Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans

Vacuolar proton-translocating ATPase (V-ATPase) is a central regulator of cellular pH homeostasis, and inactivation of all V-ATPase function has been shown to prevent infectivity in Candida albicans. V-ATPase subunit a of the V-o domain (V(o)a) is present as two fungal isoforms: Stv1p (Golgi) and Vph1p (vacuole). To delineate the individual contribution of Stv1p and Vph1p to C. albicans physiology, we created stv1 Delta/Delta and vph1 Delta/Delta mutants and compared them to the corresponding reintegrant strains (stv1 Delta/Delta R and vph1 Delta/Delta R). V-ATPase activity, vacuolar physiology, and in vitro virulence-related phenotypes were unaffected in the stv1 Delta/Delta mutant. The vph1 Delta/Delta mutant exhibited defective V1Vo assembly and a 90% reduction in concanamycin A-sensitive ATPase activity and proton transport in purified vacuolar membranes, suggesting that the Vph1p isoform is essential for vacuolar V-ATPase activity in C. albicans. The vph1 Delta/Delta cells also had abnormal endocytosis and vacuolar morphology and an alkalinized vacuolar lumen (pH(vph1)Delta/Delta = 6.8 versus pH(vph1) Delta/Delta(R) = 5.8) in both yeast cells and hyphae. Secreted protease and lipase activities were significantly reduced, and M199-induced filamentation was impaired in the vph1 Delta/Delta mutant. However, the vph1 Delta/Delta cells remained competent for filamentation induced by Spider media and YPD, 10% FCS, and biofilm formation and macrophage killing were unaffected in vitro. These studies suggest that different virulence mechanisms differentially rely on acidified vacuoles and that the loss of both vacuolar (Vph1p) and non-vacuolar (Stv1p) V-ATPase activity is necessary to affect in vitro virulence-related phenotypes. As a determinant of C. albicans pathogenesis, vacuolar pH alone may prove less critical than originally assumed.