Molecular Analysis of the Candida albicans Homolog of Saccharomyces cerevisiae MNN9, Required for Glycosylation of Cell Wall Mannoproteins

Candida albicans is a dimorphic, opportunistic fungal pathogen that is responsible for the majority of fungal infections in immunocompromised hosts (33). A great deal of attention has focused on understanding host-C. albicans interactions, in particular the elements that promote the virulence of the organism (14, 47). Putative virulence factors include morphogenesis (56), proteinase production (32), phenotypic switching (57), and adherence to host cells (8, 10). A number of systems which allow C. albicans to adhere to host tissues have been identified (reviewed in references 8 and 10). In most instances, cell wall-associated mannoproteins bind to specific components on host cells (8–10, 27). Cell wall mannan has also been implicated in other pathogenicity-related processes, including immunogenicity (10). Therefore, understanding the molecular biology of glycosylation, as well as that of other processes that relate to biosynthesis of the Candida cell wall, is critical to elucidating the pathogenicity of C. albicans. In turn, such investigations may facilitate the identification of genes which encode potential targets for improved antifungal therapy. To this end, information obtained from studies of these systems in Saccharomyces cerevisiae has proven essential in facilitating such research in Candida. The biosynthesis and structure of the mannan outer chain in S. cerevisiae have been studied extensively by Ballou and coworkers (2). Those investigations led to the isolation of a number of S. cerevisiae mannoprotein, or mnn, mutants (reviewed in references 2, 3, and 23), several of which show defects in glycosylation of secreted proteins (2–4). These mutants also exhibit phenotypes characteristic of defects in cell wall biosynthesis and/or assembly, including poor cell growth, flocculation in liquid media, clumpy growth on solid media, osmotic sensitivity, and aberrant sporulation. Of these mutants, the mnn9 strain suffers the most serious glycosylation defect. In this mutant, N-linked chains in which one α-1,6-mannose is attached to the core oligosaccharide are formed but further addition is blocked. Limited addition of α-1,2- and α-1,3-linked mannose residues results in the formation of a Man13GlcNAc2 structure (60). The MNN9 gene has been cloned (64), and an MNN9 gene family in S. cerevisiae has been identified based on sequence homology. The other members of this family include VAN1 and ANP1. VAN1 was isolated by complementation of vanadate-resistant mutant van1-18 (28) and has been shown to be allelic to vrg7, another vanadate-resistant mutant isolated in an independent screen (5). That screen also identified vrg mutants allelic to mnn9 (vrg6), as well as other mnn mutants. Like the mnn9 strain, van1 strains underglycosylate secreted invertase (5, 29). In addition, mnn9 and van1 strains have similar growth and sporulation defects. Both strains also show sensitivity to the aminoglycoside antibiotic hygromycin B, as well as resistance to sodium orthovanadate (5, 15, 28, 29). The third member of the MNN9 gene family is ANP1, which was originally identified within a cluster of genes whose deletion resulted in sensitivity to the chloramphenicol breakdown product amino nitrophenol propanediol (39). ANP1 has since been shown to complement gem3. The gem3 mutant was isolated in a screen for mutants that mislocalize a reporter protein that should reside in an early Golgi compartment (12). The anp1 (gem3) strain also grows slowly, is osmotically sensitive, exhibits clumpy growth in liquid and solid media, is sensitive to hygromycin and resistant to vanadate, and is defective in outer-chain glycosylation (12). All three genes encode putative type II membrane proteins. Disruption of the individual genes does not result in lethality (5, 12), but deletion of both ANP1 and VAN1 renders the doubly disrupted strain inviable (12). Recently, Jungmann and Munro (26) have shown that Mnn9p, Van1p, and Anp1p colocalize in the cis Golgi apparatus subcompartment in two separate complexes, both of which contain Mnn9p (26). The separate complexes were isolated by immunoprecipitation, and both were shown to have mannosyltransferase activity. One of the isolated complexes contains Van1p and Mnn9p alone, while the other complex contains Mnn9p, Anp1p, and two other tightly associated proteins, Hoc1p and an uncharacterized protein encoded by open reading frame (ORF) YJL183w (26). Hoc1p (45) is a Golgi apparatus protein with homology to Och1p (43), the mannosyltransferase which initiates mannan backbone synthesis. However, deletion of HOC1 does not lead to defects in protein glycosylation (45). The Yjl183w protein has clear homologies to Mnn10p (16) and an α-1,2-galactosyltransferase in Schizosaccharomyces pombe (13) and has since been renamed Mnn11p (26). The enzymatic studies with the partially purified complexes show they synthesize both α-1,6- (backbone) and α-1,2 (side chain)-mannose linkages, suggesting that the complexes have multiple enzymatic activities (26). While the specific catalytic functions of each of these proteins are yet to be resolved, it is suggested that in addition to the role they play in outer-chain synthesis one or more of the enzymes in these complexes may be involved in other processes responsible for maintaining the organization and function of the secretory pathway (25, 46). Additionally, MNN9 gene family members have been identified in screens for genes involved in the cell cycle-regulated progression of polarized growth in S. cerevisiae (41, 54). The C. albicans genome is diploid, yet no sexual cycle has been reported for this organism (31). Accordingly, genetic manipulation of this pathogen has been difficult in the past. In recent years, techniques developed for targeted gene disruption in Saccharomyces (1) have been adapted for use in Candida (18). This methodology has led to the successful disruption of many C. albicans genes, including genes involved in glycosylation (7, 53, 59), maintenance of cellular integrity (44, 48), and assembly and function of the cell wall (6, 20, 36, 40). Here, we report the identification of an MNN9 gene family in C. albicans and describe the isolation and characterization of one gene member of that family, C. albicans MNN9. Disruption of both copies of C. albicans MNN9 results in a phenotype suggestive of severe cell wall defects, including poor growth, flocullation in liquid medium, abnormal hyphal formation, and sensitivity to β-glucanase. Additionally, C. albicans Δmnn9 strains are sensitive to hygromycin B and resistant to vanadate. These results suggest that C. albicans MNN9 encodes a protein involved in glycosylation and/or secretion of cell wall-associated mannoproteins. In turn, the severe phenotypes exhibited by the CaΔmnn9 strain and the fact that no mammalian homologs of MNN9 gene family members have been identified suggest that C. albicans MNN9 would represent an attractive target for new antifungal drugs.