Morphogenesis, adhesive properties, and antifungal resistance depend on the Pmt6 protein mannosyltransferase in the fungal pathogen candida albicans

Abstract

Protein mannosyltransferases (Pmt proteins) initiate O glycosylation of secreted proteins in fungi. We have characterized PMT6, which encodes the second Pmt protein of the fungal pathogen Candida albicans. The residues of Pmt6p are 21 and 42% identical to those of C. albicans Pmt1p and S. cerevisiae Pmt6p, respectively. Mutants lacking one or two PMT6 alleles grow normally and contain normal Pmt enzymatic activities in cell extracts but show phenotypes including a partial block of hyphal formation (dimorphism) and a supersensitivity to hygromycin B. The morphogenetic defect can be suppressed by overproduction of known components of signaling pathways, including Cek1p, Cph1p, Tpk2p, and Efg1p, suggesting a specific Pmt6p target protein upstream of these components. Mutants lacking both PMT1 and PMT6 are viable and show pmt1 mutant phenotypes and an additional sensitivity to the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The lack of Pmt6p significantly reduces adherence to endothelial cells and overall virulence in a mouse model of systemic infection. The results suggest that Pmt6p regulates a more narrow subclass of proteins in C. albicans than Pmt1p, including secreted proteins responsible for morphogenesis and antifungal sensitivities.

FIG. 1

Sequential disruption of PMT6 alleles. (A) Schematic representation of the construction of the different alleles. The wild-type PMT6 gene and the PMT6 alleles disrupted by the hisG-URA3-hisG cassette or by hisG are shown. H, HindIII; N, NcoI; X, XhoI, S, SalI; P, PstI; B, BglII; A, Asp718. The fragment marked by asterisks was used as a probe for Southern analysis. (B and C) Southern blots of HindIII-BglII-digested chromosomal DNA of the following strains: SC5314 (PMT6/PMT6; lane 1 [B and C]); CAP2-2 (PMT6/pmt6Δ::hisG-URA3-hisG; lane 2 [B]); CAP2-23 (PMT6/pmt6Δ::hisG; lane 3 [B]); CAP2-239 (pmt6Δ::hisG-URA3-hisG/pmt6Δ::hisG; lane 4 [B]); CAP2-2391 (pmt6Δ::hisG/pmt6Δ::hisG; lane 5 [B]); CPP1 (PMT6/pmt6Δ::hisG-URA3-hisG; lane 2 [C]); CPP11 (PMT6/pmt6Δ::hisG; lane 3 [C]); CPP117 (pmt6Δ::hisG-URA3-hisG/pmt6Δ::hisG; lane 4 [C]); CPP1171 (pmt6Δ::hisG/pmt6Δ::hisG; lane 5 [C]).

FIG. 2

Sensitivities of C. albicans strains. The wild-type strain SC5314 (PMT1/PMT1 PMT6/PMT6) was compared with strain CAP2-2 (PMT1/PMT1 PMT6/pmt6), strains CAP2-234 and CAP2-2341 (PMT1/PMT1 pmt6/pmt6), strain CPP1 (pmt1/pmt1 PMT6/pmt6), and strains CPP117 and CPP1171 (pmt1/pmt1 pmt6/pmt6). Plasmid pCT34 carries PMT6, and plasmid pCT30 carries PMT1. Strains were grown on YPD medium without or with hygromycin B (200 μg/ml) or on SD medium without or with EDDHA (300 μM). The plates were incubated for 2 days at 30°C.

FIG. 3

Hypha formation of C. albicans strains. Shown are sections of colonies grown for 3 days on Spider medium at 37°C. (A) Phenotypes of pmt single mutants. The indicated pmt6/pmt6 strains were complemented with a plasmid carrying PMT6 (pCT34) or PMT1 (pCT30); as controls a pmt1/pmt1 strain complemented by PMT6 (pCT35) and a wild-type strain (SC5314) were analyzed. (B) Phenotypes of pmt double mutants. The indicated pmt1/pmt1 pmt6/pmt6 double mutants were transformed with plasmid pCT30 (PMT1) or pCT35 (PMT6). (C) Suppression of the pmt6 phenotype by genes encoding signaling components. Strain CAP2-2391 (pmt6/pmt6) was transformed with plasmids carrying EFG1, CEK1, CPH1, or TPK2 genes (Table 1). As a control, an efg1/efg1 mutant strain transformed with pCT35 (PMT6) was analyzed.

FIG. 4

PMT6 and PMT1 transcripts during hyphal induction. The wild-type strain CAI4(pRC2312) was induced to form hyphae in the presence of 2.5 mM GlcNAc. At the indicated times the percentages of hypha-forming cells were determined and total RNA was prepared and analyzed by Northern blotting using probes for the indicated genes.

FIG. 5

Virulence of C. albicans strains. Strains CAP2-239 (●; pmt6/pmt6) and the PMT6-reconstituted strain CAP2-2391(pCT35) (⧫; pmt6/pmt6 [PMT6]) were compared. The survival of mice (n = 12) injected with 10⁵ C. albicans cells in the tail vein was determined.