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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 β-1,3-glucanase, β mercaptoethanol or dithothreitol, or mild alkali, but not by saturated hydrogen fluoride (HF) in pyridine, suggesting that the complex is bound to wall β-1,3 glucans, and to proteins by disulfide bonds, but not to β-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 β 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.

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 β-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 β-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.

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.

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.

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.

A total of 6047 open reading frames in theCandida albicansgenome were screened forZn(II)2C6-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 theCysX2CysX6CysX5-16CysX2CysX6-8Cysmotif and a putative nuclear localization signal. Using this approach, 70 putativeZn(II)2C6transcription factors have been found in the genome ofC. albicans.

CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.; International audience; CandidaDB is a database dedicated to the genome of the most prevalent systemic fungal pathogen of humans, Candida albicans. CandidaDB is based on an annotation of the Stanford Genome Technology Center C.albicans genome sequence data by the European Galar Fungail Consortium. CandidaDB Release 2.0 (June 2004) contains information pertaining to Assembly 19 of the genome of C.albicans strain SC5314. The current release contains 6244 annotated entries corresponding to 130 tRNA genes and 5917 protein-coding genes. For these, it provides tentative functional assignments along with numerous pre-run analyses that can assist the researcher in the evaluation of gene function for the purpose of specific or large-scale analysis. CandidaDB is based on GenoList, a generic relational data schema and a World Wide Web interface that has been adapted to the handling of eukaryotic genomes. The interface allows users to browse easily through genome data and retrieve information. CandidaDB also provides more elaborate tools, such as pattern searching, that are tightly connected to the overall browsing system. As the C.albicans genome is diploid and still incompletely assembled, CandidaDB provides tools to browse the genome by individual supercontigs and to examine information about allelic sequences obtained from complementary contigs. CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.

After screening a Candida albicans genome database the product of an open reading frame (ORF) (CA2880) with 49% homology to the product of Saccharomyces cerevisiae YPL133c, a putative transcriptional factor, was identified. The disruption of the C. albicans gene leads to a major sensitivity to calcofluor white and Congo red, a minor sensitivity to sodium dodecyl sulfate, a major resistance to zymolyase, and an alteration of the chemical composition of the cell wall. For these reasons we called it CaCWT1 (for C. albicans cell wall transcription factor). CaCwt1p contains a putative Zn(II) Cys(6) DNA binding domain characteristic of some transcriptional factors and a PAS domain. The CaCWT1 gene is more expressed in stationary phase cells than in cells growing exponentially. To our knowledge, this is the first Zn(II) Cys(6) transcriptional factor-encoding gene implicated in the cell wall architecture.

After screening of aCandida albicansgenome database, the product of an ORF (IPF 3054) that has 62 % homology withSaccharomyces cerevisiaeSsr1p, an internal cell-wall protein, was identified and named CaSsr1p. The deduced amino acid sequence shows that CaSsr1p contains an N-terminal hydrophobic signal peptide, is rich in Ser and Thr amino acids and has a potential glycosylphosphatidylinositol-attachment signal. CaSsr1p is released following degradation of isolated cell walls by zymolyase (mainly a 1,3-β-glucanase) and therefore seems to be covalently linked to theβ-glucan of the cell walls. Both disruption and overexpression of theCaSSR1gene caused an increased sensitivity to calcofluor white, Congo red and zymolyase digestion. These results suggest that CaSsr1p has a structural role associated with the cell-wallβ-glucan.

Several cDNA fragments corresponding to transcripts differentially expressed under conditions that favor mycelial growth of Candida albicans were identified by the “differential display” technique. One of these was cloned and used as a probe to rescue the full gene from a genomic library of the fungus. The sequence identified a single, uninterrupted open reading frame of 1395 nucleotides encoding a putative protein of 465 residues and a theoretical molecular weight of 50.3 kDa, present in the genome as a single copy located at chromosome 2 in different strains. The gene product showed high homology with subtilisin-like proteases, mainly PRB1, the vacuolar B protease from Saccharomyces cerevisiae, and for this reason it was designated as a putative CaPRB1. Expression of the gene was not directly related to fungal morphogenesis, but to the initial response to inducers: Heat shock and the presence of N-acetyl glucosamine. It was also subject to nitrogen, but not to carbon catabolite repression, although glucose inhibited the GlcNAc stimulatory effect. The gene was, in our hands, unable to complement PRB1 mutation in S. cerevisiae. C. albicans prb null mutants did not show any distinct alteration in the phenotype. CaPRB1 is the first gene coding for a putative vacuolar serine protease cloned from C. albicans.

The synthesis of β-1,3-glucan, the structural component of the yeast cell wall which gives shape to the cell, occurs at the plasma membrane and is the result of the activity of at least a two-component complex. Fks1p is the catalytic subunit directly responsible for the synthesis of β-1,3-glucan, whilst the second subunit, Rho1p, has a GTP-dependent regulatory role. FKS1 has been characterized in Saccharomyces cerevisiae, where its function is at least partially redundant with that of FKS2/GSC2. FKS homologues have also been identified in several other fungal species, including Candida albicans, Schizosaccharomyces pombe, Aspergillus nidulans, Cryptococcus neoformans and Paracoccidiodes brasiliensis. In this work, we have used degenerate oligonucleotides derived from the conserved regions of Fks1ps to isolate the possible FKS homologue genes of the strictly aerobic non-conventional yeast Yarrowia lipolytica. Using this approach, we have isolated a single FKS homologue which we have named YlFKS1; this codes a 1961 amino acid protein that shows a high degree of homology with other Fksps. Expression analysis of YlFKS1 under different conditions affecting the cell wall did not reveal significant differences. Finally, attempts to obtain a Y. lipolytica strain containing a disrupted YlFKS1 allele failed, despite having used two different techniques. Taken together, these results suggest that, unlike S. cerevisiae, YlFKS1 is the only FKS1 homologue in Y. lipolytica and is essential for growth. Copyright © 2002 John Wiley & Sons, Ltd.

Urea or hot sodium dodecyl sulphate extracted a significant amount of the same proteins from the matrix of the cell wall of the yeast form and mycelial cells of Candida albicans. Gel filtration analysis of the urea-extracted proteins revealed that they occurred in the form of large complexes which were unaffected by up to 8 M urea. Among them, proteins en route to becoming covalently associated within the wall scaffold were identified by their reaction with specific antibodies. When urea was removed by dialysis, some of these proteins specifically reassociated into large aggregates which bound strongly with ConA, whereas others remained soluble in smaller associated products. The ability of some of these proteins to bind to the insoluble wall polysaccharides was also assessed. No self-assembling proteins were able to bind to glucans and/or chitin. Specificity of the binding to polysaccharides made of beta-bound glucosyl or N-acetylglucosaminyl residues was determined by the competitive effect of several disaccharides. Whereas laminaribiose and diacetylchitobiose were strong inhibitors of protein binding to both glucan and chitin, lactose, maltose and sucrose were ineffective.

A Candida albicans expression library was constructed from RNA isolated from regenerating protoplasts. A 1.4-kb cDNA clone was used to isolate a genomic fragment. Sequence analysis revealed an open reading frame of 593 amino acids with an overall identity of 63.6% with the phenylalanyl-tRNA synthetase beta subunit (FRS1) of Saccharomyces cerevisiae. We named it CaFRS1. It is located in a single copy in chromosome R, SfiI fragment M. Its expression showed a decrease during the cell wall regeneration process in protoplasts of both yeast and mycelial cells of C. albicans, suggesting its requirement thereof in initial steps of the cell wall synthesis.

Summary. We compared two phenotypic methods, colony morphotyping on Sabouraud-tripheniltetrazolium agar (STTZ) and serotyping, with two genotypic methods, karyotyping and Random Amplified Polymorphic DNA bands obtained by PCR amplification (RAPD-PCR), for strain delineation in 33 Candida clinical isolates and two C. albicans strains from culture collections. Analysis of isolates on STTZ showed 11 different morphotypes. In two patients there was a switch in the morphotype coincidential with a change in the susceptibility of the isolates to azole antifungals. C. albicans isolates were divided into two serotypes. Sixteen and 18 different patterns were identified among the Candida isolates by karyotyping and RAPD-PCR, respectively. No relationship was found between any of the typing methods used and the source of the isolates. The combination of karyotyping and morphotyping on STTZ yielded useful epidemiological information, since it allowed the differentiation among the Candida species studied and the discrimination of clusters within C. albicans as well as to check the capacity of a strain to generate variants with different susceptibility to some antifungals. Zusammenfassung. Wir haben zur Stammdifferenzierung bei 33 klinischen Candida-Isolaten zwei phanotypische Methoden — Kolonie-Morphotypisierung auf Sabouraud-Triphenyltetrazolium-Agar (STTZ) und Serotypisierung — mit zwei genotypischen Methoden — Karyotypisierung und ‘random amplified polymorphic’ DNA, welche mittels der Polymerase-Kettenreaktion erhalten wurden (RAPD-PCR) — verglichen. Die Analyse der Isolate auf STTZ ergab 11 verschiedene Morphotypen. Bei zwei Patienten fand ein Switch des Morphotyps im Zusammenhang mit einem Wechsel der Sensibilitat gegenuber Antimyzetika der Azolgruppe statt. C. albicans-Isolate wurden in zwei verschiedene Serotypen unterteilt. Sechzehn und 18 verschiedene Muster wurden bei C. albicans jeweils mittels Karyotypisierung und RAPD-PCR identifiziert. Es konnte kein Zusammenhang zwischen irgendeiner der angewandten Typisierungsmethoden und dem Ursprung der Isolate ermittelt werden. Die Kombination von Karyotypisierung und Morphotypisierung auf STTZ ergab brauchbare epidemiologische Information, da sie erlaubte, die untersuchten Candida-Arten zu differenzieren, Unterarten von C. albicans zu identifizieren sowie die Fahigkeit eines Stammes zu prufen, Varianten zu entwickeln, welche gegenuber Antimyzetika verschiedene Sensibilitat aufweisen.

We have isolated a 4·0-kb fragment from a genomic library of Candida albicans which contained two open reading frames (ORFs). One of them is homologous to a prolyl-tRNA synthetase that catalyses the charging of a specific tRNA by proline (CaPRS). A deduced sequence of 575 amino acids representing a polypeptide of 66·2 kDa was determined. A FASTA search indicated that the CaPRSp had an overall similarity of 54·4% with the product of a Saccharomyces cerevisiae ORF (YER087) and 43·8% with the prolyl-tRNA synthetase of Escherichia coli (COLIPRO). Consensus Class II aminoacyl-tRNA synthetase sequences were identified by the PROSITE program. CaPRS was localized to chromosome R of the C. albicans genome and CaPRS DNA hybridized to a major RNA transcript of 1·7 kb under all conditions tested. The CaPRS sequence submitted to the EMBL data library is available under Accession Number U86341.© 1997 John Wiley & Sons, Ltd.

Summary Cell wall regeneration of individual Candida albicans yeast and mycelial protoplasts was studied with confocal and electron microscopy using polyclonal antibodies and leetins. Quantitative measurements of the fluorescence emitted by individual protoplasts during the process of regeneration indicate that chitin is the first polymer to be laid down, whereas β(1,3)- and β(1,6)glucan are incorporated at a later stage. Mannoproteins were found on the surface of fresh protoplasts and those newly synthesized were then deposited with time. During the first steps of wall regeneration, the proteins that interacted covalently with chitin or glucan were different, but the same species were found linked to each polymer in yeast and mycelial regenerating forms. The aggregates formed by regenerating protoplasts were shown to be due to the chitin and mannoprotein network initially laid.

Screening cDNA libraries with polyclonal antibody preparations against Candida albicans yeast or mycelial cell walls resulted in isolation of several positive clones. Some of them encoded enolase; others encoded a protein of the 70 kDa heat-shock protein family (Hsp70p), etc. The presence of these cytosolic proteins in the cell wall of actively growing C. albicans was discovered by analytical (SDS-PAGE and Western blot) and cytological (indirect immunofluorescence) experiments. Supplementation of cell cultures with papulacandin B, an antibiotic that inhibits formation of the -glucan skeleton, resulted in the release of enolase to the supernatant fluids; this release was prevented when 0.6 M KCI was present as an osmotic stabilizer. The cell wall of C. albicans incorporated exogenously added proteins (enolase and Escherichia coli and C albicans cytosolic proteins). The presence in the C. albicans cell wall of enolase, Hsp70p, and probably other intracellular proteins that are highly immunogenic might help the fungal cells to evade the host defences, and consequently could represent a survival mechanism for C. albicans in vivo.

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 β-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 β-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.

Candida albicans cell wall components were analyzed by ethylenediamine (EDA) treatment. Based on their different solubility properties, the cell wall components produced three fractions (A, B, and C). Fractions B (EDA-soluble, water-insoluble) and C (EDA-insoluble) contained glucan, chitin, and protein in different proportions. After zymolyase (mainly a beta-glucanase complex) or chitinase treatment of fractions B and C, more polysaccharides and proteins were solubilized by a second EDA treatment, suggesting that the solubility of the polymers in EDA depends on the degree of polymer interactions. Western blot analysis using two monoclonal antibodies (1B12 and 4C12) revealed electrophoretic patterns that were similar in mycelial and yeast morphologies, except that in material obtained from mycelial walls, an additional band was detected with MAb 1B12. Fluorescence microscopy of cell wall fractions treated with FITC-labeled Con-A, Calcofluor white, and FITC-labeled agglutinin showed that glucan and mannoproteins are uniformly distributed in fractions B and C, while chitin is restricted to distinct patches. Transmission electron microscopy demonstrated that fraction C maintained the original shape of the cells, with an irregular thickness generally wider than the walls. When fraction C was treated with chitinase, the morphology was still present and was maintained by an external glucan layer, with an internal expanded fibrillar material covering the entire cellular lumen. Degradation of the glucan skeleton of fraction C with zymolyase resulted in the loss of the morphology.

Following synthesis of its individual components, the cell wall of Candida albicans is assembled extracellularly in two steps. First, a viscoelastic composite is formed by noncovalent interactions between mannoproteins and other wall components. Second, the initial network is consolidated by formation of covalent cross-linkages among the wall polymers. In both processes, specific proteins may regulate the final yeast or mycelial morphology. These proteins might carry out part of what could be called a morphogenetic code. Experimental results have shown that some mannoproteins form supramolecular complexes. They are secreted independently, but released together from cell walls by hydrolases. In C. albicans cell walls a transglutaminase activity has been detected that could be responsible for the formation of covalent bonds between structural proteins. Key words: fungal cell wall, construction, morphogenesis, protein interactions, noncovalent linkages, covalent linkages.

The use of a novel monoclonal antibody (mAb) that reacts with (1,6)-beta-glucan has permitted the study of the different covalent linkages between glucan and mannoproteins in the cell wall of Candida albicans. The mAb JRR1 was originally raised by immunization with Zymolyase extracts from C. albicans cell walls, but it soon became apparent that it reacted with a (1,6)-beta-glucan epitope. By using this antibody, we show the existence of glucan-mannoprotein complexes between the (1,6)-beta-glucan epitope recognized by the antibody and cell wall mannoproteins. The topology of the (1,6)-beta-glucan in the cell wall of C. albicans has also been studied.

Immunoscreening of a mycelial expression library with polyclonal antibodies raised against mycelial cell wall resulted in the detection of a cDNA encoding a heat shock protein of Candida albicans. Sequence analysis of a 0.8-kb cDNA subclone, 2M-1, revealed an open reading frame encoding 244 amino acids. Southern blot analysis with this fragment as a probe demonstrated hybridization to C. albicans DNA. Northern analysis showed a substantial increase in 2M RNA expression levels after cells were subjected to heat shock. Western blot analysis with 2M monospecific antibodies recognized a 70-kDa protein which was present in membrane particles and cytosolic fractions.

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 β-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.

Two cDNA libraries were constructed from mRNAs obtained from yeast cells and germ-tubes of Candida albicans in lambda gt11. Immunoscreening with polyclonal antibodies raised against cell wall components allowed the detection of 29 positive clones. Two of these clones were selected for their specific reactivity with antisera either from yeast (clone 11Y) or germ-tubes (clone 24M). cDNA fragments were isolated by the digestion of lambda DNA with EcoRI. Southern blot analysis with these fragments as probes demonstrated homology with C. albicans DNA, and by Northern analysis two mRNAs transcripts were detected with sizes of approximately 1·5 kb for 11Y and 1·1 kb for 24M. Both transcripts were present in yeast cells as well as in germ-tubes. The whole genes were isolated from a C. albicans genomic library in the YRp7 vector by hybridization with the cDNA probes. Monospecific antibodies were purified from polyclonal antisera by affinity for the fusion proteins. Western blot analysis with 11Y-specific antibodie...

Mild alkaline solutions (β-elimination), after removing the non-covalently bonded wall materials by hot SDS, released 13% and 26% of remaining wall proteins from mycelial and yeast cells of Candida albicans, respectively. When the β-elimination was carried out after digestion of the walls with chitinase, four-fold more proteinaceous materials were released from mycelium and a similar amount in yeast walls. The solubilized materials were shown to be highly polydisperse, and endo-glycosidase H reduced their polydispersity and molecular masses, revealing different electrophoretic patterns in yeast and mycelial cell walls. The solubilized mycelial proteins carried N-glycosidic sugar chains and the epitopes recognized by two monoclonal antibodies were preserved, although showing a different behaviour in yeast walls. These results are consistent with the idea that significant amounts of intrinsic O-glycosylated mannoproteins are interconnected in the walls of C. albicans.

Iranzo, M., Marcilla, A., Elorza, M. V., Mormeneo, S., and Sentandreu, R. 1994. Preparation of anti-protein and anti-mannan antisera against fungal cell wall by affinity chromatography. Experimental Mycology 18, 159-167. A novel and easy chromatographic method has been developed for the isolation of anti-protein and anti-mannan antisera from a population of polyclonal antibodies obtained against Candida albicans and Yarrowia lipolytica cell wall mannoproteins. The technique is based on the immobilization of mannan (to be used as immunoadsorbent) by Affi-Prep H z resin after the oxidation of neighboring hydroxyl groups of the polysaccharide with sodium periodate. For Y. lipolytica polyclonal antiserum, a single chromatographic step using the homologous mannan was sufficient to obtain an antiprotein antibody preparation free of antimannan antibodies. For C. albicans , three chromatographic processes using homologous and heterologous mannan were needed to obtain a satisfactory antiprotein antiserum. The potential application of the anti-protein antiserum obtained has been demonstrated by indirect immunofluorescence assays of whole cells and electrophoretic analysis of wall proteins in C. albicans and Saccharomyces cerevisiae .

The kinectics of incorporation of two precursor mannoproteins into the regenerating cell wall of Candida albicans protoplasts have been followed at 28°C and 37°C using two monoclonal antibodies specific for protein epitopes (MAb 1B12 and 4C12) as probes. Both molecules were secreted from the beginning of the regeneration process, and their incorporation was retarded significantly. Analysis of the secreted materials by Western immunoblotting with MAb 1B12 allowed the identification of two closely migrating bands at apparent Mr higher than 170 kDa and significant amounts of a highly polydisperse material of even greater molecular mass. Some of these mannoproteinaceous species carried both N- and O-glycosidically linked mannose residues, as deduced from their drop in apparent Mr when synthesized in the presence of tunicamycin and by their reactivity with Concanavalin A. Following secretion, the molecules reacting with MAb 1B12 were incorporated into the regenerating walls by covalent binding. Then, when the regenerating walls by covalent binding. Then, when the antigen molecules were solubilized from partially regenerated walls, their mobility differed when regeneration took place at 28°C (blastoconidia) or 37°C (mycelial cells).

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.

Aculeacin A and papulacandin B block cell wall regeneration in Candida albicans protoplasts at an intermediate step in which the protoplasts have not yet synthesized the rigid structure of the cell wall and are therefore still osmotically sensitive. In the presence of the antibiotics, total synthesis of glucan is not significantly lowered with respect to control cells, although most of it appears either in the culture medium or in the regenerating wall as alkali-soluble glucan. Thus, it is proposed that echinocandins (such as aculeacin A) and papulacandins may not inhibit glucan synthesis per se but instead inhibit its incorporation into the supramolecular organization of the cell wall.

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) from C. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen, in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processes in vivo and in vitro or, alternatively, that both enzyme activities (active in vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml-1 of digitonin and 64 micrograms ml-1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 micrograms ml-1 of protease, whereas activity strongly decreases in the presence of 64 micrograms ml-1 of trypsin. Digitonin does not produce zymogen activation per se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.

Determination of Candida species represents an important problem derived from the clinical implications of the species belonging to this genus. DNA probes have already been used for the epidemiology of Candida albicans, as well as for taxonomic analysis of Candida and other genera, although these probes are based on non-species-specific DNA sequences. In this work we carried out a 48-h assay, allowing the identification of C. albicans from clinical isolates, using DNA probes based on C. albicans LEU2 and URA3 genes. Another probe related to C. albicans SEC18 gene was shown not to be C. albicans specific.

Development of new effective antifungal drugs is limited by the absence of specific target sites in the fungal cells. Knowledge of the fungal cell wall structure and biosynthesis is of interest in searching for a potential target site for new chemotherapeutic agents. Our group has demonstrated that the fungal cell wall is a metabolically active structure where interaction between distinct components occurs to give rise to the mature cell wall structure. Mannoproteins play an essential role in the cell wall organization, and there is evidence for the formation of covalent bonds between these molecules and the structural polymers (glucans and chitin) outside the plasma membrane. Such interactions, which specifically occur at the fungal cell wall, are of great interest in defining target sites for potential new chemotherapeutic agents, which may inhibit the interactions and, thus, lead to a defective cell wall formation and cell death.

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 β1,6-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. Copyright © 2009 John Wiley & Sons, Ltd.

Cell surface hydrophobicity (CSH) of blastoconidia and blastoconidia bearing germ tubes of Candida albicans ATCC 26555 was monitored by assessing attachment of polystyrene microspheres to the cell surface, and we found that mature hyphae were significantly hydrophobic. Treatment of intact cells with low concentrations of beta-glucanase (Zymolyase 20T) or proteases abolished or significantly reduced attachment of latex beads to hyphae. This effect paralleled an obvious reduction in CSH of the entire cell population, as measured by an aqueous-hydrocarbon biphasic partitioning assay. Analysis of the cell wall material released by Zymolyase and adsorbed on polystyrene microspheres indicated that germ tube-specific cell wall proteins and mannoproteins with apparent molecular masses of 20 to 67 kDa may be responsible for the hydrophobicity of hyphae. Zymolyase released from blastoconidia cell walls a different set of proteins and mannoproteins that were able to adsorb to polystyrene microbeads. Such molecular species might in turn be responsible for the CSH exhibited by blastoconidium populations as determined by the biphasic partitioning assay, although these probably hydrophobic components can be masked on the surface of blastoconidia, as the latter had no or very few latex microspheres attached to their surfaces. Treatment of cells of both C. albicans morphologies with 2-mercaptoethanol released qualitatively distinct species of polystyrene-adsorbed proteins and mannoproteins from yeast and mycelial cells. These observations suggested that hydrophobic proteins and mannoproteins that could be associated with CSH are bound to the cell wall structure through diverse types of linkages.

The effect of a lipase activity (EC 3.1.1.3) on the chitin synthetase from Candida albicans has been studied, both on the active and the trypsin activated enzyme. Removal of fatty acids from acylglycerides by lipase has an inhibitory effect on the activity as well as on the ‘in vitro’ activation process by trypsin in the membrane-bound enzyme and in the chitosomes. This would indicate that an adequate lipid environment is required for both the activation process and proper function of the synthetase activity.

Hyphal development in Candida albicans was selectively blocked by the ornithine decarboxylase competitive inhibitor 1,4-diaminobutanone (DAB). Inhibition of hyphal development required DAB during both yeast inoculum growth and subsequent incubation at 37 degrees C to induce mycelial growth. This effect was not due to general growth inhibition since DAB did not inhibit yeast growth, and reduced protein synthesis by 30% at most. Moreover, protein synthesis was unaffected by DAB when cells were pre-grown in drug-containing media. Since DAB inhibited dimorphic transition at 37 degrees C, morphology- and temperature-dependent protein synthesis could be distinguished. DAB stimulated the synthesis of several yeast wall-proteins, irrespective of morphology or growth temperature, and two at 37 degrees C only, but it inhibited the synthesis of a single mycelial-specific glycoprotein species.

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.

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.

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.

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.

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.

The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-beta-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional high-molecular-mass, highly polydispersed band was also detected. Beta-elimination of each fraction resulted in the disappearance of all antigen bands, suggesting that they are highly O-glycosylated. In addition, the electrophoretic mobility of the high-molecular-mass, highly polydispersed bands increased after digestion with endoglycosidase H, indicating that they are also N-glycosylated. New antigen bands were released when remnants of the cell walls extracted with 1,3-beta-glucanase or chitinase were digested with chitinase or 1,3-beta-glucanase. These results are consistent with the notion that, after secretion, parts of the O-glycosylated antigen molecules are transferred to an N-glycosylated protein(s). This molecular complex, as well as the remaining original 70 and 80 kDa antigen molecules, next bind to 1,3-beta-glucan or chitin, probably via 1,6-beta-glucan, and, in an additional step, to chitin or 1,3-beta-glucan. This process results in the final molecular product of each antigen, and their distribution in the cell walls.

A cDNA encoding a 37 kDa protein was isolated from an expression library using antibodies raised against mycelial cell walls fromCandida albicans.The 37 kDa protein has over 60% sequence identity with the 37 kDa laminin-binding protein (LBP) from humans and over 80% identity with the Yst proteins ofSaccharomyces cerevisiae. TheC. albicansprotein was named CaYst1. It was found in membrane and ribosome fractions but surprisingly, was not found in cell walls. Unlike the human LBP, CaYst1p does not bind laminin. These data indicate that CaYst1p is not a cell-surface receptor for laminin as has been proposed for the human LBP. Instead, like theS. cerevisiaeYst proteins, it appears to be a ribosomal protein. This conclusion is supported by the finding thatCaYST1-cDNA complements the lethal phenotype linked to the disruption of bothYSTgenes inS. cerevisiae.

The Candida albicans MKC1 gene encodes a mitogen-activated protein (MAP) kinase, which has been cloned by complementation of the lytic phenotype associated with Saccharomyces cerevisiae slt2 (mpk1) mutants. In this work, the physiological role of this MAP kinase in the pathogenic fungus C. albicans was characterized and a role for MKC1 in the biogenesis of the cell wall suggested based on the following criteria. First, C. albicans mkc1Δ/mkc1Δ strains displayed alterations in their cell surfaces under specific conditions as evidenced by scanning electron microscopy. Second, an increase in specific cell wall epitopes (O-glycosylated mannoprotein) was shown by confocal microscopy in mkc1Δ/mkc1Δ mutants. Third, the sensitivity to antifungals which inhibit (1,3)-β-glucan and chitin synthesis was increased in these mutants. In addition, evidence for a role for the MKC1 gene in morphological transitions in C. albicans is presented based on the impairment of pseudohyphal formation of mkc1Δ/mkc1Δ strains on Spider medium and on the effect of its overexpression on Sacch. cerevisiae colony morphology on SLADH medium. Using the two-hybrid system, it was also demonstrated that MKC1 is able to interact specifically with Sacch. cerevisiae Mkk1p and Mkk2p, the MAP-kinase kinases of the PKC1-mediated route of Sacch. cerevisiae, and to activate transcription in Sacch. cerevisiae when bound to a DNA-binding element. These results suggest a role for this MAP kinase in the construction of the cell wall of C. albicans and indicate its potential relevance for the development of novel antifungals.

Candida albicans is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of C. albicans was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designated PRA1 , for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The PRA1 protein was homologous to surface antigens of Aspergillus spp. which react with serum from aspergillosis patients, suggesting that the PRA1 protein may have a role in the host-parasite interaction during candidal infection.

In the course of an analysis of the functions and assembly of the cell wall of Candida albicans, we have cloned and characterized a gene, which we designated CSP37 (cell surface protein), encoding a 37-kDa polypeptide which is a membrane-associated protein. The gene was isolated by immunological screening of a DNA library constructed from mycelial cells with a polyclonal serum raised against cell walls of this morphology. Analysis of the nucleotide sequence of a corresponding genomic DNA fragment revealed a single open reading frame which encodes a predicted protein of 321 amino acids with no significant homology to others in the databases. Disruption of the CSP37 gene by the method described by Fonzi and Irwin (Genetics 134:717-728, 1993) eliminated expression of the Csp37 protein. The mutant strains showed no apparent defect in cell viability, growth, or cell wall assembly but displayed attenuated virulence in systemic infections induced in mice and reduced the ability to adhere to polystyrene.

The topological distribution of two epitopes in the cell wall of Candida albicans, the kinetics of their incorporation into the regenerating protoplast wall, and the effect of different antibiotics upon their incorporation and localization have been studied. To do so, two monoclonal antibodies that react against an O-glycosylated mannoprotein (1B12) and against a 1,6-beta-glucan epitope (JRR1) were used. The results show that the JRR1 epitope is localized in an internal layer of the cell wall, in contrast to the 1B12 epitope, which is superficial, and that the incorporation of the JRR1 epitope into walls of regenerating protoplasts precedes that of the 1B12 epitope. The JRR1 epitope is normally found in the culture medium of control cells, but not in that of papulacandin-B-treated cells, and tunicamycin interferes with the incorporation of the 1B12 epitope into the cell walls. Finally, the results support the hypothesis that mannoproteins are not 1,6-beta-glycosylated before their secretion.

Activity of the enzyme glutaminyl-peptide--glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N epsilon-(gamma-glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms of Candida albicans. Higher levels of enzymatic activity were observed in the cell wall fraction, whereas the cytosol contained only trace amounts of activity. Cystamine, a highly specific inhibitor of the enzyme, was used to analyze a possible role of transglutaminase in the organization of the cell wall structure of the fungus. Cystamine delayed protoplast regeneration and inhibited the yeast-to-mycelium transition and the incorporation of proteins into the cell wall. The incorporation of covalently bound high-molecular-weight proteins into the wall was sensitive to cystamine. Proteic epitopes recognized by two monoclonal antibodies, one of which is specific for the mycelial walls of the fungus, were also sensitive to cystamine. These data suggest that transglutaminase may be involved in the formation of covalent bonds between different cell wall proteins during the final assembly of the mature cell wall.

In invasive candidiasis, the identification of Candida organisms in tissue samples or in normally sterile fluids is essential for an accurate diagnosis. Species identification is an important clue for the source of infection and in epidemiological studies. In this article, the authors have tested the value of a new monoclonal antibody (1B12) to detect C albicans in culture by immunofluorescence, and in tissue samples by immunohistochemistry. MAb 1B12 was found to specifically recognize C albicans , does not cross-react with other Candida species or other structurally similar fungi, and is very sensitive and specific in paraffin-embedded tissue, having no reactivity in normal human tissues or necrotic areas. Therefore, MAb may be a valuable tool in the evaluation of fungal infections in paraffin-embedded tissue, particularly when Candida species identification is needed.

The organization of the components of the cell wall from Candida albicans was studied by means of sequential treatment with hot SDS, anhydrous ethylenediamine (EDA) and lytic enzymes, followed by chemical and microscopic analyses of the different separated fractions. The EDA-insoluble fraction retained the original morphology of the wall, which was destroyed by beta-glucanase, but not by chitinase treatments. Staining with fluorescent lectins revealed distinct distributions of mannoproteins, glucans and chitin in the wall. Amino acid analysis of SDS-extracted walls, and the EDA-soluble and -resistant fractions gave similar results, with seven amino acids making up about 70% of the total protein weight. Treatment of the EDA-insoluble fraction with Zymolyase or chitinase released fragments of variable size whose susceptibility to these and other hydrolases suggests that they are made of glucan, chitin and mannan oligomers associated with proteins. Treatment of the Zymolyase-insoluble residue with chitinase released a series of low-molecular-mass oligomers made of neutral sugars, GlcNAc and amino acids, mainly lysine. It is suggested that they represent fragments of the core making up the scaffold of the cell wall of the fungus.