Identification and differential gene expression of adhesin-like wall proteins in Candida glabrata biofilms.

An important initial step in biofilm development and subsequent establishment of fungal infections by the human pathogen Candida glabrata is adherence to a surface. Adherence is mediated through a large number of differentially regulated cell wall-bound adhesins. The fungus can modify the incorporation of adhesins in the cell wall allowing crucial adaptations to new environments. In this study, expression and cell wall incorporation of C. glabrata adhesins were evaluated in biofilms cultured in two different media: YPD and a semi-defined medium SdmYg. Tandem mass spectrometry of isolated C. glabrata cell walls identified 22 proteins including six adhesins: the novel adhesins Awp5 and Awp6, Epa3 and the previously identified adhesins Epa6, Awp2 and Awp4. Regulation of expression of these and other relevant adhesin genes was investigated using real-time qPCR analysis. For most adhesin genes, significant up-regulation was observed in biofilms in at least one of the culturing media. However, this was not the case for EPA6 and AWP2, which is consistent with their gene products already being abundantly present in planktonic cultures grown in YPD medium. Furthermore, most of the adhesin genes tested also show medium-dependent differential regulation. These results underline the idea that many adhesins in C. glabrata are involved in biofilm formation and that their expression is tightly regulated and dependent on environmental conditions and growth phase. This may contribute to its potential to form resilient biofilms and cause infection in various host tissues.

CandidaDB: a genome database for Candida albicans pathogenomics.

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.

Cell wall dynamics in yeast.

The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level. It is a dynamic and highly regulated structure. This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall.

Identification of SPT14/CWH6 as the yeast homologue of hPIG-A, a gene involved in the biosynthesis of GPI anchors.

Cwh6 is a temperature-sensitive cell wall mutant of Saccharomyces cerevisiae. CWH6 was found to be identical to SPT14, a gene that is highly homologous to both human PIG-A and to RFAK from Salmonella typhimurium. PIG-A and RFAK are involved in transferring N-acetylglucosamine to, respectively, a GPI anchor precursor and to lipopolysaccharides. Because cell walls of cwh6 are greatly reduced in mannose, and because some cell wall proteins are known to be incorporated into the cell wall through a GPI-anchor dependent mechanism, we propose that Spt14p/Cwh6p is involved in transferring N-acetylglucosamine to a precursor of GPI anchors. We further propose that the majority of cell wall proteins are incorporated into the cell wall through a GPI anchor.

The contribution of cell wall proteins to the organization of the yeast cell wall.

Our knowledge of the yeast cell wall has increased rapidly in the past few years, allowing for the first time a description of its structure in molecular terms. Two types of cell wall proteins (CWPs) have been identified that are covalently linked to beta-glucan, namely GPI-CWPs and Pir-CWPs. Both define a characteristic supramolecular complex or structural unit. The GPI building block has the core structure GPI-CWP-->beta1,6-glucan-->beta1,3-glucan, which may become extended with one or more chitin chains. The Pir building block is less well characterized, but preliminary evidence points to the structure, Pir-CWP-->beta1,3-glucan, which probably also may become extended with one or more chitin chains. The molecular architecture of the cell wall is not fixed. The cell can make considerable adjustments to the composition and structure of its wall, for example, during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH. When the cell wall is defective, dramatic changes can occur in its molecular architecture, pointing to the existence of cell wall repair mechanisms that compensate for cell damage. Finally, evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for the cell wall of the Ascomycetes.

A new tool for studying the molecular architecture of the fungal cell wall: one-step purification of recombinant trichoderma beta-(1-6)-glucanase expressed in Pichia pastoris.

The fungal cell wall is a supramolecular network of glycoproteins and polysaccharides. Its analysis is seriously hampered by the lack of easily available hydrolytic enzymes in a pure form. Here we describe a simple and efficient purification procedure of a recombinant beta-(1-6)-glucanase from Trichoderma harzianum expressed in Pichia pastoris. Transformed cells efficiently secreted the enzyme into the induction medium. We purified the enzyme using a one-step method based on hydrophobic interaction chromatography. The yield was 80%. SDS-PAGE of the purified enzyme revealed a single band with an apparent molecular mass of 43 kDa. The isoelectric point of the enzyme was 5.8, and it showed maximal enzyme activity and stability at pH 5.0. As beta-(1-6)-glucan is an important component of fungal cell walls, the easy availability of pure beta-(1-6)-glucanase will highly facilitate studies of the molecular organization of the fungal cell wall.

The retention mechanism of cell wall proteins in Saccharomyces cerevisiae. Wall-bound Cwp2p is beta-1,6-glucosylated.

It has been proposed that the cell wall proteins of Saccharomyces cerevisiae are anchored by means of a beta-1,6-glucose-containing side chain. Recently, we have identified three cell wall mannoproteins. Two of these mannoproteins are recognized in their cell wall bound form by an antiserum raised against beta-1,6-glucan but the third, Cwp2p, is not. This could indicate the existence of alternative retention mechanisms for cell wall proteins. Western analysis of a fusion protein consisting of Cwp2p and the reporter enzyme alpha-galactosidase revealed that this protein is glycosyl phosphatidylinositol-anchored in the intracellular precursor form and is recognized by an anti beta-1,6-glucan antiserum in the cell wall bound form. The cell wall bound forms of fusion proteins consisting of the anchor regions of Sed1p or Flo1p and alpha-galactosidase were also recognized by an anti beta-1,6-glucan antiserum. This is consistent with the existence of a general anchoring mechanism of proteins to the cell wall by means of a beta-1,6-glucose-containing carbohydrate chain. Western analysis of a yeast strain producing c-myc epitope tagged Cwp2p revealed that this protein is only detectable if fatty acid chains are present on the protein, indicating that the lack of recognition of Cwp2p by an anti beta-1,6-glucan antiserum is caused by a blotting artefact of the mature protein.

Characterization of agglutinin-like sequence genes from non-albicans Candida and phylogenetic analysis of the ALS family.

The ALS (agglutinin-like sequence) gene family of Candida albicans encodes cell-surface glycoproteins implicated in adhesion of the organism to host surfaces. Southern blot analysis with ALS-specific probes suggested the presence of ALS gene families in C. dubliniensis and C. tropicalis; three partial ALS genes were isolated from each organism. Northern blot analysis demonstrated that mechanisms governing expression of ALS genes in C. albicans and C. dubliniensis are different. Western blots with an anti-Als serum showed that cross-reactive proteins are linked by beta 1,6-glucan in the cell wall of each non-albicans Candida, suggesting similar cell wall architecture and conserved processing of Als proteins in these organisms. Although an ALS family is present in each organism, phylogenetic analysis of the C. albicans, C. dubliniensis, and C. tropicalis ALS genes indicated that, within each species, sequence diversification is extensive and unique ALS sequences have arisen. Phylogenetic analysis of the ALS and SAP (secreted aspartyl proteinase) families show that the ALS family is younger than the SAP family. ALS genes in C. albicans, C. dubliniensis, and C. tropicalis tend to be located on chromosomes that also encode genes from the SAP family, yet the two families have unexpectedly different evolutionary histories. Homologous recombination between the tandem repeat sequences present in ALS genes could explain the different histories for co-localized genes in a predominantly clonal organism like C. albicans.

Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae.

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.

Immobilizing proteins on the surface of yeast cells.

Yeast has a rigid cell wall comprising an outer layer of glycoproteins and an internal skeletal layer of glucan; heterologous proteins can be targeted to the glycoprotein layer and become covalently linked to the glucan skeleton. Yeast is a eukaryote that has 'generally regarded as safe' (GRAS) status, and is easy to cultivate, so it seems ideally suited for applications including the manufacture of recyclable, immobilized, biocatalysts, whole-cell vaccines, the presentation of peptide or antibody libraries, and the presentation of adhesion or metal-binding proteins.

Identification of beta-1,6-glucosylated cell wall proteins in yeast and hyphal forms of Candida albicans.

Several cell wall proteins released from yeast and hyphal cells of Candida albicans by laminarinase reacted with an affinity-purified antiserum raised against beta-1,6-glucan. Binding of the antiserum was competitively inhibited by beta-1,6-glucan, but not by beta-1,3-glucan or isolated N-chains. Immunodetection was completely abolished when the proteins were treated with periodate. These results demonstrate that the laminarinase-released wall proteins of C. albicans possess an epitope consisting of beta-1,6-linked glucose residues. The yeast form of C. albicans contained four beta-1,6-glucosylated wall proteins, an Endo H-resistant protein of 125 kDa and three glycoproteins which became only detectable after Endo H digestion and had a molecular mass of 320, 170 and 44 kDa, respectively. As for the hyphal form, a different set of beta-1,6-glucosylated wall proteins was found consisting of two Endo H-resistant glycoproteins of 125 and 80 kDa, respectively, and two glycoproteins that after Endo H digestion had a molecular mass of 320 and 38 kDa, respectively. Sodium dodecyl sulfate-extractable wall proteins and medium proteins did not react with the beta-1,6-glucan antiserum. The beta-1,6-glucan epitope could be removed by aqueous hydrofluoric acid indicating that the epitope is phosphodiester-linked to the cell wall proteins. It is speculated that the epitope forms part of a GPI-anchor and might be involved in the anchoring of mannoproteins into the cell wall.

Glucosylation of chimeric proteins in the cell wall of Saccharomyces cerevisiae.

Extension of a reporter protein with the carboxyterminal thirty amino acids of the cell wall mannoprotein alpha-agglutinin of Saccharomyces cerevisiae resulted in incorporation of the chimeric protein in the cell wall. By Western analysis it was shown that the incorporated protein contained beta-1,6-glucan similar to endogenous cell wall proteins, whereas excreted reporter protein was not glucosylated. This suggests that beta-1,6-glucan is involved in anchoring mannoproteins in the cell wall.

Identification of two cell cycle regulated genes affecting the beta 1,3-glucan content of cell walls in Saccharomyces cerevisiae.

The Calcofluor white-hypersensitive mutants cwh52 and cwh53 are severely reduced in beta 1,3-glucan. CWH52 was equivalent to GAS1. CWH53 represented a new gene, located on the right arm of chromosome XII, and predicted to encode a 215 kDa protein with multiple transmembrane domains. The transcription of CWH53 was cell cycle-dependent and, similar to GAS1/CWH52, increased in late G1, indicating that the formation of beta-glucan is cell cycle-regulated. Further, in some mutant alleles of both gas1/cwh52 and cwh53 lethal concentrations of Calcofluor induced growth arrest at a specific phase of the cell cycle.

Green fluorescent protein-cell wall fusion proteins are covalently incorporated into the cell wall of Saccharomyces cerevisiae.

Tagging of two cell wall mannoproteins, Cwp1p and Cwp2p, in Saccharomyces cerevisiae with the green fluorescent protein from Aequorea victoria resulted in incorporation of fluorescent fusion proteins into the cell wall. Both living cells and isolated cell walls became brightly labeled. Intriguingly, the incorporation patterns of both fusion proteins differed. Western analysis of enzymatically released fusion proteins showed that they were covalently linked to the beta-1,6-glucan part of the cell wall. Removal of the glycosylphosphatidyl-inositol anchor signal sequence of the green fluorescent protein-cell wall protein fusion proteins resulted in secretion of the proteins into the culture medium. These results indicate that green fluorescent protein-cell wall protein fusion proteins can be used as a convenient fluorescent marker to study the incorporation of specific cell wall proteins and the control mechanisms involved.

Glucosylation of cell wall proteins in regenerating spheroplasts of Candida albicans.

The cell wall of Candida albicans contains mannoproteins that are covalently associated with beta-1,6-glucan. When spheroplasts were allowed to regenerate a new cell wall, initially non-glucosylated cell wall proteins accumulated in the medium. While the spheroplasts became osmotically stable, beta-1,6-glucosylated proteins could be identified in their cell wall by SDS-extraction or beta-1,3-glucanase digestion. At later stages of regeneration, beta-1,3-glucosylated proteins were also found. Hence, incorporation of proteins into the cell wall is accompanied by extracellular coupling to beta-1,6-/beta-1,3-glucan. The SDS-extractable glucosylated proteins probably represent degradation products of wall proteins rather than their precursors. Tunicamycin delayed, but did not prevent the formation of beta-1,6-glucosylated proteins, demonstrating that beta-1,6-glucan is not attached to N-glycosidic side-chains of wall proteins.

Transcription of multiple cell wall protein-encoding genes in Saccharomyces cerevisiae is differentially regulated during the cell cycle.

The yeast cell wall consists of an internal skeletal layer and an outside protein layer. The synthesis of both beta-1,3-glucan and chitin, which together from the cell wall skeleton, is cell cycle-regulated. We show here that the expression of five cell wall protein-encoding genes (CWP1, CWP2, SED1, TIP1 and TIR1) is also cell cycle-regulated. TIP1 is expressed in G1 phase, CWP1, CWP2 and TIR1 are expressed in S/G2 phase, and SED1 in M phase. The data suggest that these proteins fulfil distinct functions in the cell wall.

The beta-1, 6-glucan containing side-chain of cell wall proteins of Saccharomyces cerevisiae is bound to the glycan core of the GPI moiety.

Cell wall proteins of Saccharomyces cerevisiae are anchored by means of a beta-1, 6-glucan-containing side-chain. It is not known whether this chain is linked to the protein part (e.g. through carbohydrate side-chains) or to the glycosylphosphatidylinositol (GPI) moiety of cell wall proteins. An IgA protease recognition site was introduced in Cwp2p, a beta-1, 6-glucosylated cell wall protein, immediately N-terminal from the omega amino acid (the attachment site of the GPI moiety). Proteolytic cleavage of this site revealed that the beta-1, 6-glucan epitope was not linked to the protein part. We conclude that neither N-or O-glycosylation is involved in beta-glucosylation of cell wall proteins. This confirms that the glycan core of the GPI moiety is the probable beta-1, 6-glucan attachment site.

Molecular and cellular mechanisms that lead to Candida biofilm formation.

Fungal infections in the oral cavity are mainly caused by C. albicans, but other Candida species are also frequently identified. They are increasing in prevalence, especially in denture-wearers and aging people, and may lead to invasive infections, which have a high mortality rate. Attachment to mucosal tissues and to abiotic surfaces and the formation of biofilms are crucial steps for Candida survival and proliferation in the oral cavity. Candida species possess a wide arsenal of glycoproteins located at the exterior side of the cell wall, many of which play a determining role in these steps. In addition, C. albicans secretes signaling molecules that inhibit the yeast-to-hypha transition and biofilm formation. In vivo, Candida species are members of mixed biofilms, and subject to various antagonistic and synergistic interactions, which are beginning to be explored. We believe that these new insights will allow for more efficacious treatments of fungal oral infections. For example, the use of signaling molecules that inhibit biofilm formation should be considered. In addition, cell-wall biosynthetic enzymes, wall cross-linking enzymes, and wall proteins, which include adhesins, proteins involved in biofilm formation, fungal-bacterial interactions, and competition for surface colonization sites, offer a wide range of potential targets for therapeutic intervention.

Glycosylated seryl residues in wall protein of elongating pea stems.

The protein content of salt-washed cell walls isolated from etiolated stems of Pisum sativum L. approximately doubled during elongation. In the same period the concentration in the wall of hydroxyproline, hydrazine-labile (= presumably glycosylated) serine, valine, tyrosine, lysine, and histidine increased markedly in comparison with other amino acids. After elongation was completed both the amino acid composition and the protein content of the cell wall changed only slightly. The ratio for the wall of hydrazine-labile seryl residues to hydroxyprolyl residues remained constant during and after elongation and was found to be 0.20. A linear relationship was established between the rate of elongation and the concentration in the wall of the hydroxyproline-rich glycoprotein both in vivo and in cut sections incubated in buffer.