Poacic acid, a ß-1,3-glucan-binding antifungal agent, inhibits cell-wall remodeling and activates transcriptional responses regulated by the cell-wall integrity and high-osmolarity glycerol pathways in yeast.

As a result of the relatively few available antifungals and the increasing frequency of resistance to them, the development of novel antifungals is increasingly important. The plant natural product poacic acid (PA) inhibits ß-1,3-glucan synthesis in Saccharomyces cerevisiae and has antifungal activity against a wide range of plant pathogens. However, the mode of action of PA is unclear. Here, we reveal that PA specifically binds to ß-1,3-glucan, its affinity for which is ~30-fold that for chitin. Besides its effect on ß-1,3-glucan synthase activity, PA inhibited the yeast glucan-elongating activity of Gas1 and Gas2 and the chitin-glucan transglycosylase activity of Crh1. Regarding the cellular response to PA, transcriptional co-regulation was mediated by parallel activation of the cell-wall integrity (CWI) and high-osmolarity glycerol signaling pathways. Despite targeting ß-1,3-glucan remodeling, the transcriptional profiles and regulatory circuits activated by caspofungin, zymolyase, and PA differed, indicating that their effects on CWI have different mechanisms. The effects of PA on the growth of yeast strains indicated that it has a mode of action distinct from that of echinocandins, suggesting it is a unique antifungal agent.

Spanish microbiology in an era of constant advances: a view from the battleground.

It has been 75 years since a group of pioneers founded the Spanish Society of Microbiology (SEM). Among them were, in addition to university professors and research scientists, microbiology practitioners from clinical laboratories, public health, and industry. This account describes the trajectory of the SEM, as a focal point of cooperation for the development of Microbiology in Spain during the more than seven decades of its existence. The efforts of its members resulted in everything from promoting a scientific environment to leadership of some of the activities of the Federation of European Microbiological Societies (FEMS). My interest was to frame the development of Microbiology in Spain in the context of the continuous advances of microbial studies in the world. Indeed, since the 1940s, description of and experimentation with microbial species has provided a deep understanding. Furthermore, it has furnished model systems used to establish basic concepts in Life Sciences, as well as the most appropriate technological instruments for the advancement of Omics, Systems Biology, and Synthetic Biology. Those who 75 years ago dreamed of the progress of our discipline in Spain perhaps did not imagine that the Spanish microbiologist Francisco Mojica would contribute to essential knowledge, making the acronym "CRISPR" the most used across the world of Life Sciences and Biomedicine.

Rlm1 mediates positive autoregulatory transcriptional feedback that is essential for Slt2-dependent gene expression.

Activation of the yeast cell wall integrity (CWI) pathway induces an adaptive transcriptional programme that is largely dependent on the transcription factor Rlm1 and the mitogen-activated protein kinase (MAPK) Slt2. Upon cell wall stress, the transcription factor Rlm1 is recruited to the promoters of RLM1 and SLT2, and exerts positive-feedback mechanisms on the expression of both genes. Activation of the MAPK Slt2 by cell wall stress is not impaired in strains with individual blockade of any of the two feedback pathways. Abrogation of the autoregulatory feedback mechanism on RLM1 severely affects the transcriptional response elicited by activation of the CWI pathway. In contrast, a positive trans-acting feedback mechanism exerted by Rlm1 on SLT2 also regulates CWI output responses but to a lesser extent. Therefore, a complete CWI transcriptional response requires not only phosphorylation of Rlm1 by Slt2 but also concurrent SLT2- and RLM1-mediated positive-feedback mechanisms; sustained patterns of gene expression are mainly achieved by positive autoregulatory circuits based on the transcriptional activation of Rlm1.

Cooperation between SAGA and SWI/SNF complexes is required for efficient transcriptional responses regulated by the yeast MAPK Slt2.

The transcriptional response of Saccharomyces cerevisiae to cell wall stress is mainly mediated by the cell wall integrity (CWI) pathway through the MAPK Slt2 and the transcription factor Rlm1. Once activated, Rlm1 interacts with the chromatin remodeling SWI/SNF complex which locally alters nucleosome positioning at the target promoters. Here we show that the SAGA complex plays along with the SWI/SNF complex an important role for eliciting both early induction and sustained gene expression upon stress. Gcn5 co-regulates together with Swi3 the majority of the CWI transcriptional program, except for a group of genes which are only dependent on the SWI/SNF complex. SAGA subunits are recruited to the promoter of CWI-responsive genes in a Slt2, Rlm1 and SWI/SNF-dependent manner. However, Gcn5 mediates acetylation and nucleosome eviction only at the promoters of the SAGA-dependent genes. This process is not essential for pre-initiation transcriptional complex assembly but rather increase the extent of the remodeling mediated by SWI/SNF. As a consequence, H3 eviction and Rlm1 recruitment is completely blocked in a swi3Δ gcn5Δ double mutant. Therefore, SAGA complex, through its histone acetylase activity, cooperates with the SWI/SNF complex for the mandatory nucleosome displacement required for full gene expression through the CWI pathway.

Proteomics unravels extracellular vesicles as carriers of classical cytoplasmic proteins in Candida albicans.

The commensal fungus Candida albicans secretes a considerable number of proteins and, as in different fungal pathogens, extracellular vesicles (EVs) have also been observed. Our report contains the first proteomic analysis of EVs in C. albicans and a comparative proteomic study of the soluble secreted proteins. With this purpose, cell-free culture supernatants from C. albicans were separated into EVs and EV-free supernatant and analyzed by LC-MS/MS. A total of 96 proteins were identified including 75 and 61 proteins in EVs and EV-free supernatant, respectively. Out of these, 40 proteins were found in secretome by proteomic analysis for the first time. The soluble proteins were enriched in cell wall and secreted pathogenesis related proteins. Interestingly, more than 90% of these EV-free supernatant proteins were classical secretory proteins with predicted N-terminal signal peptide, whereas all the leaderless proteins involved in metabolism, including some moonlighting proteins, or in the exocytosis and endocytosis process were exclusively cargo of the EVs. We propose a model of the different mechanisms used by C. albicans secreted proteins to reach the extracellular medium. Furthermore, we tested the potential of the Bgl2 protein, identified in vesicles and EV-free supernatant, to protect against a systemic candidiasis in a murine model.

Genomic profiling of fungal cell wall-interfering compounds: identification of a common gene signature.

BACKGROUND: The fungal cell wall forms a compact network whose integrity is essential for cell morphology and viability. Thus, fungal cells have evolved mechanisms to elicit adequate adaptive responses when cell wall integrity (CWI) is compromised. Functional genomic approaches provide a unique opportunity to globally characterize these adaptive mechanisms. To provide a global perspective on these CWI regulatory mechanisms, we developed chemical-genomic profiling of haploid mutant budding yeast cells to systematically identify in parallel those genes required to cope with stresses interfering the cell wall by different modes of action: ß-1,3 glucanase and chitinase activities (zymolyase), inhibition of ß-1,3 glucan synthase (caspofungin) and binding to chitin (Congo red). RESULTS: Measurement of the relative fitness of the whole collection of 4786 haploid budding yeast knock-out mutants identified 222 mutants hypersensitive to caspofungin, 154 mutants hypersensitive to zymolyase, and 446 mutants hypersensitive to Congo red. Functional profiling uncovered both common and specific requirements to cope with different cell wall damages. We identified a cluster of 43 genes highly important for the integrity of the cell wall as the common "signature of cell wall maintenance (CWM)". This cluster was enriched in genes related to vesicular trafficking and transport, cell wall remodeling and morphogenesis, transcription and chromatin remodeling, signal transduction and RNA metabolism. Although the CWI pathway is the main MAPK pathway regulating cell wall integrity, the collaboration with other signal transduction pathways like the HOG pathway and the invasive growth pathway is also required to cope with the cell wall damage depending on the nature of the stress. Finally, 25 mutant strains showed enhanced caspofungin resistance, including 13 that had not been previously identified. Only three of them, wsc1Δ, elo2Δ and elo3Δ, showed a significant decrease in ß-1,3-glucan synthase activity. CONCLUSIONS: This work provides a global perspective about the mechanisms involved in cell wall stress adaptive responses and the cellular functions required for cell wall integrity. The results may be useful to uncover new potential antifungal targets and develop efficient antifungal strategies by combination of two drugs, one targeting the cell wall and the other interfering with the adaptive mechanisms.

Serum antibody signature directed against Candida albicans Hsp90 and enolase detects invasive candidiasis in non-neutropenic patients.

Invasive candidiasis (IC) adds significantly to the morbidity and mortality of non-neutropenic patients if not diagnosed and treated early. To uncover serologic biomarkers that alone or in combination could reliably detect IC in this population, IgG antibody-reactivity profiles to the Candida albicans intracellular proteome were examined by serological proteome analysis (SERPA) and data mining procedures in a training set of 24 non-neutropenic patients. Despite the high interindividual molecular heterogeneity, unsupervised clustering analyses revealed that serum 22-IgG antibody-reactivity patterns differentiated IC from non-IC patients. Univariate analyses further highlighted that 15 out of the 22 SERPA-identified IgG antibodies could be useful candidate IC biomarkers. The diagnostic performance of one of these candidates (anti-Hsp90 IgG antibodies) was validated using an ELISA prototype in a test set of 59 non-neutropenic patients. We then formulated an IC discriminator based on the combined immunoproteomic fingerprints of this and another SERPA-detected and previously validated IC biomarker (anti-Eno1 IgG antibodies) in the training set. Its consistency was substantiated using their ELISA prototypes in the test set. Receiver-operating-characteristic curve analyses showed that this two-biomarker signature accurately identified IC in non-neutropenic patients and provided better IC diagnostic accuracy than the individual biomarkers alone. We conclude that this serum IgG antibody signature directed against C. albicans Hsp90 and Eno1, if confirmed prospectively, may be useful for IC diagnosis in non-neutropenic patients.

Prediction of the clinical outcome in invasive candidiasis patients based on molecular fingerprints of five anti-Candida antibodies in serum.

Better prognostic predictors for invasive candidiasis (IC) are needed to tailor and individualize therapeutic decision-making and minimize its high morbidity and mortality. We investigated whether molecular profiling of IgG-antibody response to the whole soluble Candida proteome could reveal a prognostic signature that may serve to devise a clinical-outcome prediction model for IC and contribute to known IC prognostic factors. By serological proteome analysis and data-mining procedures, serum 31-IgG antibody-reactivity patterns were examined in 45 IC patients randomly split into training and test sets. Within the training cohort, unsupervised two-way hierarchical clustering and principal-component analyses segregated IC patients into two antibody-reactivity subgroups with distinct prognoses that were unbiased by traditional IC prognostic factors and other patients-related variables. Supervised discriminant analysis with leave-one-out cross-validation identified a five-IgG antibody-reactivity signature as the most simplified and accurate IC clinical-outcome predictor, from which an IC prognosis score (ICPS) was derived. Its robustness was confirmed in the test set. Multivariate logistic-regression and receiver-operating-characteristic curve analyses demonstrated that the ICPS was able to accurately discriminate IC patients at high risk for death from those at low risk and outperformed conventional IC prognostic factors. Further validation of the five-IgG antibody-reactivity signature on a multiplexed immunoassay supported the serological proteome analysis results. The five IgG antibodies incorporated in the ICPS made biologic sense and were associated either with good-prognosis and protective patterns (those to Met6p, Hsp90p, and Pgk1p, putative Candida virulence factors and antiapoptotic mediators) or with poor-prognosis and risk patterns (those to Ssb1p and Gap1p/Tdh3p, potential Candida proapoptotic mediators). We conclude that the ICPS, with additional refinement in future larger prospective cohorts, could be applicable to reliably predict patient clinical-outcome for individualized therapy of IC. Our data further provide insights into molecular mechanisms that may influence clinical outcome in IC and uncover potential targets for vaccine design and immunotherapy against IC.

Cell surface shaving of Candida albicans biofilms, hyphae, and yeast form cells.

We used a brief trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS to identify the surface proteins on live Candida albicans organisms growing in biofilms and planktonic yeast cells and hyphae. One hundred thirty-one proteins were present in at least two of the three replicates of one condition and distributed in various combinations of the three growth conditions. Both previously reported and new surface proteins were identified and these were distributed between covalently attached proteins and noncovalently attached proteins of the cell wall.

Systematic Identification of Essential Genes Required for Yeast Cell Wall Integrity: Involvement of the RSC Remodelling Complex.

Conditions altering the yeast cell wall lead to the activation of an adaptive transcriptional response mainly governed by the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway. Two high-throughput screenings were developed using the yTHC collection of yeast conditional mutant strains to systematically identify essential genes related to cell wall integrity, and those required for the transcriptional program elicited by cell wall stress. Depleted expression of 52 essential genes resulted in hypersensitivity to the dye Calcofluor white, with chromatin organization, Golgi vesicle transport, rRNA processing, and protein glycosylation processes, as the most highly representative functional groups. Via a flow cytometry-based quantitative assay using a CWI reporter plasmid, 97 strains exhibiting reduced gene-reporter expression levels upon stress were uncovered, highlighting genes associated with RNA metabolism, transcription/translation, protein degradation, and chromatin organization. This screening also led to the discovery of 41 strains displaying a basal increase in CWI-associated gene expression, including mainly putative cell wall-related genes. Interestingly, several members of the RSC chromatin remodelling complex were uncovered in both screenings. Notably, Rsc9 was necessary to regulate the gene expression of CWI-related genes both under stress and non-stress conditions, suggesting distinct requirements of the RSC complex for remodelling particular genes.

Quantitative proteome and acidic subproteome profiling of Candida albicans yeast-to-hypha transition.

Candida albicans yeast-to-hypha morphological transition is involved in the virulence strategy of this opportunistic fungal pathogen. Changes in relative abundance of the Candida proteome related to this process were analyzed using different two-dimensional differential in-gel electrophoresis (2D-DIGE)-based approaches. First, a comparative analysis of yeast and hyphal cytoplasmic proteins allowed the detection of 106 protein spots with significant variation in abundance. Sixty-one of them, corresponding to 46 proteins, were identified. As most of the differentially abundant proteins had an acidic isoelectric point, a large-scale prefractionation approach to analyze the acidic C. albicans subproteome was carried out. Ninety acidic C. albicans proteins were identified by either gel-based or nongel-based approaches. Additionally, different workflows combining preparative isoelectric focusing, Cy labeling, and narrow pH gradient 2-DE gels were tested to analyze the differences in relative protein abundance between yeast and hyphal acidic subproteomes. It was possible to identify 21 differentially abundant acidic proteins; 10 of them were not identified in the previous 2D-DIGE gels. Functional and network interaction analyses of the 56 differentially abundant proteins identified by both approaches rendered an integrated view of metabolic and cellular process reorganization during the yeast-to-hypha transition. With these results, we propose a model of metabolic reorganization.

Genome-wide survey of yeast mutations leading to activation of the yeast cell integrity MAPK pathway: novel insights into diverse MAPK outcomes.

BACKGROUND: The yeast cell wall integrity mitogen-activated protein kinase (CWI-MAPK) pathway is the main regulator of adaptation responses to cell wall stress in yeast. Here, we adopt a genomic approach to shed light on two aspects that are only partially understood, namely, the characterization of the gene functional catalog associated with CWI pathway activation and the extent to which MAPK activation correlates with transcriptional outcomes. RESULTS: A systematic yeast mutant deletion library was screened for constitutive transcriptional activation of the CWI-related reporter gene MLP1. Monitoring phospho-Slt2/Mpk1 levels in the identified mutants revealed sixty-four deletants with high levels of phosphorylation of this MAPK, including mainly genes related to cell wall construction and morphogenesis, signaling, and those with unknown function. Phenotypic analysis of the last group of mutants suggests their involvement in cell wall homeostasis. A good correlation between levels of Slt2 phosphorylation and the magnitude of the transcriptional response was found in most cases. However, the expression of CWI pathway-related genes was enhanced in some mutants in the absence of significant Slt2 phosphorylation, despite the fact that functional MAPK signaling through the pathway was required. CWI pathway activation was associated to increased deposition of chitin in the cell wall - a known survival compensatory mechanism - in about 30% of the mutants identified. CONCLUSION: We provide new insights into yeast genes related to the CWI pathway and into how the state of activation of the Slt2 MAPK leads to different outcomes, discovering the versatility of this kind of signaling pathways. These findings potentially have broad implications for understanding the functioning of other eukaryotic MAPKs.

The Sko1 protein represses the yeast-to-hypha transition and regulates the oxidative stress response in Candida albicans.

Cells respond to environmental changes triggering adaptive responses which are, in part, mediated by a transcriptional response. These responses are complex and are dependent on different transcription factors. The present work reports the implication of the Sko1 protein in several processes relevant to the physiology of Candida albicans. First, Sko1 acts as transcriptional repressor of genes involved in pathogenesis and hyphal formation, which results in increased expression of the hyphal related genes ECE1 and HWP1 without significant changes in the virulence using a mouse model of systemic infection. Second Sko1 is involved in the response to oxidative stress and sko1 mutants increase the sensitivity of hog1 to the myelomonocytic cell line HL-60. Genome-wide transcriptional analysis after hydrogen peroxide treatment revealed that sko1 mutants were able to generate an adaptive response similar to wild type strains, although important differences were detected in the magnitude of the transcriptional response. Collectively, these results implicate Sko1 as an important mediator of the oxidative stress response in C. albicans.

Fluconazole at subinhibitory concentrations induces the oxidative- and nitrosative-responsive genes TRR1, GRE2 and YHB1, and enhances the resistance of Candida albicans to phagocytes.

OBJECTIVES: To analyse the oxidative and nitrosative stress response in Candida albicans generated by fluconazole at subinhibitory concentrations, and the functional consequences of such a response for the interaction with phagocytic cells. METHODS: The C. albicans CAI-4 strain carrying transcriptional fusions of the TRR1p, YHB1p and GRE2p genes to the Renilla reniformis luciferase LUC gene was pre-treated with subinhibitory concentrations of fluconazole and incubated with oxidants (diamide and hydrogen peroxide) or with the myelomonocytic cell line HL-60. RESULTS: Fluconazole induced oxidative and nitrosative stress in a time- and dose-dependent manner as determined using oxidative- and nitrosative-specific gene reporters. At subinhibitory concentrations, fluconazole was able to induce protection in vitro to subsequent challenges with oxidants in both liquid and solid media, and also induced partial protection against the oxidative-mediated killing mechanisms of the myelocytic HL-60 cells. CONCLUSIONS: Subinhibitory concentrations of fluconazole protect against oxidants and killing mediated by phagocytes.

The high-osmolarity glycerol (HOG) and cell wall integrity (CWI) signalling pathways interplay: a yeast dialogue between MAPK routes.

Two mitogen-activated protein kinase (MAPK) pathways, viz. the high-osmolarity glycerol (HOG) and the cell wall integrity (CWI) pathways, regulate stress responses in the yeast Saccharomyces cerevisiae. Whereas the former is mainly involved in adaptation of yeast cells to hyperosmotic stress, the latter is activated under conditions leading to cell wall instability. Although MAPK signalling specificity can be conceived as requiring insulation of the different pathways, it is also becoming clear that the two pathways do not compete with each other but can be positively coordinated to regulate many stress responses. This review highlights our current knowledge about the collaboration between these two MAPK pathways to counteract different kinds of environmental stress.

Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes.

The intracellular trafficking/survival strategies of the opportunistic human pathogen Candida albicans are poorly understood. Here we investigated the infection of RAW264.7 macrophages with a virulent wild-type (WT) filamentous C. albicans strain and a hyphal signalling-defective mutant (efg1Delta/cph1Delta). A comparative analysis of the acquisition by phagosomes of actin, and of early/late endocytic organelles markers of the different fungal strains was performed and related to Candida's survival inside macrophages. Our results show that both fungal strains have evolved a similar mechanism to subvert the 'lysosomal' system, as seen by the inhibition of the phagosome fusion with compartments enriched in the lysobisphosphatidic acid and the vATPase, and thereby the acquisition of a low pH from the outset of infection. Besides, the virulent WT strain displayed additional specific survival strategies to prevent its targeting to compartmentsdisplaying late endosomal/lysosomal features, such as induction of active recycling out of phagosomes of the lysosomal membrane protein LAMP-1, the lysosomal protease cathepsin D and preinternalized colloidal gold. Finally, both virulent and efg1Delta/cph1Delta mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO.

Proteomics of RAW 264.7 macrophages upon interaction with heat-inactivated Candida albicans cells unravel an anti-inflammatory response.

Murine macrophages (RAW 264.7) were allowed to interact with heat-inactivated cells of Candida albicans SC5314 during 45 min. The proteomic response of the macrophages was then analyzed using 2-D gel electrophoresis. Many proteins having differential expression with respect to control macrophages were identified, and their functions were related to important processes, such as cytoskeletal organization, signal transduction, metabolism, protein biosynthesis, stress response and protein fate. Several of these proteins have been described as being involved in the process of inflammation, such as Erp29, Hspa9a, AnxaI, Ran GTPase, P4hb, Clic1 and Psma1. The analysis of the consequences of their variation unravels an overall anti-inflammatory response of macrophages during the interaction with heat-inactivated cells. This result was corroborated by the measurement of TNF-alpha and of ERK1/2 phosphorylation levels. This anti-inflammatory effect was contrary to the one observed with live C. albicans cells, which induced higher TNF-alpha secretion and higher ERK1/2 phosphorylation levels with respect to control macrophages.

Proteomic analysis of cytoplasmic and surface proteins from yeast cells, hyphae, and biofilms of Candida albicans.

Candida albicans is a human commensal and opportunistic pathogen that participates in biofilm formation on host surfaces and on medical devices. We used DIGE analysis to assess the cytoplasmic and non-covalently attached cell-surface proteins in biofilm formed on polymethylmethacrylate and planktonic yeast cells and hyphae. Of the 1490 proteins spots from cytoplasmic and 580 protein spots from the surface extracts analyzed, 265 and 108 were differentially abundant respectively (>or=1.5-fold, p <0.05). Differences of both greater and lesser abundance were found between biofilms and both planktonic conditions as well as between yeast cells and hyphae. The identity of 114 cytoplasmic and 80 surface protein spots determined represented 73 and 25 unique proteins, respectively. Analyses showed that yeast cells differed most in cytoplasmic profiling while biofilms differed most in surface profiling. Several processes and functions were significantly affected by the differentially abundant cytoplasmic proteins. Particularly noted were many of the enzymes of respiratory and fermentative pentose and glucose metabolism, folate interconversions and proteins associated with oxidative and stress response functions, host response, and multi-organism interaction. The differential abundance of cytoplasmic and surface proteins demonstrated that sessile and planktonic organisms have a unique profile.

Proteopathogen, a protein database for studying Candida albicans--host interaction.

There exist, at present, public web repositories for management and storage of proteomic data and also fungi-specific databases. None of them, however, is focused to the specific research area of fungal pathogens and their interactions with the host, and contains proteomics experimental data. In this context, we present Proteopathogen, a database intended to compile proteomics experimental data and to facilitate storage and access to a range of data which spans proteomics workflows from description of the experimental approaches leading to sample preparation to MS settings and peptides supporting protein identification. Proteopathogen is currently focused on Candida albicans and its interaction with macrophages; however, data from experiments concerning different pathogenic fungi species and other mammalian cells may also be found suitable for inclusion into the database. Proteopathogen is publicly available at http://proteopathogen.dacya.ucm.es.

Analysis of Candida albicans plasma membrane proteome.

The opportunistic human fungal pathogen Candida albicans causes a wide variety of infections including deep systemic syndromes. The C. albicans plasma membrane is an important interface in the host-pathogen relationship. The plasma membrane proteins mediate a variety of functions, including sensing and signalling to the external environment, in which the glycosylphosphatidylinositol (GPI)-anchored membrane proteins play a crucial role. A subproteomic approach to obtain a global picture of the protein composition of the C. albicans plasma membrane was developed, and different strategies were tested in order to extract the largest number of yeast plasma membrane proteins and GPI-anchored membrane proteins. These methods involved: (i) protoplast generation, (ii) mechanical disruption, (iii) ultracentrifugation in sucrose gradients, and (iv) Na(2)CO(3) treatments. To isolate GPI-anchored proteins two additional steps were performed: two-phase separation and phosphatidylinositol-phospholipase C treatment. After LC-MS/MS analysis using both a MALDI-TOF/TOF and a linear ion trap quadrupole, a total of 214 membrane proteins were identified, including 41 already described as plasma membrane proteins, 20 plasma membrane associated proteins, and 22 proteins with unknown membrane localisation. Bioinformatic analysis revealed that this set of C. albicans membrane proteins is highly enriched in proteins involved in biopolymer biosynthesis or transport processes. Furthermore, after phosphatidylinositol-phospholipase C treatment, 12 GPI-anchored membrane proteins were released and identified; most of them are associated with cell wall beta-glucan synthesis and maintenance or are virulence factors, such as phospholipases or aspartyl proteinases.