The cell wall of the human pathogen Candida glabrata: differential incorporation of novel adhesin-like wall proteins.

The cell wall of the human pathogen Candida glabrata governs initial host-pathogen interactions that underlie the establishment of fungal infections. With the aim of identifying species-specific features that may directly relate to its virulence, we have investigated the cell wall of C. glabrata using a multidisciplinary approach that combines microscopy imaging, biochemical studies, bioinformatics, and tandem mass spectrometry. Electron microscopy revealed a bilayered wall structure in which the outer layer is packed with mannoproteins. Biochemical studies showed that C. glabrata walls incorporate 50% more protein than Saccharomyces cerevisiae walls and, consistent with this, have a higher mannose/glucose ratio. Evidence is presented that C. glabrata walls contain glycosylphosphatidylinositol (GPI) proteins, covalently bound to the wall 1,6-beta-glucan, as well as proteins linked through a mild-alkali-sensitive linkage to 1,3-beta-glucan. A comprehensive genome-wide in silico inspection showed that in comparison to other fungi, C. glabrata contains an exceptionally large number, 67, of genes encoding adhesin-like GPI proteins. Phylogenetically these adhesin-like proteins form different clusters, one of which is the lectin-like EPA family. Mass spectrometric analysis identified 23 cell wall proteins, including 4 novel adhesin-like proteins, Awp1/2/3/4, and Epa6, which is involved in adherence to human epithelia and biofilm formation. Importantly, the presence of adhesin-like proteins in the wall depended on the growth stage and on the genetic background used, and this was reflected in alterations in adhesion capacity and cell surface hydrophobicity. We propose that the large repertoire of adhesin(-like) genes of C. glabrata contributes to its adaptability and virulence.

Mass spectrometry-based proteomics of fungal wall glycoproteins.

The manifold functions of fungal wall glycoproteins include maintenance of cell wall integrity, homotypic and heterotypic adhesion, biofilm formation, acquisition of iron and sterols, protein degradation and coping with oxidative stress. Transcriptome studies indicate that the expression levels of most cell wall glycoproteins can vary widely and are tightly controlled. However, owing to their complex and variable glycosylation, fungal wall glycoproteins are difficult to analyze using traditional proteomics approaches. Recent advances in mass spectrometry-based proteomics have enabled rapid and sensitive identification and quantitation of fungal wall glycoproteins; this will be particularly useful for studying the dynamics of the subproteome of fungal wall glycoproteins, and for the development of novel vaccines and diagnostic tools.

Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae.

The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 x 10(3), 44 x 10(3), 38 x 10(3), 11 x 10(3) and 6.5 x 10(3) of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 x 10(3) copies microm(-2). For relative quantitation, we compared wild-type cells to gas1Delta cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi.

Mass spectrometric identification of covalently bound cell wall proteins from the fission yeast Schizosaccharomyces pombe.

The cell wall of Schizosaccharomyces pombe is bilayered, consisting of an inner layer of mainly polysaccharides and an outer layer of galactomannoproteins. We present a detailed analysis of the cell wall proteome. Six covalently-bound cell wall proteins (CWPs) were identified using tandem mass spectrometry, including four predicted GPI-dependent CWPs (Gas1p, Gas5p, Ecm33p and Pwp1p) and two alkali-sensitive CWPs (Psu1p and Asl1p). Gas1p and Gas5p belong to glycoside hydrolase family 72, and are believed to be involved in 1,3-beta-glucan elongation. Ecm33p belongs to a ubiquitous fungal protein family with an unknown but crucial function in cell wall integrity. Pwp1p is an abundant protein with an unknown but probably non-enzymatic function. All four CWPs were present in HF-pyridine extracts, indicating that they are linked via a phosphodiester bridge to the glucan network. Psu1p is a homologue of the Saccharomyces cerevisiae Sun family, whereas Asl1p has no homologues in S. cerevisiae but is related to Aspergillus fumigatus and Ustilago maydis proteins. Finally, although the protein content of Sz. pombe cell walls is only slightly less than in S. cerevisiae and Candida albicans, the amount of carbohydrate added to the proteins was found to be two- to three-fold decreased, consistent with earlier reported differences in outer chain N-glycosylation.

Identification of cell wall-associated proteins from Phytophthora ramorum.

The oomycete genus Phytophthora comprises a large group of fungal-like plant pathogens. Two Phytophthora genomes recently have been sequenced; one of them is the genome of Phytophthora ramorum, the causal agent of sudden oak death. During plant infection, extracellular proteins, either soluble secreted proteins or proteins associated with the cell wall, play important roles in the interaction with host plants. Cell walls of P. ramorum contain 1 to 1.5% proteins, the remainder almost exclusively being accounted for by glucan polymers. Here, we present an inventory of cell-wall-associated proteins based on mass spectrometric sequence analysis of tryptic peptides obtained by proteolytic digestion of sodium dodecyl sulfate-treated mycelial cell walls. In total, 17 proteins were identified, all of which are authentic secretory proteins. Functional classification based on homology searches revealed six putative mucins or mucin-like proteins, five putative glycoside hydrolases, two transglutaminases, one annexin-like protein, the elicitin protein RAM5, one protein of unknown function, and one Kazal-type protease inhibitor. We propose that the cell wall proteins thus identified are important for pathogenicity.

The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH.

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

Comprehensive proteomic analysis of Saccharomyces cerevisiae cell walls: identification of proteins covalently attached via glycosylphosphatidylinositol remnants or mild alkali-sensitive linkages.

The cell wall of yeast contains proteins that are covalently bound to the glycan network. These cell wall proteins (CWPs) mediate cell-cell interactions and may be involved in cell wall biosynthesis. Using tandem mass spectrometry, we have identified 19 covalently bound CWPs of Saccharomyces cerevisiae. Twelve of them are shown for the first time to be covalently incorporated into the cell wall. The identified proteins include 12 predicted glycosylphosphatidylinositol-modified CWPs, all four members of the Pir protein family, and three additional proteins (Scw4p, Scw10p, and Tos1p) that are, like Pir proteins, connected to the cell wall glycan network via an alkali-sensitive linkage. However, Scw4p, Scw10p, and Tos1p do not contain internal repeat sequences shown to be essential for Pir protein incorporation and may represent a separate class of CWPs. Strikingly, seven of the identified proteins (Gas1p, Gas3p, Gas5p, Crh1p, Utr2p, Scw4p, and Scw10p) are classified as glycoside hydrolases. Phenotypic analysis of deletion mutants lacking the corresponding CWP-encoding genes indicated that most of them have altered cell wall properties, which reinforces the importance of the identified proteins for proper cell wall formation. In particular, gas1Delta and ecm33Delta were highly sensitive to Calcofluor White and high temperature, whereas gas1Delta, scw4Delta, and tos1Delta were highly resistant to incubation with beta-1,3-glucanase. The CWP identification method developed here relies on directly generating tryptic peptides from isolated cell walls and is independent of the nature of the covalent linkages between CWPs and cell wall glycans. Therefore, it will probably be equally effective in many other fungi.