The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by <i>VMA2</i> , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

Abstract

Candida albicans vacuoles are central to many critical biological processes, including filamentation and in vivo virulence. The V-ATPase proton pump is a multisubunit complex responsible for organellar acidification and is essential for vacuolar biogenesis and function. To study the function of the V₁B subunit of C. albicans V-ATPase, we constructed a tetracycline-regulatable VMA2 mutant, tetR-VMA2. Inhibition of VMA2 expression resulted in the inability to grow at alkaline pH and altered resistance to calcium, cold temperature, antifungal drugs, and growth on nonfermentable carbon sources. Furthermore, V-ATPase was unable to fully assemble at the vacuolar membrane and was impaired in proton transport and ATPase-specific activity. VMA2 repression led to vacuolar alkalinization in addition to abnormal vacuolar morphology and biogenesis. Key virulence-related traits, including filamentation and secretion of degradative enzymes, were markedly inhibited. These results are consistent with previous studies of C. albicans V-ATPase; however, differential contributions of the V-ATPase Vo and V₁ subunits to filamentation and secretion are observed. We also make the novel observation that inhibition of C. albicans V-ATPase results in increased susceptibility to osmotic stress. Notably, V-ATPase inhibition under conditions of nitrogen starvation results in defects in autophagy. Lastly, we show the first evidence that V-ATPase contributes to virulence in an acidic in vivo system by demonstrating that the tetR-VMA2 mutant is avirulent in a Caenorhabditis elegans infection model. This study illustrates the fundamental requirement of V-ATPase for numerous key virulence-related traits in C. albicans and demonstrates that the contribution of V-ATPase to virulence is independent of host pH.