Role of Protein O -Mannosyltransferase Pmt4 in the Morphogenesis and Virulence of Cryptococcus neoformans

Abstract

Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during motherdaughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans.Protein O glycosylation is a fundamentally important protein modification in eukaryotes, in which glycosyl residues are covalently attached to secreted proteins. In fungi, O glycosylation is often referred to as O mannosylation. In the yeast Saccharomyces cerevisiae, protein O mannosylation is essential for cell viability, cell wall formation, cell integrity, morphology, and budding, and the secretion, localization, and function of many proteins (6,22,35,36). In the human fungal pathogen, Candida albicans, O mannosylation is involved in morphogenic transitions and virulence (16,23). Furthermore, decreased O mannosylation of proteins in these fungi also results in cell wall changes leading to increased sensitivity to certain antifungal compounds and cell wall destabilizing agents (25,32,38,39).Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmts) that catalyze the transfer of mannose from the sugar donor, dolichol phosphate-mannose, to the hydroxyl groups of serine and threonine residues in the secreted acceptor protein. The Pmt enzymes have been extensively studied in S. cerevisiae, for which seven Pmt proteins have been described previously (37). Recently, five Pmts were described for C. albicans and three for the fission yeast Schizosaccharomyces pombe (32,46). Two PMT genes in S. pombe, OMA1 and OMA4, are not essential for viability. However, oma1⌬ and oma4⌬ mutants exhibit abnormal cell morphology, altered cell wall structure, and partial cell separation defects (46). The third S. pombe PMT gene,

TheO-Mannosyltransferase PMT4 Is Essential for Normal Appressorium Formation and Penetration inUstilago maydis

“…Pmt1 and pmt4 mutants did not exhibit enhanced sensitivity to any of these agents or conditions (see Supplemental Figure 7 online; data not shown) except for increased sensitivity to SDS by the Dpmt4 strain ( Figure 3E). SDS sensitivity has also been observed in C. neoformans pmt4 mutants and was linked to a failure of cell wall integrity (Olson et al, 2007).…”

“…Similarly, we found regions with homology to the MIR domain submotifs in all three Um PMTs (with amino acid similarity of between 48 and 64%). Um11220, Um10749, and Um05433 all possess the conserved initial Leu-His-Ser-His sequence of submotif A, while Um05433 also has a specific seven-amino acid insertion (Tyr-Asn-Asn-Gly-Arg-Ile-Ser) within submotif A, which is present in Sc Pmt4p, Sp OGM4, and Cn PMT4 (Olson et al, 2007;see Supplemental Figure 2 online).…”

“…By contrast, only three members have been found in the U. maydis genome, one for each of these subfamilies. Three PMT family members have also been described in S. pombe (Willer et al, 2005), C. neoformans (Olson et al, 2007;Willger et al, 2009), and Aspergillus fumigatus (Zhou et al, 2007). The increased number of PMT proteins in S. cerevisiae probably originates from a duplication event during evolution (reviewed in Dujon, 2006).…”

“…In order to determine other PMT targets, in silico studies based on the general characteristics of O-mannosylated proteins have been initiated (Hutzler et al, 2007). Mutations in PMT family proteins have been linked to defects in S. pombe morphogenesis (Willer et al, 2005), reduced pathogenicity of C. albicans (Timpel et al, 2000;Prill et al, 2005) and Cryptococcus neoformans (Olson et al, 2007;Willger et al, 2009), or, in the case of humans, to the WalterWarburg syndrome (Beltran-Valero de Bernabe et al, 2002;Lengeler et al, 2008).…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…This process mediates the import of secretory proteins into the ER and is highly conserved across eukaryotic organisms. PMTs have been well characterized for many fungal species and are involved in many important cellular processes, including the morphogenetic transitions of both C. albicans (469,580) and C. neoformans (434). In A. fumigatus, Pmt1 acts as an O-mannosyltransferase, and a ⌬pmt1 mutant strain has defects in cell wall integrity as well as cellular morphology (654).…”