Surface stress induces a conserved cell wall stress response in the pathogenic fungus Candida albicans.

The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using (15)N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the ß-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.

Mass spectrometric quantification of the adaptations in the wall proteome of Candida albicans in response to ambient pH.

The mucosal layers colonized by the pathogenic fungus Candida albicans differ widely in ambient pH. Because the properties and functions of wall proteins are probably pH dependent, we hypothesized that C. albicans adapts its wall proteome to the external pH. We developed an in vitro system that mimics colonization of mucosal surfaces by growing biomats at pH 7 and 4 on semi-solid agarose containing mucin as the sole nitrogen source. The biomats expanded radially for at least 8 days at a rate of ~30 µm h(-1). At pH 7, hyphal growth predominated and growth was invasive, whereas at pH 4 only yeast and pseudohyphal cells were present and growth was noninvasive. Both qualitative mass spectrometric analysis of the wall proteome by tandem mass spectrometry and relative quantification of individual wall proteins (pH 7/pH 4), using Fourier transform mass spectrometry (FT-MS) and a reference mixture of (15)N-labelled yeast and hyphal walls, identified similar sets of >20 covalently linked wall proteins. The adhesion proteins Als1 and Als3, Hyr1, the transglucosidase Phr1, the detoxification enzyme Sod5 and the mammalian transglutaminase substrate Hwp1 (immunological detection) were only present at pH 7, whereas at pH 4 the level of the transglucosidase Phr2 was >35-fold higher than at pH 7. Sixteen out of the 22 proteins identified by FT-MS showed a greater than twofold change. These results demonstrate that ambient pH strongly affects the wall proteome of C. albicans, show that our quantitative approach can give detailed insights into the dynamics of the wall proteome, and point to potential vaccine targets.

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

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

Hypoxic conditions and iron restriction affect the cell-wall proteome of Candida albicans grown under vagina-simulative conditions.

Proteins that are covalently linked to the skeletal polysaccharides of the cell wall of Candida albicans play a major role in the colonization of the vaginal mucosal surface, which may result in vaginitis. Here we report on the variability of the cell-wall proteome of C. albicans as a function of the ambient O(2) concentration and iron availability. For these studies, cells were cultured at 37 degrees C in vagina-simulative medium and aerated with a gas mixture consisting of 6 % (v/v) CO(2), 0.01-7 % (v/v) O(2) and N(2), reflecting the gas composition in the vaginal environment. Under these conditions, the cells grew exclusively in the non-hyphal form, with the relative growth rate being halved at approximately 0.02 % (v/v) O(2). Using tandem MS and immunoblot analysis, we identified 15 covalently linked glycosylphosphatidylinositol (GPI) proteins in isolated walls (Als1, Als3, Cht2, Crh11, Ecm33, Hwp1, Pga4, Pga10, Phr2, Rbt5, Rhd3, Sod4, Ssr1, Ywp1, Utr2) and 4 covalently linked non-GPI proteins (MP65, Pir1, Sim1/Sun42, Tos1). Five of them (Als3, Hwp1, Sim1, Tos1, Utr2) were absent in cells grown in rich medium. Immunoblot analysis revealed that restricted O(2) availability resulted in higher levels of the non-GPI protein Pir1, a putative beta-1,3-glucan cross-linking protein, and of the GPI-proteins Hwp1, an adhesion protein, and Pga10 and Rbt5, which are involved in iron acquisition. Addition of the iron chelator ferrozine at saturating levels of O(2) resulted in higher cell wall levels of Hwp1 and Rbt5, suggesting that the responses to hypoxic conditions and iron restriction are related.

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.