Molecular and Phenotypic Analysis ofCaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter

Abstract

Cell surface mannan is implicated in almost every aspect of pathogenicity of Candida albicans. In Saccharomyces cerevisiae, the Vrg4 protein acts as a master regulator of mannan synthesis through its role in substrate provision. The substrate for mannosylation of proteins and lipids in the Golgi apparatus is GDP-mannose, whose lumenal transport is catalyzed by Vrg4p. This nucleotide sugar is synthesized in the cytoplasm by pathways that are highly conserved in all eukaryotes, but its lumenal transport (and hence Golgi apparatusspecific mannosylation) is a fungus-specific process. To begin to study the role of Golgi mannosylation in C. albicans, we isolated the CaVRG4 gene and analyzed the effects of loss of its function. CaVRG4 encodes a functional homologue of the S. cerevisiae GDP-mannose transporter. CaVrg4p localized to punctate spots within the cytoplasm of C. albicans in a pattern reminiscent of localization of Vrg4p in the Golgi apparatus in S. cerevisiae. Like partial loss of ScVRG4 function, partial loss of CaVRG4 function resulted in mannosylation defects, which in turn led to a number of cell wall-associated phenotypes. While heterozygotes displayed no growth phenotypes, a hemizygous strain, containing a single copy of CaVRG4 under control of the methioninerepressible MET3 promoter, did not grow in the presence of methionine and cysteine, demonstrating that CaVRG4 is essential for viability. Mutant Candida vrg4 strains were defective in hyphal formation but exhibited a constitutive polarized mode of pseudohyphal growth. Because the VRG4 gene is essential for yeast viability but does not have a mammalian homologue, it is a particularly attractive target for development of antifungal therapies.Candida albicans is the most important human fungal pathogen. It causes infections that range from superficial colonization of oral and vaginal tissues to life-threatening infections in severely immunocompromised hosts. An essential step for the colonization and infection of host tissues is adhesion, which is initiated by the outermost components of the fungal cell wall. Cell wall-associated mannosylated proteins (mannans) on the external layer of the cell wall have been implicated as key determinants that mediate these initial and critical interactions between the fungus and its host (for reviews, see references 8, 10, and 11). The mannose branches on these glycoproteins, attached by N-and O-glycosidic linkages, are the structures recognized during the immune response against the pathogen (for reviews, see references 3, 14, and 15). Therefore, the enzymes that regulate addition of the mannose molecules are the focus of intense research for understanding fungal biology and mechanisms of host defense and as potential antifungal drug targets.Biogenesis of mannoproteins in Saccharomyces cerevisiae has been well characterized (for a review see reference 40). Following the initial glycosylation steps in the endoplasmic reticulum, yeast mannoproteins are elongated by addition of mannose or mannosylphosphate in the Golgi...

Outer Chain N-Glycans Are Required for Cell Wall Integrity and Virulence of Candida albicans

“…The carbohydrate structure consists of the Man 8 GlcNAc 2 core that is extensively elaborated in the Golgi by the addition of the highly branched outer chain. Previous studies have demonstrated the overall importance of glycosylation to the cell wall structure and virulence (25)(26)(27). Loss of O-mannan has been shown to result in reduced adherence and the attenuation of virulence, suggesting that O-mannan may act as a ligand in the interaction with host surfaces (12, 14, 28 -31).…”

“…Null Mutant-Previously, the electrophoretic mobility of secreted acid phosphatase on native gels has been used as a maker of N-glycosylation status (13,25,27). However, no acid phosphatase activity was detected in zymograms of the Caoch1⌬ null mutant.…”

“…The side chains and positions within the core are also modified by the addition of phosphomannan, consisting of a chain of ␤1,2-linked mannose residues attached via a phosphodiester bond (24). Previous studies in C. albicans have demonstrated the overall importance of glycosylation for cellular viability and virulence (25)(26)(27). Analysis of the PMT and MNT gene families required for O-glycosylation has demonstrated its relative importance in the host-fungal interaction and virulence (12, 14, 28 -31).…”

Biosynthesis of the Fungal Cell Wall Polysaccharide Galactomannan Requires Intraluminal GDP-mannose

“…GDP-mannose (GDP-Man) transporters have been studied in various organisms, such as the protozoan parasite Leishmania (20), S. cerevisiae (21), plants (22), and various fungi (23)(24)(25), including two Aspergilli (26,27). Mammalian cells do not possess any * This work was supported by the Deutsche Forschungsgemeinschaft.…”

“…In fungi, GDP-Man transport is indispensable for modification of protein N-and O-glycans (28 -30), glycolipid biosynthesis (31), and GPI anchor substitution (32,33). It has been reported to be essential in Saccharomyces (21), Candida (23), and Aspergillus niger (26).…”