Loss of Cell Wall Mannosylphosphate in Candida albicans Does Not Influence Macrophage Recognition

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The outer layer of the Candida albicans cell wall is enriched in highly glycosylated mannoproteins that are the immediate point of contact with the host and strongly influence the host-fungal interaction. N-Glycans are the major form of mannoprotein modification and consist of a core structure, common to all eukaryotes, that is further elaborated in the Golgi to form the highly branched outer chain that is characteristic of fungi. In yeasts, outer chain branching is initiated by the action of the ␣1,6-mannosyltransferase Och1p; therefore, we disrupted the C. albicans OCH1 homolog to determine the importance of outer chain N-glycans on the host-fungal interaction. Loss of CaOCH1 resulted in a temperature-sensitive growth defect and cellular aggregation. Outer chain elongation of N-glycans was absent in the null mutant, demonstrated by the lack of the ␣1,6-linked polymannose backbone and the underglycosylation of N-acetylglucosaminidase. A null mutant lacking OCH1 was hypersensitive to a range of cell wall perturbing agents and had a constitutively activated cell wall integrity pathway. These mutants had near normal growth rates in vitro but were attenuated in virulence in a murine model of systemic infection. However, tissue burdens for the Caoch1⌬ null mutant were similar to control strains with normal N-glycosylation, suggesting the host-fungal interaction was altered such that high burdens were tolerated. This demonstrates the importance of N-glycan outer chain epitopes to the host-fungal interaction and virulence.Candida albicans is a commensal organism carried by a significant proportion of healthy individuals. It is the most common opportunistic fungal pathogen of humans causing superficial infections of the mucosa and in the immunocompromised host life-threatening systemic infections (1-4). The cell wall is the immediate point of contact between fungus and host and plays an important role in adherence, antigenicity, and the modulation of the host immune response (5-9). The outer layer of the cell wall is enriched in highly glycosylated mannoproteins (10), and both the protein and carbohydrate components have been implicated in the host-fungal interaction (5, 6, 11). The study of glycosylation in C. albicans therefore has its own relevance in identifying the carbohydrate epitopes involved in pathogenesis.Cell surface mannoproteins contain both O-and N-linked oligosaccharides. The O-linked oligosaccharides, attached to serine or threonine, consist of a linear chain of one to five ␣1,2-linked mannose residues (12)(13)(14) and are known to be required for full virulence (14). The process of N-glycosylation has been studied extensively in Saccharomyces cerevisiae. N-Linked glycosylation is initiated in the endoplasmic reticulum with the transfer of the Glc 3 Man 9 GlcNAc 2 oligosaccharide precursor to the protein target (15, 16). The oligosaccharide precursor is then processed by endoplasmic reticulum-resident glucosidases and a mannosidase to yield the mature triantennary Man 8 GlcNAc 2 core (17). Outer chain ...

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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

Bates

Hughes

Munro

et al. 2006

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277

“…Phosphomannan has been implicated in the interaction of C. albicans with macrophages. However, deletion of the C. albicans MNN4 gene, which is required for mannosyl phosphate transfer, demonstrated that phosphomannan is not required for macrophage interactions or virulence of C. albicans (26). Genes such as…”

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confidence: 99%

Candida albicans Pmr1p, a Secretory Pathway P-type Ca2+/Mn2+-ATPase, Is Required for Glycosylation and Virulence

Bates

MacCallum

Bertram

et al. 2005

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213

The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn 2؉ as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca 2؉ and Mn 2؉ ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn 2؉ -dependent mannosyltransferases. The Capmr1⌬ null mutant was viable in vitro and had no growth defect even on media containing low Ca 2؉ /Mn 2؉ ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.

“…Furthermore, an interesting observation is that members of the BMT gene family are present in only a limited number of known fungal species, but among these is the prominent fungal pathogen A. fumigatus (60). Despite these findings, recently published papers have contradicted this notion and led to the conclusion that ␤-Man may actually have little effect on C. albicans virulence (20,21). However, the strategy followed in these papers did not specifically target ␤-Man, but rather was based on the inactivation of C. albicans genes, homologous to S. cerevisiae genes, which prevented downstream association of ␤-Man to PPM (20,21).…”

Section: Discussionmentioning

confidence: 99%

Identification of a New Family of Genes Involved in β-1,2-Mannosylation of Glycans in Pichia pastoris and Candida albicans

Mille

Bobrowicz

Trinel

et al. 2008

Structural studies of cell wall components of the pathogenic yeast Candida albicans have demonstrated the presence of ␤-1,2-linked oligomannosides in phosphopeptidomannan and phospholipomannan. During C. albicans infection, ␤-1,2-oligomannosides play an important role in host/pathogen interactions by acting as adhesins and by interfering with the host immune response. Despite the importance of ␤-1,2-oligomannosides, the genes responsible for their synthesis have not been identified. The main reason is that the reference species Saccharomyces cerevisiae does not synthesize ␤-linked mannoses. On the other hand, the presence of ␤-1,2-oligomannosides has been reported in the cell wall of the more genetically tractable C. albicans relative, P. pastoris. Here we present the identification, cloning, and characterization of a novel family of fungal genes involved in ␤-mannose transfer. Employing in silico analysis, we identified a family of four related new genes in P. pastoris and subsequently nine homologs in C. albicans. Biochemical, immunological, and structural analyses following deletion of four genes in P. pastoris and deletion of four genes acting specifically on C. albicans mannan demonstrated the involvement of these new genes in ␤-1,2-oligomannoside synthesis. Phenotypic characterization of the strains deleted in ␤-mannosyltransferase genes (BMTs) allowed us to describe the stepwise activity of Bmtps and acceptor specificity. For C. albicans, despite structural similarities between mannan and phospholipomannan, phospholipomannan ␤-mannosylation was not affected by any of the CaBMT1-4 deletions. Surprisingly, depletion in mannan major ␤-1,2-oligomannoside epitopes had little impact on cell wall surface ␤-1,2-oligomannoside antigenic expression.