The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a gene-reconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acid-stable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of beta-eliminated (O-linked), acid-hydrolyzed (acid-labile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the beta-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual oligomannosyl residues inCandida mannoprotein, the major antigenic determinant located on the outer surface of the yeast cell wall. The single terminal aldehydes of oligomannosyl residues released by hydrolysis were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling was not biased by oligomannoside length; therefore, band fluorescence intensity was directly related to the relative abundance of individual oligomannoside moieties in heterogeneous samples. FACE analysis revealed the major oligomannosides released by acid hydrolysis and β-elimination of Fehling-precipitated mannan from Candida albicans, which were the same as those previously reported in studies based on mass and nuclear magnetic spectroscopic analysis. FACE was also amenable to the analysis of samples obtained by direct hydrolysis of whole yeast cells. Whole-cell acid hydrolysis and whole-cell β-elimination of two isolates each ofC. albicans, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis,C. rugosa, C. stellatoidea, and C. tropicalis resulted in oligomannoside gel banding patterns that were species and strain specific for the 16 isolates surveyed. Whereas some bands were specific for an individual isolate or species, other bands were shared by two or three species in various groupings. Differences in the mannoprotein composition of C. albicans A9 and four spontaneous cell surface mutants were also detected. Mannan “fingerprints,” or banding pattern profiles, derived from the electrophoretic mobilities of individual bands relative to the migration of acid-hydrolyzed dextran (relative migration index) yielded profiles characteristic of individual isolates not revealed by standard assimilation and biochemical profiles. FACE represents an accessible, sensitive, and quantitative analytical tool enabling the characterization of yeast mannan complexity.

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The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a gene-reconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acid-stable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of beta-eliminated (O-linked), acid-hydrolyzed (acid-labile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the beta-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.