Candida albicansVMA3Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation

Abstract

ABSTRACTThe vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems inSaccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogenCandida albicans, we created a conditional mutant in whichVMA3was placed under the control of a tetracycline-regulated promoter (tetR-VMA3strain). Repression ofVMA3in the tetR-VMA3strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss ofC. albicansV-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-VMA3strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-VMA3strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in anin vitromacrophage killing model. Repression ofVMA3suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression ofRIM8,MDS3,EFG1,CST20, orUME6, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits inC. albicans.