PHR1 and PHR2 of Candida albicans Encode Putative Glycosidases Required for Proper Cross-Linking of β-1,3- and β-1,6-Glucans

Abstract

PHR1 and PHR2 encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen Candida albicans. These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo. Deletion of either gene results in a pH-conditional defect in cell morphology and virulence. Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and β-galactosidases. Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity. By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH. The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution. The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between β-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in β-1,3-glucan-associated β-1,6-glucans and, within several generations, a fivefold increase in the chitin content of the walls. The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis. Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between β-1,6- and β-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process β-1,3-glucans and make available acceptor sites for the attachment of β-1,6-glucans.

The role of the histidine kinase [) gene in the regulation of cell wall mannan and glucan biosynthesis

“…Of importance, we are currently measuring temporal events related to cell wall biosynthesis in CHK21, since compensatory changes occur as a consequence of deletions in genes encoding cell wall regulatory or structural genes. For example, Fonzi [28] has shown that deletions in PHR1, which processes L-1,3 glucans to facilitate attachment of L-1,6 glucans, resulted in an increased cell wall content of chitin that was preceded by an increase in crosslinking of chitin between L-1,6 glycosylated mannoproteins and chitin. Therefore, temporal studies with the chk1 mutant may provide observations on the cause^e¡ect relationship between glucan and mannan that we described in the current study.…”

“…All cultures were incubated at 37 ‡C for 24 h. For dry-weight determinations, cell samples in triplicate were collected on preweighed nitrocellulose ¢lters, and dried in vacuo. For hexose analysis of cell wall fractions, we followed the procedures of Fonzi [28] and Boone et al [32]. Cell samples were collected in triplicate from each culture by centrifugation, washed with ice-cold, sterile distilled water and stored at 370 ‡C until needed.…”

Virulence genes in the pathogenic yeast Candida albicans

“…PHR1 and PHR2 are glycosyl phosphatidyl inositol (GPI)-anchored cell wall proteins that exhibit a pH-dependent pattern of expression [31,110], being expressed at complementary pH (neutral or alkaline for PHR1 and acidic for PHR2). At alkaline pH, C. albicans phr1 mutants display a cell wall whose amount of chitin increases as the 1,6-L-D-glucan portion decreases [111], a result that can be explained in terms of PHR1 and PHR2 encoding glycosidases required for crosslinking of the L-glucan fractions of the cell wall [112]. phr1 null mutants showed a reduced virulence in systemic infection of BALB/c mice [113].…”

Generating cell surface diversity inCandida albicansand other fungal pathogens

“…Als3, expressed on hyphae, acts as an invasin, promoting uptake of C. albicans by host endothelium (Phan et al, 2007) (Sundstrom et al, 2002). Other GPI-CWPs act in cell wall biogenesis and remodelling, for example, the Gas/Phr family (Fonzi, 1999;Eckert et al, 2006), the Crh family and Ecm33 (Martinez-Lopez et al, 2006). GPI-anchored aspartyl proteinases or yapsins Sap9 and Sap10 also contribute to cell wall integrity (Albrecht et al, 2006).…”