Outer chain N-glycans are required for cell wall integrity and virulence of Candida albicans

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated mannoproteins that are the immediate point of contact with the host and strongly influence the host-fungal interaction. N-Glycans are the major form of mannoprotein modification and consist of a core structure, common to all eukaryotes, that is further elaborated in the Golgi to form the highly branched outer chain that is characteristic of fungi. In yeasts, outer chain branching is initiated by the action of the alpha1,6-mannosyltransferase Och1p; therefore, we disrupted the C. albicans OCH1 homolog to determine the importance of outer chain N-glycans on the host-fungal interaction. Loss of CaOCH1 resulted in a temperature-sensitive growth defect and cellular aggregation. Outer chain elongation of N-glycans was absent in the null mutant, demonstrated by the lack of the alpha1,6-linked polymannose backbone and the underglycosylation of N-acetylglucosaminidase. A null mutant lacking OCH1 was hypersensitive to a range of cell wall perturbing agents and had a constitutively activated cell wall integrity pathway. These mutants had near normal growth rates in vitro but were attenuated in virulence in a murine model of systemic infection. However, tissue burdens for the Caoch1delta null mutant were similar to control strains with normal N-glycosylation, suggesting the host-fungal interaction was altered such that high burdens were tolerated. This demonstrates the importance of N-glycan outer chain epitopes to the host-fungal interaction and virulence.     

PEPped up: induction of Candida albicans virulence by bacterial cell wall fragments

In this issue, Xu et al. (2008) report that Candida albicans, a prevalent fungal pathogen, expresses virulence-related attributes in response to bacterial peptidoglycan components, muramyl dipeptides, in human serum. Conserved elements in human and fungal detection of muramyl dipeptides highlight a common ability to perceive proximal bacterial populations.     

Phosphatidylserine synthase and phosphatidylserine decarboxylase are essential for cell wall integrity and virulence in Candida albicans

Phospholipid biosynthetic pathways play crucial roles in the virulence of several pathogens; however, little is known about how phospholipid synthesis affects pathogenesis in fungi such as Candida albicans. A C. albicans phosphatidylserine (PS) synthase mutant, cho1Δ/Δ, lacks PS, has decreased phosphatidylethanolamine (PE), and is avirulent in a mouse model of systemic candidiasis. The cho1Δ/Δ mutant exhibits defects in cell wall integrity, mitochondrial function, filamentous growth, and is auxotrophic for ethanolamine. PS is a precursor for de novo PE biosynthesis. A psd1Δ/Δ psd2Δ/Δ double mutant, which lacks the PS decarboxylase enzymes that convert PS to PE in the de novo pathway, has diminished PE levels like those of the cho1Δ/Δ mutant. The psd1Δ/Δ psd2Δ/Δ mutant exhibits phenotypes similar to those of the cho1Δ/Δ mutant; however, it is slightly more virulent and has less of a cell wall defect. The virulence losses exhibited by the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutants appear to be related to their cell wall defects which are due to loss of de novo PE biosynthesis, but are exacerbated by loss of PS itself. Cho1p is conserved in fungi, but not mammals, so fungal PS synthase is a potential novel antifungal drug target.     

Some biological features of Candida albicans mutants for genes coding fungal proteins containing the CFEM domain

Several biological features of Candida albicans genes (PGA10, RBT5 and CSA1) coding for putative polypeptide species belonging to a subset of fungal proteins containing an eight-cysteine domain referred as common in several fungal extracellular membrane (CFEM) are described. The deletion of these genes resulted in a cascade of pleiotropic effects. Thus, mutant strains exhibited higher cell surface hydrophobicity levels and an increased ability to bind to inert or biological substrates. Confocal scanning laser microscopy using concanavalin A-Alexafluor 488 (which binds to mannose and glucose residues) and FUN-1 (a cytoplasmic fluorescent probe for cell viability) dyes showed that mutant strains formed thinner and more fragile biofilms. These apparently contained lower quantities of extracellular matrix material and less metabolically active cells than their parental strain counterpart, although the relative percentage of mycelial forms was similar in all cases. The cell surface of C. albicans strains harbouring deletions for genes coding CFEM-domain proteins appeared to be severely altered according to atomic force microscopy observations. Assessment of the relative gene expression within individual C. albicans cells revealed that CFEM-coding genes were upregulated in mycelium, although these genes were shown not to affect virulence in animal models. Overall, this study has demonstrated that CFEM domain protein-encoding genes are pleiotropic, influencing cell surface characteristics and biofilm formation.     

Biofilm formation by Candida albicans mutants 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.     

Molecular organization of the cell wall of Candida albicans and its relation to pathogenicity

Candida albicans is one of the most important opportunistic pathogenic fungi. Weakening of the defense mechanisms of the host, and the ability of the microorganism to adapt to the environment prevailing in the host tissues, turn the fungus from a rather harmless saprophyte into an aggressive pathogen. The disease, candidiasis, ranges from light superficial infections to deep processes that endanger the life of the patient. In the establishment of the pathogenic process, the cell wall of C. albicans (as in other pathogenic fungi) plays an important role. It is the outer structure that protects the fungus from the host defense mechanisms and initiates the direct contact with the host cells by adhering to their surface. The wall also contains important antigens and other compounds that affect the homeostatic equilibrium of the host in favor of the parasite. In this review, we discuss our present knowledge of the structure of the cell wall of C. albicans, the synthesis of its different components, and the mechanisms involved in their organization to give rise to a coherent composite. Furthermore, special emphasis has been placed on two further aspects: how the composition and structure of C. albicans cell wall compare with those from other fungi, and establishing the role of some specific wall components in pathogenesis. From the data presented here, it becomes clear that the composition, structure and synthesis of the cell wall of C. albicans display both subtle and important differences with the wall of different saprophytic fungi, and that some of these differences are of utmost importance for its pathogenic behavior.     

Proteomic analysis of Candida albicans yeast and hyphal cell wall and associated proteins

Candida albicans is an important human pathogen that causes systemic infections, predominantly among populations with weakened immune systems. The morphological transition from the yeast to the hyphal state is one of the key factors in C. albicans pathogenesis. Owing to their location at the host-pathogen interface, the cell wall and associated proteins are of interest, especially with respect to the yeast to hyphal transition. This study entailed the proteomic analysis of differentially regulated proteins involved in this transition. The protein profiles of C. albicans DTT/SDS-extractible proteins and the cyanogen bromide (CNBr)/trypsin-extractable proteins of a cell wall-enriched fraction from yeast and hyphae were compared. In total, 107 spots were identified from the DTT/SDS-extractible cell wall-enriched fraction, corresponding to 82 unique proteins. Of these DTT/SDS-extractible proteins, 14 proteins were upregulated and 10 were downregulated in response to hyphal induction. Approximately 6-9% of total cell wall-protein-enriched fraction was found to be resistant to DTT/SDS extraction. Analysis of the DTT/SDS-resistant fraction using a CNBr/trypsin extraction resulted in the identification of 29 proteins. Of these, 17 were identified only in the hyphae, four were identified only in the yeast, and eight were identified in both the yeast and hyphae.     

PalI domain proteins of Saccharomyces cerevisiae and Candida albicans

The Rim9/PalI groups of proteins are members of the Sur7 family, all of which contain a signal sequence and a block of three potential trans-membrane helices. Multi-protein sequence comparisons among fungi suggest that there are two classes of Rim9/PalI proteins; longer proteins like PalI that contain a Sur7 domain and a C-terminal extension, and shorter proteins like Rim9 that contain essentially only the Sur7 domain. We have examined possible roles of the longer, PalI-like proteins of both Saccharomyces cerevisiae (Yol019w) and Candida albicans (Orf19.1510/Srd1), two species that also contain short Rim9 proteins required for alkaline-associated stress responses. Deletions of the long form genes did not create any significant stress response phenotype in either S. cerevisiae or C. albicans, nor did the deletions enhance any of the rim9 deletion effects when combined in a double mutant. Furthermore, challenges in C. albicans show RIM9 but not SRD1 is important for proper response and hyphal formation. It appears that in fungal species such as Aspergillus nidulans containing only a long-form PalI-like protein, this element functions in the process of stress response, while in fungi with both versions the response to stress function is limited to the short-form protein.     

Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages

Glycosylphosphatidylinositol (GPI)-anchored proteins are involved in cell wall integrity and cell-cell interactions. We disrupted the Candida albicans homologue of the Saccharomyces cerevisiae GPI7/LAS21 gene, which encodes a GPI anchor-modifying activity. In the mutant and on solid media, the yeast-to-hyphae transition was blocked, whereas chlamydospore formation was enhanced. However, the morphogenetic switch was normal in liquid medium. Abnormal budding patterns, cytokinesis and cell shape were observed in both liquid and solid media. The cell wall structure was also modified in the mutants, as shown by hypersensitivity to Calcofluor white. In vitro and in vivo assays revealed that the mutant interacted with its host in a modified way, resulting in reduced virulence in mice and reduced survival in the gastrointestinal environment of mice. The mitogen-activated protein (MAP) kinase pathway of macrophages was downregulated by the wild-type cells but not by the DeltaCagpi7 null strains. In agreement with this abnormal behaviour, mutant cells were more sensitive to the lytic action of macrophages. Our results indicate that a functional GPI anchor is required for full hyphal formation in C. albicans, and that perturbation of the GPI biosynthesis results in hypersensitivity to host defences.     

The Sur7 protein regulates plasma membrane organization and prevents intracellular cell wall growth in Candida albicans

The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains.     

The Golgi GDPase of the fungal pathogen Candida albicans affects morphogenesis,glycosylation, and cell wall properties

Cell wall mannoproteins are largely responsible for the adhesive properties and immunomodulation ability of the fungal pathogen Candida albicans. The outer chain extension of yeast mannoproteins occurs in the lumen of the Golgi apparatus. GDP-mannose must first be transported from the cytosol into the Golgi lumen, where mannose is transferred to mannans. GDP is hydrolyzed by a GDPase, encoded by GDA1, to GMP, which then exits the Golgi lumen in a coupled, equimolar exchange with cytosolic GDP-mannose. We isolated and disrupted the C. albicans homologue of the Saccharomyces cerevisiae GDA1 gene in order to investigate its role in protein mannosylation and pathogenesis. CaGda1p shares four apyrase conserved regions with other nucleoside diphosphatases. Membranes prepared from the C. albicans disrupted gda1/gda1 strain had a 90% decrease in the ability to hydrolyze GDP compared to wild type. The gda1/gda1 mutants showed a severe defect in O-mannosylation and reduced cell wall phosphate content. Other cell wall-related phenotypes are present, such as elevated chitin levels and increased susceptibility to attack by β-1,3-glucanases. Our results show that the C. albicans organism contains β-mannose at their nonreducing end, differing from S. cerevisiae, which has only α-linked mannose residues in its O-glycans. Mutants lacking both alleles of GDA1 grow at the same rate as the wild type but are partially blocked in hyphal formation in Lee solid medium and during induction in liquid by changes in temperature and pH. However, the mutants still form normal hyphae in the presence of serum and N-acetylglucosamine and do not change their adherence to HeLa cells. Taken together, our data are in agreement with the hypothesis that several pathways regulate the yeast-hypha transition. Gda1/gda1 cells offer a model for discriminating among them.     

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants

In Candida albicans wild-type cells, the beta1, 6-glucanase-extractable glycosylphosphatidylinositol (GPI)-dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI-CWPs, including Als1p and Als3p, are attached via beta1,6-glucan to beta1,3-glucan. The remaining GPI-CWPs are linked through beta1,6-glucan to chitin. The beta1,6-glucanase-resistant protein fraction is small and consists of Pir-related CWPs, which are attached to beta1,3-glucan through an alkali-labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Delta and pmt1Delta mutant strains, which are defective in N- and O-glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI-CWPs through beta1,6-glucan to chitin. In these cells, the level of Pir-CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a beta1, 6-glucan-deficient hemizygous kre6Delta mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.     

Candida albicans cell wall ssa proteins bind and facilitate import of salivary histatin 5 required for toxicity

Fungicidal activity of Hst 5 is initiated by binding to cell surface proteins on Candida albicans, followed by intracellular transport to cytoplasmic effectors leading to cell death. As we identified heat shock 70 proteins (Ssa1p and/or Ssa2p) from C. albicans lysates that bind Hst 5, direct interactions between purified recombinant Ssa proteins and Hst 5 were tested by pull-down and yeast two-hybrid assays. Pulldown of both native complexes and those stabilized by cross-linking demonstrated higher affinity of Hst 5 for Ssa2p than for Ssa1p, in agreement with higher levels of interactions between Ssa2p and Hst 5 measured by yeast two-hybrid analyses. C. albicans ssa1Delta and ssa2Delta mutants were constructed to examine Hst 5 binding, translocation, and candidacidal activities. Both ssa1Delta and ssa2Delta mutants were indistinguishable from wild-type cells in growth and hyphal formation. However, C. albicans ssa2Delta mutants were highly resistant to the candidacidal activity of Hst 5, although the ssa1Delta mutant did not have any significant reduction in killing by Hst 5. Total cellular binding of 125I-Hst 5 in the ssa2Delta mutant was reduced to one-third that of wild-type cells, in contrast to the ssa1Delta mutant whose total cellular binding of Hst 5 was similar to the wild-type strain. Intracellular transport of Hst 5 was significantly impaired in the ssa2Delta mutant strain, but only mildly so in the ssa1Delta mutant. Thus, C. albicans Ssa2p facilitates fungicidal activity of Hst 5 in binding and intracellular translocation, whereas Ssa1p appears to have a lesser functional role in Hst 5 toxicity.     

Cloning and analysis of a Candida albicans gene that affects cell surface hydrophobicity

The opportunistic pathogenic yeast Candida albicans exhibits growth phase-dependent changes in cell surface hydrophobicity, which has been correlated with adhesion to host tissues. Cell wall proteins that might contribute to the cell surface hydrophobicity phenotype were released by limited glucanase digestion. These proteins were initially characterized by their rates of retention during hydrophobic interaction chromatography--high-performance liquid chromatography and used as immunogens for monoclonal antibody production. The present work describes the cloning and functional analysis of a C. albicans gene encoding a 38-kDa protein recognized by the monoclonal antibody 6C5-H4CA. The 6C5-H4CA antigen was resolved by two-dimensional electrophoresis, and a partial protein sequence was determined by mass spectrometry analysis of tryptic fragments. The obtained peptides were used to identify the gene sequence from the unannotated C. albicans DNA database. The antibody epitope was provisionally mapped by peptide display panning, and a peptide sequence matching the epitope was identified in the gene sequence. The gene sequence encodes a novel open reading frame (ORF) of unknown function that is highly similar to several other C. albicans ORFs and to a single Saccharomyces cerevisiae ORF. Knockout of the gene resulted in a decrease in measurable cell surface hydrophobicity and in adhesion of C. albicans to fibronectin. The results suggest that the 38-kDa protein is a hydrophobic surface protein that meditates binding to host target proteins.     

Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulence

The cell wall of Candida albicans consists of an internal skeletal layer and an external protein coat. This coat has a mosaic-like nature, containing c . 20 different protein species covalently linked to the skeletal layer. Most of them are GPI proteins. Coat proteins vary widely in function. Many of them are involved in the primary interactions between C. albicans and the host and mediate adhesive steps or invasion of host cells. Others are involved in biofilm formation and cell–cell aggregation. They further include iron acquisition proteins, superoxide dismutases, and yapsin-like aspartic proteases. In addition, several covalently linked carbohydrate-active enzymes are present, whose precise functions remain hitherto largely elusive. The expression levels of the genes that encode covalently linked cell wall proteins (CWPs) can vary enormously. They depend on the mode of growth and the combined inputs of several signaling pathways that sense environmental conditions. This is reflected in the unusually long intergenic regions of most of these genes. Finally, the precise location of several covalently linked CWPs is temporally and spatially regulated. We conclude that covalently linked CWPs of C. albicans play a crucial role in fitness and virulence and that their expression is tightly controlled.     

Proteolytic cleavage of covalently linked cell wall proteins by Candida albicans Sap9 and Sap10

The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.     

Functional analysis of Candida albicans GPI-anchored proteins: roles in cell wall integrity and caspofungin sensitivity

The outer layer of the Candida albicans cell wall is enriched in highly glycosylated proteins. The major class, the GlycosylPhosphatidylInositol (GPI)-anchored proteins are tethered to the wall by GPI-anchor remnants and include adhesins, glycosyltransferases, yapsins and superoxide dismutases. In silico analysis suggested that C. albicans possesses 115 putative GPI anchored proteins (GpiPs), almost twice the number reported for Saccharomyces cerevisiae. A global approach to characterise in silico predicted GpiPs has been initiated by generating a library of 45 mutants. This library was subjected to a screen for cell wall modifications by testing the cell wall integrity (SDS and Calcofluor White sensitivity) and response to caspofungin. We showed that, when caspofungin sensitivity was modified, in more than half of the cases the susceptibility can be correlated to the level of chitin and cell wall thickness: sensitive strains have low level of chitin and a thin cell wall. We also identified, for the first time, genes that when deleted lead to decreased caspofungin sensitivity: DFG5, PHR1, PGA4 and PGA62. The role of two unknown GpiPs, Pga31 and Pga62 in the cell wall structure and composition was clearly demonstrated during this study.     

Comprehensive characterization of secreted aspartic proteases encoded by a virulence gene family in Candida albicans

Candida albicans is a commensal organism, but causes life-threatening infections in immunocompromised patients. Certain factors such as yeast-hyphae transition and hydrolytic enzymes are suggested as virulence attributes of C. albicans. Among them, 10 types of secreted aspartic protease (SAP) genes have received particular attention as a major virulence gene family. However, their full functional repertoire, including its biochemical properties, remains to be elucidated. Hence, we purified all Sap isozymes using Pichia pastoris and comprehensively determined and compared their biochemical properties. While optimum pH of Sap7 was 6.5 and that of Sap8 was 2.5, presence of other Sap isozymes functioning within a broad range of optimum pH could allow C. albicans to survive and cause infections in various tissues. The substrate specificities of Sap isozymes were analysed by using FRETS-25Xaa libraries. Sap7 and Sap10 showed high substrate specificity, while other Sap isozymes had broad substrate specificities. Principal component analysis revealed that the 10 Sap isozymes were clustered into 3 distinct groups in terms of their substrate specificities. Interestingly, Sap4-6, which are coproduced in the hyphal form, were clustered as the same group, indicating that they may target similar host proteins. These results will lead to further understanding of C. albicans pathogenicity.