A novel role for a glycosylphosphatidylinositol‐anchored aspartyl protease, CgYps1, in the regulation of pH homeostasis in Candida glabrata

Abstract

Proteases, key virulence factors of many bacterial and fungal pathogens, are pivotally important for nutrient acquisition, invasion and adherence to host cells and evasion/escape from host immune cells. In this study, we report a novel role for CgYps1, member of a family of 11 GPI-linked aspartyl proteases, in a human opportunistic fungal pathogen, Candida glabrata, in the regulation of pH homeostasis under acidic environmental conditions. We show that CgYps1 is required to survive low-external-pH environment and the inability of Cgyps1Δ mutant to maintain pH homeostasis results in intracellular acidification and increased reactive oxygen species (ROS) production. We also provide evidence that the reduced intracellular pH in Cgyps1Δ mutant under acidic conditions is, partly, owing to the diminished activity of a plasma membrane proton pump, CgPma1, an orthologue of a key component of pH homeostasis machinery in Saccharomyces cerevisiae, Pma1. In addition, we have examined C. glabrata's response to low environmental pH via genome-wide expression analysis and several genes required for protein folding/modification and stress response pathways including seven of the CgYPS genes were found to be upregulated. Lastly, we show that C. glabrata responds to acidic environment by reducing total β-glucan levels in the cell wall in a CgYps1-dependent manner.

Crz1p Regulates pH Homeostasis in Candida glabrata by Altering Membrane Lipid Composition

“…Changes in lipid composition of the Cgcrz1⌬ mutant might affect the activity of membrane proteins essential for barrier function (50,51). The deletion of CgCRZ1 might have the following effects: (i) a reduction of CgYPS1 mRNA levels, thus affecting Pma1p and pH homeostasis under acidic conditions (52,53), (ii) a decrease in ELO3 mRNA levels, thus affecting biosynthesis and maturation of very-long-chain (C 26 ) fatty acids that control Pma1p biogenesis (54), and (iii) a decrease in the activity of membrane H ϩ -ATPase, which pumps protons out of yeast cells at a low pH (55). Our results suggest that manipulating the plasma membrane lipid composition is a novel strategy for improving growth under acid stress conditions.…”

The Rho1 GTPase-activating Protein CgBem2 Is Required for Survival of Azole Stress in Candida glabrata

“…Cell wall isolation and ␤-glucan was measured as described previously (32). van den Hazel's protocol for Rhodamine-6G uptake assay was used (33).…”

“…Fluconazole-induced viability loss in Cgbem2⌬ mutant is partially rescued by inhibition of CgPkc1 signaling. A, ␤-glucan content of wild-type and Cgbem2 mutants grown either in YPD or in YPD medium containing 16 g/ml fluconazole (FLC) was determined as described previously (32) and was expressed as g of glucan/mg of dry weight of cell wall. Total amount of ␤-glucan quantified from YPD grown wild-type cells was normalized to 100%, and data presented are relative to the ␤-glucan levels in WT cell wall under standard growth conditions.…”

Heterogeneous Expression of the Virulence-Related Adhesin Epa1 between Individual Cells and Strains of the Pathogen Candida glabrata

“…Treating cells with 100 M ketoconazole and 2 M myriocin, which inhibit biosynthesis of major lipid raft components (sterols and sphingolipids) (29), had no discernible effect on Epa1-HA expression heterogeneity, suggesting that the heterogeneity is not lipid raft related (data not shown). Yps1 and Yps7 are extracellular GPI-anchored aspartyl proteases which cleave Epa1 from the cell surface of C. glabrata (4,25). The possibility that these protein activities determine heterogeneous cellular Epa1-HA levels was discounted with the observation that YPS1 and/or YPS7 deletion had no significant effect on heterogeneity (data not shown).…”