A family of glycosylphosphatidylinositol-linked aspartyl proteases is required for virulence of Candida glabrata

Abstract

Candida glabrata is a yeast pathogen of humans. We have established a tissue culture model to analyze the interaction of C. glabrata with macrophages. Transcript profiling of yeast ingested by macrophages reveals global changes in metabolism as well as increased expression of a gene family (YPS genes) encoding extracellular glycosylphosphatidylinositol-linked aspartyl proteases. Eight of these YPS genes are found in a cluster that is unique to C. glabrata. Genetic analysis shows that the C. glabrata YPS genes are required for cell wall integrity, adherence to mammalian cells, survival in macrophages and virulence. By monitoring the processing of a cell wall adhesin, Epa1, we also show that Yps proteases play an important role in cell wall re-modeling by removal and release of glycosylphosphatidylinositol-anchored cell wall proteins.

Fig. 1.

Macrophage-induced transcriptions of C. glabrata YPS genes. (A) Schematic representation of C. glabrata YPS gene loci. (B) Relative mRNA abundance of YPS genes in C. glabrata coincubated with J774A.1 macrophages for 6 h (filled bars) and in cells grown in DMEM alone (open bars), as measured by quantitative real-time PCR. Results are the means ± SD of two biological duplicate experiments, each performed in triplicate.

Fig. 2.

Phenotypic characterization of C. glabrata ypsΔ strains. (A) C. glabrata ypsΔ strains display sensitivity to drugs causing cell wall stress. Equal number of cells were spotted in 10-fold serial dilutions onto YPD plates alone or supplemented with different compounds. Plates were photographed after 2 days at 30°C. (B) C. glabrata ypsΔ strains lose viability in stationary phase. Cells were grown in liquid YPD at 30°C, and viable cells as a percentage of total cells were determined as a function of time over 96 h.

Fig. 3.

C. glabrata ypsΔ mutants are compromised for virulence. Groups of 10 mice were infected with each C. glabrata strain via tail vein injection and killed 7 days after infection. Recovered CFUs from three target organs are indicated for individual mice as a diamond, and the geometric mean is shown as a bar.

Fig. 4.

Epal is stabilized on the cell surface in ypsΔ strains. (A) FACS analysis of surface expressed Epa1 in C. glabrata wild-type and ypsΔ strains. Epa1 surface expression during a 10-h time course in YPD was assessed by an anti-Epa1 antibody and a FITC-conjugated secondary antibody. The geometric mean of fluorescence is indicated. (B) Western blot analysis of Epa1 protein in the cell wall and media fractions of the wild-type and ypsΔ strains grown in YPD. The samples were resolved on a 3–8% SDS/PAGE gel and labeled with anti-Epa1 antibody. The locations of molecular weight markers are indicated.