PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of beta-1,3- and beta-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 beta-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 beta-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in beta-1,3-glucan-associated beta-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 beta-1,6- and beta-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process beta-1,3-glucans and make available acceptor sites for the attachment of beta-1,6-glucans.

FIG. 1

Alignment of Phr1p homologs and β-galactosidase sequences. The sequences of Phr1p, Phr2p, and seven related proteins were compared with β-galactosidases from 12 different microorganisms. Only four of the sequences are shown for simplicity. Boxed residues were present in at least 14 of the sequences. Asterisks indicate amino acids that were present in all 21 sequences. The dashes indicate gaps introduced to optimize the alignment. The underlined regions indicate conserved blocks identified by MACAW analysis. Each of the blocks had a P value of 0 and a search space (N) of 1.34⁶⁰. S. xylosus, Staphylococcus xylosus.

FIG. 2

Effect of site-specific mutagenesis on the function of PHR1. Mutant alleles of PHR1 were created by site-specific mutagenesis to introduce a silent mutation, an E169Q missense mutation, or an E270Q missense mutation. These mutants were tested for their ability to restore morphological development to a phr1Δ strain. The strains were induced to form hyphae in Medium 199 as previously described (27, 41).

FIG. 3

Elution profile of [¹⁴C]glucose-labeled Zymolyase-soluble material fractionated by molecular sieve chromatography. (A) A Phr1⁺ control strain or a Phr1⁻ null mutant was cultured at the restrictive pH for 60, 120, or 240 min and pulse-labeled for 60 min with [¹⁴C]glucose. The Zymolyase-solubilized wall material was prepared and fractionated on a 1.5- by 100-cm column of Bio-Gel A 5m. Since identical profiles were obtained for all three time points of the control strain, only one is shown. (B) Zymolyase-soluble material was prepared from a Phr2⁺ control strain or a Phr2⁻ mutant cultured 240 min at the restrictive pH and then labeled for 60 min. The material was chromatographed as described for panel A.

FIG. 4

Pulse-chase analysis of Zymolyase-soluble fraction. The Zymolyase-soluble fraction was prepared from control cells (A) or Phr1⁻ cells (B) and chromatographed on a 1.5- by 100-cm column of Bio-Gel A 5m. The cells were pulse-labeled after 4 h at the restrictive pH, and label was chased for 4 h at the permissive pH.

FIG. 5

Effect of alkali extraction on chitinase-solubilized material. A cell wall fraction was prepared from the phr2Δ mutant cultured 4 h at the restrictive pH. Half of the material was extracted with alkali prior to sequential digestion with Zymolyase and chitinase. The other half was not extracted prior to digestion. The chitinase-solubilized material was fractionated on a column of HW-55S, and the hexose in each fraction was measured as described in reference .