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.Secreted proteins in fungi can get modified by the attachment of short glycosyl chains consisting of one to seven mannoses to serine or threonine residues (reviewed in reference 38). The first mannosylation step in O glycosylation occurs in the endoplasmic reticulum, presumably cotranslationally, and is mediated by protein mannosyltransferases (Pmt proteins). In the yeast Saccharomyces cerevisiae seven PMT genes are known (20,24,28,37); their paralogous gene products, by their degree of homology, can be grouped in at least two subclasses consisting of either the Pmt1 and Pmt5 proteins or the Pmt2, Pmt3, and Pmt6 proteins (10). We recently isolated and characterized the PMT1 gene of the important human fungal pathogen Candida albicans (41). Pmt homologues in Drosophila melanogaster (25) and humans (21) have also been described, and Pmt homologues deduced from "expressed sequence tags" occur in nematodes, plants, and mammals, although their enzymatic functions as Pmt proteins have not yet been demonstrated. In C. albicans, O-glycosyl chains initiated by Pmt proteins are extended further by mannosyltransferases including Mnt1p (3).Although the molecular details of target protein recognition by Pmt proteins are unknown, it appears that Pmt proteins can have a preference for certain glycosylation targets. Thus, the lack of Pmt1 and Pmt2 proteins in S. cerevisiae mutants affects O glycosylation of a set of secreted proteins overlapping with, but different from, the set affected in mutants lacking Pmt4p (16), and certain cell wall proteins are affected differently by mutations in PMT genes (28). Recently, the Axl2p protein, involved in axial budding, was recognized as a specific target of mannosylation by Pmt4p (29). Unknown O-glycosylated proteins are needed for gener...