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

Abstract

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.

Regulation of Intracellular Manganese Homeostasis by Kufor-Rakeb Syndrome-associated ATP13A2 Protein

“…A number of P-type ATPase ion channels have been suggested to transport Mn 2ϩ in yeast, Caenorhabditis elegans, and other lower organisms (31)(32)(33)(34)(35). Interestingly, ATP13A2 is a P5-type ATPase localized in lysosomal membranes and is expressed ubiquitously in various tissues with particularly high levels in brain (1,36).…”

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

“…Strains were tested for sensitivity to cell wall stressing agents by microdilution testing as described previously (27). Standardized inocula were prepared from 24-h YEPD cultures.…”

“…Cells Cell Wall Analysis and Sensitivity Testing-Alcian blue binding was assayed essentially as described previously (27,32). A suspension of ϳ1 ϫ 10 7 stationary phase yeast cells was washed and resuspended in 1 ml of 30 g/ml Alcian blue in 0.02 M HCl.…”

“…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).…”

Putative PmrA and PmcA are important for normal growth, morphogenesis and cell wall integrity, but not for viability in Aspergillus nidulans

“…The elevated Ca 2+ causes continuous activation of the Ca 2+ signalling pathway, whilst Mn 2+ is toxic to some cytosolic enzymes (Lauer Jú nior et al, 2008;Park et al, 2001;Wuytack et al, 2003). At the same time, insufficient supply of Mn 2+ to the Golgi apparatus impairs the glycosylation procedure, because many glycosyltransferases use it as the cofactor (Bates et al, 2005). In S. cerevisiae, Pmr1 and Pmc1 cannot be deleted simultaneously for it to survive in standard medium (Cunningham & Fink, 1994).…”