasts. Disruption of PGA26 caused hypersensitivity to cell wall-perturbing compounds (Calcofluor white and Congo red) and to zymolyase, which degrades the cell wall β-1,3-glucan network. However, susceptibility to caspofungin, an inhibitor of β-1,3-glucan synthesis, was decreased. In addition, pga26Δ mutants show increased susceptibility to antifungals (fluconazol, posaconazol or amphotericin B) that target the plasma membrane and have altered sensitivities to environmental (heat, osmotic and oxidative) stresses. Except for a threefold increase in β-1,6-glucan and a slightly widened outer mannoprotein layer, the cell wall composition and structure was largely unaltered. Therefore, Pga26 is important for proper cell wall integrity, but does not seem to be directly involved in the synthesis of cell wall components. Deletion of PGA26 further leads to hyperfilamentation, increased biofilm formation and reduced virulence in a mouse model of disseminated candidiasis. We propose that deletion of PGA26 may cause an imbalance in the morphological switching ability of Candida, leading to attenuated dissemination and infection.

Construction of Candida albicans Tet‐on tagging vectors with a Ura‐blaster cassette

It has been difficult to develop molecular tools for studying the fungal pathogen Candida albicans because this species uses a non-standard genetic code and is diploid without a complete sexual cycle. Vector systems with regulatable promoters to produce conditional mutants, epitope tags for protein detection and recyclable selection markers are useful for functional study of genes. However, most currently available vectors contain only a subset of desired properties, which limits their application. To combine several useful properties in one vector, the vector pTET25 was initially modified into pTET25M, so that the URA3 gene flanked by dpl200 could be used repetitively. To enable more choices for cloning, a multiple cloning site was introduced at both ends of GFP in pTET25M. GFP expression was induced by doxycycline in a dose- and time-dependent manner when the plasmid was introduced into C. albicans with or without URA3. The applicability of the vectors was verified by constructing strains capable of expressing either the N-terminal GFP fusion of Cdc10 or the C-terminal GFP fusion of Cdc11. Additionally, by replacing the GFP gene of pTET25M with DNA sequence encoding Cdc10 with an epitope tag of six histidine residues at the C-terminus, doxycycline-induced expression of CDC10 was achieved when the expression vector was introduced into C. albicans. This new system allows for inducible expression of a desired C. albicans gene with the advantage of convenience of cloning. It also allows the presence of a recyclable URA3 marker and the detectable expression of fusion or epitope-tagged protein.

Correction: Impact of Genetic Background on Allele Selection in a Highly Mutable Candida albicans Gene, PNG2

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Impact of Genetic Background on Allele Selection in a Highly Mutable Candida albicans Gene, PNG2

In many microbes rapid mutation of highly mutable contingency genes continually replenishes a pool of variant alleles from which the most suitable are selected, assisting in rapid adaptation and evasion of the immune response. In some contingency genes mutability is achieved through DNA repeats within the coding region. The fungal human pathogen Candida albicans has 2600 repeat-containing ORFs. For those investigated (ALS genes, HYR1, HYR2, CEK1, RLM1) many protein variants with differing amino acid repeat regions exist, as expected for contingency genes. However, specific alleles dominate in different clades, which is unexpected if allele variation is used for short-term adaptation. Generation of new alleles of repeat-containing C. albicans ORFs has never been observed directly. Here we present evidence for restrictions on the emergence of new alleles in a highly mutable C. albicans repeat-containing ORF, PNG2, encoding a putative secreted or cell surface glycoamidase. In laboratory cultures new PNG2 alleles arose at a rate of 2.8×10−5 (confidence interval 3.3×10−6−9. 9×10−5) per cell per division, comparable to rates measured for contingency genes. Among 80 clinical isolates 17 alleles of different length and 23 allele combinations were distinguishable; sequence differences between repeat regions of identical size suggest the existence of 36 protein variants. Specific allele combinations predominated in different genetic backgrounds, as defined by DNA fingerprinting and multilocus sequence typing. Given the PNG2 mutation rate, this is unexpected, unless in different genetic backgrounds selection favors different alleles. Specific alleles or allele combinations were not preferentially associated with C. albicans isolates from particular body sites or geographical regions. Our results suggest that the mutability of PNG2 is not used for short-term adaptation or evasion of the immune system. Nevertheless the large number of alleles observed indicates that mutability of PNG2 may assist C. albicans strains from different genetic backgrounds optimize their interaction with the host in the long term.

Glucose promotes stress resistance in the fungal pathogen Candida albicans.

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

Genome-wide analysis of Candida albicans gene expression patterns during infection of the mammalian kidney

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

Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae

One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.

Gpm1p Is a Factor H-, FHL-1-, and Plasminogen-binding Surface Protein of Candida albicans

The human pathogenic yeast Candida albicans utilizes host complement regulators for immune evasion. Here we identify the first fungal protein that binds Factor H and FHL-1. By screening a protein array of 4088 proteins of Saccharomyces cerevisiae, phosphoglycerate mutase (ScGpm1p) was identified as a Factor H- and FHL-1-binding protein. The homologous C. albicans Gpm1p (CaGpm1p) was cloned and recombinantly expressed as a 36-kDa His-tagged protein. Purified CaGpm1p binds the host complement regulators Factor H and FHL-1, but not C4BP. The CaGpm1p binding regions in the host proteins were localized; FHL-1 binds via short consensus repeats (SCRs) 6 and 7, and Factor H utilizes two contact regions that are located in SCRs 6 and 7 and in SCRs 19 and 20. In addition, recombinant CaGpm1p binds plasminogen via lysine residues. CaGpm1p is a surface protein as demonstrated by immunostaining and flow cytometry. A C. albicans gpm1(-/-) mutant strain was generated that did not grow on glucose-supplemented but on ethanol- and glycerol-supplemented medium. Reduced binding of Factor H and plasminogen to the null mutant strain is in agreement with the presence of additional binding proteins. Attached to CaGpm1p, each of the three host plasma proteins is functionally active. Factor H and FHL-1 show cofactor activity for cleavage of C3b, and bound plasminogen is converted by urokinase-type plasminogen activator to proteolytically active plasmin. Thus, the surface-expressed CaGpm1p is a virulence factor that utilizes the host Factor H, FHL-1, and plasminogen for immune evasion and degradation of extracellular matrices.

Temporal analysis of Candida albicans gene expression during biofilm development

Microarrays were used to identify changes in gene expression associated with Candida albicans biofilm development. Two biofilm substrates (denture and catheter), and two C. albicans strains for each substrate, were tested to remove model- and strain-dependent variability from the overall dataset. Three biofilm developmental phases were examined: early (6 h), intermediate (12 h), and mature (48 h). Planktonic specimens were collected at the same time points. Data analysis focused primarily on gene expression changes over the time-course of biofilm development. Glycolytic and non-glycolytic carbohydrate assimilation, amino acid metabolism, and intracellular transport mechanisms were important during the early phase of biofilm formation. These early events increase intracellular pools of pyruvate, pentoses and amino acids, which prepare the biofilm for the large biomass increase that begins around 12 h of development. This developmental stage demands energy and utilizes specific transporters for amino acids, sugars, ions, oligopeptides and lactate/pyruvate. At mature phase (48 h), few genes were differentially expressed compared with the 12 h time point, suggesting a relative lack of initiation of new metabolic activity. Data analysis to assess biofilm model-specific gene expression showed more dynamic changes in the denture model than in the catheter model. Data analysis to identify gene expression changes that are associated with each strain/substrate combination identified the same types of genes that were identified in the analysis of the entire dataset. Collectively, these data suggest that genes belonging to different, but interconnected, functional categories regulate the morphology and phenotype of C. albicans biofilm.

TheCandida albicansgeneHGT12(orf19.7094) encodes a hexose transporter

Yeast cells of the human pathogen Candida albicans that enter the bloodstream can be engulfed by macrophage cells but survive in, and can escape from, the phagolysosome. The C. albicans gene HGT12, which is specifically expressed during macrophage infection, encodes a protein that transports fructose, glucose and mannose. Expression of this hexose transporter along with the shift from glycolysis to gluconeogenesis that occurs in these phagocytosed cells suggests a requirement for glucose that can be supplied in part by uptake from the lumen of the phagolysosome.

Integrated Proteomics and Genomics Strategies Bring New Insight into Candida albicans Response upon Macrophage Interaction

The interaction of Candida albicans with macrophages is considered a crucial step in the development of an adequate immune response in systemic candidiasis. An in vitro model of phagocytosis that includes a differential staining procedure to discriminate between internalized and non-internalized yeast was developed. Upon optimization of a protocol to obtain an enriched population of ingested yeasts, a thorough genomics and proteomics analysis was carried out on these cells. Both proteins and mRNA were obtained from the same sample and analyzed in parallel. The combination of two-dimensional PAGE with MS revealed a total of 132 differentially expressed yeast protein species upon macrophage interaction. Among these species, 67 unique proteins were identified. This is the first time that a proteomics approach has been used to study C. albicans-macrophage interaction. We provide evidence of a rapid protein response of the fungus to adapt to the new environment inside the phagosome by changing the expression of proteins belonging to different pathways. The clear down-regulation of the carbon-compound metabolism, plus the up-regulation of lipid, fatty acid, glyoxylate, and tricarboxylic acid cycles, indicates that yeast shifts to a starvation mode. There is an important activation of the degradation and detoxification protein machinery. The complementary genomics approach led to the detection of specific pathways related to the virulence of Candida. Network analyses allowed us to generate a hypothetical model of Candida cell death after macrophage interaction, highlighting the interconnection between actin cytoskeleton, mitochondria, and autophagy in the regulation of apoptosis. In conclusion, the combination of genomics, proteomics, and network analyses is a powerful strategy to better understand the complex host-pathogen interactions.

Autophagy in the pathogen Candida albicans

Autophagy is a major cellular process that facilitates the bulk degradation of eukaryotic macromolecules and organelles, through degradation within the lysosomal/vacuole compartment. This has been demonstrated to influence a diverse array of eukaryotic cell functions including adaptation, differentiation and developmental programmes. For example, in Saccharomyces cerevisiae autophagy is required for sporulation and survival of nitrogen starvation. The opportunistic pathogen Candida albicans has the ability to colonize and cause disease within a diverse range of mammalian host sites. The ability to adapt and differentiate within the host is liable to be critical for host colonization and infection. Previous results indicated that the vacuole plays an important role in C. albicans adaptation to stress, differentiation, and survival within and injury of host cells. In this study the importance of vacuole-mediated degradation through the process of autophagy was investigated. This involved identification and deletion of ATG9, a C. albicans gene required for autophagy. The deletion strain was blocked in autophagy and the closely related cytoplasm to vacuole (cvt) trafficking pathway. This resulted in sensitivity to nitrogen starvation, but no defects in growth rate, vacuole morphology or resistance to other stresses. This indicates that the mutant has specific defects in autophagy/cvt trafficking. Given the importance of autophagy in the development and differentiation of other eukaryotes, it was surprising to find that the atg9Delta mutant was unaffected in either yeast-hypha or chlamydospore differentiation. Furthermore, the atg9Delta mutant survived within and killed a mouse macrophage-like cell line as efficiently as control strains. The data suggest that autophagy plays little or no role in C. albicans differentiation or during interaction with host cells.

Biofilm formation byCandida albicansmutants for genes coding fungal proteins exhibiting the eight-cysteine-containing CFEM domain

Several features and functions of a Candida albicans gene, PGA10 (also designated as RBT51), coding for a putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as CFEM (Common in several Fungal Extracellular Membrane proteins), are described. The ORF of the gene (ORF19.5674) encoded a protein of 250 amino acids, with a predicted molecular mass of 25.17 kDa. The product of the PGA10 gene also exhibited some features reminiscent of a class II-type hydrophobin. Deletion of PGA10 resulted in a cascade of pleiotropic effects, mostly affecting cell-surface-related properties. Thus, the null pga10Delta mutant displayed an increased sensitivity to cell-wall-perturbing agents and formed fragile biofilms that appeared partially split and weakly attached to the substratum. The biofilm-forming ability of several C. albicans mutants with single, double and triple deletions of genes encoding other protein species also containing the CFEM domain (RBT5 and WAP1/CSA1) was determined. These mutants also exhibited an abnormal ability to form biofilms. Overall, the evidence presented here suggests that fungal proteins containing the CFEM domain (Pga10p/Rbt51p, Rbt5p and Wap1p/Csa1p) may play a key role in the formation, development and/or maintenance of the biofilm structure in C. albicans.