Analysis of the 3H8 antigen of Candida albicans reveals new aspects of the organization of fungal cell wall proteins.

The walls of both, yeast and mycelial cells of Candida albicans possess a species-specific antigen that is recognized by a monoclonal antibody (MAb 3H8). This antigen can be extracted in the form of a very high Mr complex, close or over 106 Da, by treatment, with ß-1,3-glucanase, ß mercaptoethanol or dithothreitol, or mild alkali, but not by saturated hydrogen fluoride (HF) in pyridine, suggesting that the complex is bound to wall ß-1,3 glucans, and to proteins by disulfide bonds, but not to ß-1,6 glucans. Through its sensitivity to trypsin and different deglycosylation procedures, it was concluded that the epitope is associated to a glycoprotein containing N-glycosidic, but not O-glycosidic mannan moieties. By means of electrophoresis in polycrylamide gradient gels, followed by mass spectrometric analysis, the epitope was pinpointed to a very high MW complex containing Agglutinin-Like Sequence (ALS) family proteins, and other cytoplasmic, membrane and secreted proteins. The components of this complex are bound by unknown covalent bonds. The material extracted with ß mercaptoethanol or dilute alkali appeared under the electron microscope as large aggregates in the form of spheroidal and mostly web-like structures of large sizes. These, and additional data, suggest that this protein complex may constitute an important part of the basic glycoprotein structure of C. albicans. The possibility that similar complexes exist in the wall of other fungi is an attractive, although yet untested possibility.

The GCA1 gene encodes a glycosidase-like protein in the cell wall of Candida albicans.

Candida albicans Gca1p is a putative glucoamylase enzyme which contains 946 amino acids, 11 putative sites for N-glycosylation and 9 for O-glycosylation. Gca1p was identified in ß-mercaptoethanol extracts from isolated cell walls of strain C. albicans SC5314 and it is involved in carbohydrate metabolism. The significance and the role of this protein within the cell wall structure were studied in the corresponding mutants. The homozygous mutant showed that GCA1 was not an essential gene for cell viability. Subsequent phenotypic analysis performed in the mutants obtained did not show significant difference in the behavior of mutant when compared with the wild strain SC5314. Zymoliase, Calcofluor White, Congo red, SDS, caffeine or inorganic compounds did not affect the integrity of the cell wall. No differences were observed when hyphal formation assays were carried out. However, an enzyme assay in the presence of substrate p-nitrophenyl-α-D-glucopyranoside enabled us to detect a significant decrease in glycosidase activity in the mutants compared with the parental strain, revealing the function of Gca1.

Identification of Candida albicans wall mannoproteins covalently linked by disulphide and/or alkali-sensitive bridges.

This paper describes the results obtained by analysing the human pathogen Candida albicans cell wall subproteome by mass spectrometry, using extraction procedures aimed at releasing proteins bound by disulphide bridges (RAE-CWP) or alkali-labile ester linkages (ALS-CWP). Ten of the total proteins released from the wall by ß-ME and/or NaOH contained a potential signal peptide, lacked a GPI cell wall hydrophobic C-terminal domain and were identified as true wall proteins by in silico analysis, whereas four additional proteins were identified as bound to the plasma membrane. The results surprisingly demonstrated that, in addition to the expected RAE-CWP and ALS-CWP proteins, 16 GPI proteins were bound to the wall by disulphide or alkali-sensitive bonds, since they were released by ß-ME and/or NaOH. The biological significance of these results is discussed in relation to the added complexity of the organization of the proteins in the C. albicans cell wall.

Pga13 in Candida albicans is localized in the cell wall and influences cell surface properties, morphogenesis and virulence.

The fungal cell wall is an essential organelle required for maintaining cell integrity and also plays an important role in the primary interactions between pathogenic fungi and their hosts. PGA13 encodes a GPI protein in the human pathogen Candida albicans, which is highly up-regulated during cell wall regeneration in protoplasts. The Pga13 protein contains a unique tandem repeat, which is present five times and is characterized by conserved spacing between the four cysteine residues. Furthermore, the mature protein contains 38% serine and threonine residues, and therefore probably is a highly glycosylated cell wall protein. Consistent with this, a chimeric Pga13-V5 protein could be localized to the cell wall, but only after deglycosylation was performed. Disruption of PGA13 led to increased sensitivity to Congo red, Calcofluor white, and zymolyase, and to a diminished ability of protoplasts to recover their cell wall. In addition, pga13Δ mutants exhibited delayed filamentation, a higher surface hydrophobicity, and increased adherence and flocculation (cell-cell interactions). Furthermore, transcript profiling showed that expression of four members of the ALS family (adhesin-encoding genes) is up-regulated in the pga13Δ null mutant. Altogether, these results indicate that Pga13 is a wall-localized protein that contributes to cell wall synthesis and is important for acquiring normal surface properties. The contribution of Pga13 to surface hydrophilicity may be important for cell dispersal during development of invasive infections, and possibly for morphological development. This is consistent with the observed reduced virulence of pga13Δ mutants in a mouse model of disseminated candidiasis.

Dosage-dependent roles of the Cwt1 transcription factor for cell wall architecture, morphogenesis, drug sensitivity and virulence in Candida albicans.

The Cwt1 transcription factor is involved in cell wall architecture of the human fungal pathogen Candida albicans. We demonstrate here that deficiency of Cwt1 leads to decreased beta1,6-glucan in the cell wall, while mannoproteins are increased in the cell wall of exponentially growing cells and are released into the medium of stationary phase cells. Hyphal morphogenesis of cwt1 mutants is reduced on the surfaces of some inducing media. Unexpectedly, the CWT1/cwt1 heterozygous strains shows some stronger in vitro phenotypes compared to the homozygous mutant. The heterozygous but not the homozygous strain is also strongly impaired for its virulence in a mouse model of systemic infection. We suggest that an intermediate dosage of Cwt1 affects phenotypes profoundly, while its complete absence may elicit compensatory responses of C. albicans.

Adhesive properties and hydrolytic enzymes of oral Candida albicans strains.

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.

A study of the Candida albicans cell wall proteome.

Considering the importance of proteins in the structure and function of the cell wall of Candida albicans, we analyzed the cell wall subproteome of this important human pathogen by LC coupled to MS (LC-MS) using different protein extraction procedures. The analyzed samples included material extracted by hydrogen fluoride-pyridine (HF-pyridine), and whole SDS-extracted cell walls. The use of this latter innovative procedure gave similar data as compared to the analysis of HF-pyridine extracted proteins. A total of 21 cell wall proteins predicted to contain a signal peptide were identified, together with a high content of potentially glycosylated Ser/Thr residues, and the presence of a GPI motif in 19 of them. We also identified 66 "atypical" cell wall proteins that lack the above-mentioned characteristics. After tryptic removal of the most accessible proteins in the cell wall, several of the same expected GPI proteins and the most commonly found "atypical" wall proteins were identified. This result suggests that proteins are located not only at the cell wall surface, but are embedded within the cell wall itself. These results, which include new identified cell wall proteins, and comparison of proteins in blastospore and mycelial walls, will help to elucidate the C. albicans cell wall architecture.

Saccharomyces cerevisiae Rds2 transcription factor involvement in cell wall composition and architecture.

Although the cell wall is very important in yeasts, relatively little is known about the relationship between its structure and function. In Saccharomyces cerevisiae, a family of 55 transcription factor proteins unique to fungi, so-called zinc cluster proteins, has been described. Of these, Rds2 has been identified as an activator/inhibitor of gluconeogenesis. However, previous studies have pointed out additional roles for this protein, specifically, in the modulation of cell-wall architecture and drug sensitivity. In this work, evidence regarding the role of Rds2 as a regulator of cell-wall architecture and composition is presented based on phenotypical analysis of the cell walls prepared from a S. cerevisiae Rds2 mutant strain. Analyses of the sensitivity of this rds2Delta mutant to different drugs and to osmotic stress showed that Rds2 is indeed involved in the drug-sensitivity response and plays a role in determining osmotic sensitivity.

Analysis of the proteins involved in the structure and synthesis of the cell wall of Ustilago maydis.

A study of the proteins involved in the synthesis and structure of the cell wall of Ustilago maydis was made by in silico analysis of the fungal genome, with reference to supporting experimental evidence. The composition of the cell wall of U. maydis shows similarities with the structural composition of the walls of Ascomycetes, but also shows important differential features. Accordingly, the enzymes involved in the synthesis of the U. maydis wall polysaccharides chitin and beta-1,6 glucans displayed some differential characteristics. The most salient difference in protein composition was the predicted absence of Pir proteins, an important class of proteins present in the Ascomycetes. Other classes of proteins that are covalently-linked to the wall in Ascomycetes, including those bound through disulfide linkages, joined by alkali-labile bonds, and GPI proteins, were predicted to be present in the U. maydis walls. The main characteristic of the exo-cellular, non-covalently-bound proteins was their relative low number, especially for hydrolytic enzymes.

Saccharomyces cerevisiae Rds2 transcription factor involvement in cell wall composition and architecture

Although the cell wall is very important in yeasts, relatively little is known about the relationship between its structure and function. In Saccharomyces cerevisiae, a family of 55 transcription factor proteins unique to fungi, so-called zinc cluster proteins, has been described. Of these, Rds2 has been identified as an activator/inhibitor of gluconeogenesis. However, previous studies have pointed out additional roles for this protein, specifically, in the modulation of cell-wall architecture and drug sensitivity. In this work, evidence regarding the role of Rds2 as a regulator of cell-wall architecture and composition is presented based on phenotypical analysis of the cell walls prepared from a S. cerevisiae Rds2 mutant strain. Analyses of the sensitivity of this rds2Delta mutant to different drugs and to osmotic stress showed that Rds2 is indeed involved in the drug-sensitivity response and plays a role in determining osmotic sensitivity (AU)

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Genomic response programs of Saccharomyces cerevisiae following protoplasting and regeneration.

Global transcription profiling during regeneration of Saccharomyces cerevisiae protoplasts was explored. DNA microarrays measured the expression of 6388 genes and wall removal resulted initially in over-expression of 861 genes that decayed later on, a behaviour expected from a transient stress response. Kinetics of expression divided the genes into 25 clusters. Transcription of the genes from clusters 14-25 was initially up-regulated, suggesting that the grouped genes permitted cell adaptation to the removal of the wall. Clustering of genes involved in "wall structure and biosynthesis" showed that most of them had initially low levels of expression that increased along the process. Analysis by use of the T-profiler showed that the group of "structural components of the wall" was up-regulated after two hours and remained as such during the process. These results evoke the likeness and difference with the process occurring in Candida albicans.

Disruption of the Candida albicans ATC1 gene encoding a cell-linked acid trehalase decreases hypha formation and infectivity without affecting resistance to oxidative stress.

In Candida albicans, the ATC1 gene, encoding a cell wall-associated acid trehalase, has been considered as a potentially interesting target in the search for new antifungal compounds. A phenotypic characterization of the double disruptant atc1Delta/atc1Delta mutant showed that it was unable to grow on exogenous trehalose as sole carbon source. Unlike actively growing cells from the parental strain (CAI4), the atc1Delta null mutant displayed higher resistance to environmental insults, such as heat shock (42 degrees C) or saline exposure (0.5 M NaCl), and to both mild and severe oxidative stress (5 and 50 mM H(2)O(2)), which are relevant during in vivo infections. Parallel measurements of intracellular trehalose and trehalose-metabolizing enzymes revealed that significant amounts of the disaccharide were stored in response to thermal and oxidative challenge in the two cell types. The antioxidant activities of catalase and glutathione reductase were triggered by moderate oxidative exposure (5 mM H(2)O(2)), whereas superoxide dismutase was inhibited dramatically by H(2)O(2), where a more marked decrease was observed in atc1Delta cells. In turn, the atc1Delta mutant exhibited a decreased capacity of hypha and pseudohypha formation tested in different media. Finally, the homozygous null mutant in a mouse model of systemic candidiasis displayed strongly reduced pathogenicity compared with parental or heterozygous strains. These results suggest not only a novel role for the ATC1 gene in dimorphism and infectivity, but also that an interconnection between stress resistance, dimorphic conversion and virulence in C. albicans may be reconsidered. They also support the hypothesis that Atc1p is not involved in the physiological hydrolysis of endogenous trehalose.

Identification of pH-regulated antigen 1 released from Candida albicans as the major ligand for leukocyte integrin alphaMbeta2.

Candida albicans is a common opportunistic fungal pathogen and is the leading cause of invasive fungal disease in immunocompromised individuals. The induction of cell-mediated immunity to C. albicans is of critical importance in host defense and the prime task of cells of the innate immune system. We previously demonstrated that the integrin alpha(M)beta(2) (CD11b/CD18) is the major leukocyte receptor involved in C. albicans recognition, mediating both adhesive and migratory responses to the fungus. In the present study, we demonstrate that various C. albicans strains release a protease-sensitive activity into their conditioned medium that supports alpha(M)beta(2)-mediated cell adhesion and migration. The isolation and characterization of this protein was undertaken by two independent approaches: 1) immunoaffinity purification on a mAb raised to conditioned medium which blocked alpha(M)beta(2)-dependent adhesion and migration; and 2) affinity chromatography on purified alpha(M)beta(2). Each approach led to the isolation of the same protein, which was unequivocally identified as pH-regulated Ag 1 (Pra1p), based on mass spectrometry and amino acid sequence analyses. C. albicans mutant strains lacking Pra1p were unable to support leukocyte adhesion or migration. In a neutrophil-mediated fungal killing assay, such mutant strains were resistant to killing and/or phagocytosis. Addition of purified Pra1p or reagents that block alpha(M)beta(2) function prevented killing of Pra1p-expressing but not Pra1p-deficient strains of C. albicans. Together, these data indicate that Pra1p is a ligand of alpha(M)beta(2) on C. albicans and that the soluble form of Pra1p may assist the fungus in escaping host surveillance.

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Genomic response programs of Candida albicans following protoplasting and regeneration.

Transcription profiling of Candida albicans cells responding to the elimination of the wall (protoplasts) and posterior regeneration was explored. DNA microarrays were used to measure changes in the expression of 6039 genes, and the upregulated genes during regeneration at 28 degrees C were assigned to fourteen categories. A total of 407 genes were upregulated during the process, of which 144 reached a maximum after 1 h. MKC1, a gene encoding a member of the regulatory pathway involved in cell wall integrity was overexpressed. Time-dependent expression divided the genes into 40 clusters. Clusters 1-19 were highly expressed initially (time 0) and downregulated following incubation, whereas transcription of the genes grouped into clusters 20-40 showed the opposite behaviour. These results suggest that the first clusters group genes permitting the cell adaptation to a sub-optimal environment due to removal of the wall, whereas the second group represents genes required for protoplasts regeneration after shifted to optimal conditions from 4 to 28 degrees C.

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.

Anchorage of Candida albicans Ssr1 to the cell wall, and transcript profiling of the null mutant.

Incorporation into the wall of Candida albicans Ssr1, a GPI-dependent protein, was investigated by construction of different truncated genes for which the three potential omega sites (S199, S215 and G216) and the corresponding omega+1 and omega+2 were eliminated or modified. Cells of the C. albicans ssr1Delta mutant were transformed with pADH-pl harboring the truncated versions of CaSSR1, pADH-DeltaCaSSR1t(217-234) (lacking a C-terminal hydrophobic stretch of 18 aa including the putative omega+2 and omega+1, omega+2 of S215 and G216) or pADH-DeltaCaSSR1t(199-201) (lacking three serine residues), and their walls were analyzed for the protein. Results suggested that the three serine residues are essential for incorporation of CaSsr1 into the wall beta-glucan. This interpretation was confirmed when the truncated protein CaSsr1pt(199-201) was found in the spent medium. The transcription profile of the 6039 genes in C. albicans ssr1Delta showed that seven genes are upregulated (1.4-fold), including SRP54 (a signal recognition particle subunit), IPF29 (a zinc finger protein) and PTR3 (a transcriptional regulator), whereas 27 genes are downregulated (0.7-fold), including IPF6318 (a beta-glucosidase) and SOU1 (a sorbitol utilization protein). Additional genes showed a reduced increase, or decreased expression, suggesting that some current orphan genes may have unknown cell wall functions. In addition, a compensatory mechanism would appear to occur, as a substantial increase in the amount of beta-1,3-glucan (2.34-fold) was detected in the cell wall of the mutant cells.

In silico analysis for transcription factors with Zn(II)(2)C(6) binuclear cluster DNA-binding domains in Candida albicans.

A total of 6047 open reading frames in the Candida albicans genome were screened for Zn(II)(2)C(6)-type zinc cluster proteins (or binuclear cluster proteins) involved in DNA recognition. These fungal proteins are transcription regulators of genes involved in a wide range of cellular processes, including metabolism of different compounds such as sugars or amino acids, as well as multi-drug resistance, control of meiosis, cell wall architecture, etc. The selection criteria used in the sequence analysis were the presence of the CysX(2)CysX(6)CysX(5-16)CysX(2)CysX(6-8)Cys motif and a putative nuclear localization signal. Using this approach, 70 putative Zn(II)(2)C(6) transcription factors have been found in the genome of C. albicans.

Role of Pir1 in the construction of the Candida albicans cell wall.

Searches in a Candida albicans database (http://genolist.pasteur.fr/CandidaDB/) identified two Individual Protein Files (IPF 15363 and 19968) whose deduced amino acid sequences showed 42 % and 45 % homology with Saccharomyces cerevisiae Pir4. The two DNA sequences are alleles of the same gene (CaPIR1) but IPF 19968 has a deletion of 117 bases. IPF 19968 encodes a putative polypeptide of 364 aa, which is highly O-glycosylated and has an N-mannosylated chain, four cysteine residues and seven repeats. Both alleles are expressed under different growth conditions and during wall construction by regenerating protoplasts. The heterozygous mutant cells are elongated, form clumps of several cells and are hypersensitive to drugs that affect cell wall assembly. CaPir1 was labelled with the V5 epitope and found linked to the 1,3-beta-glucan of the C. albicans wall and also by disulphide bridges when expressed in S. cerevisiae.

The ATC1 gene encodes a cell wall-linked acid trehalase required for growth on trehalose in Candida albicans.

After screening a Candida albicans genome data base, the product of an open reading frame (IPF 19760/CA2574) with 41% identity to Saccharomyces cerevisiae vacuolar acid trehalase (Ath1p) was identified and named Atc1p. The deduced amino acid sequence shows that Atc1p contains an N-terminal hydrophobic signal peptide and 20 potential sites for N-glycosylation. C. albicans homozygous mutants that lack acid trehalase activity were constructed by gene disruption at the two ATC chromosomal alleles. Analysis of these null mutants shows that Atc1p is localized in the cell wall and is required for growth on trehalose as a carbon source. An Atc1p endowed with acid trehalase activity was obtained by an in vtro transcription-translation coupled system. These results strongly suggest that ATC1 is the structural gene encoding cell wall acid trehalase in C. albicans. Determinations of ATC1 mRNA expression as well as acid trehalase activity in the presence and absence of glucose point out that ATC1 gene is regulated by glucose repression.