Abstract
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 Vo domain (Voa) 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 stv1Δ/Δ and vph1Δ/Δ mutants and compared them to the corresponding reintegrant strains (stv1Δ/ΔR and vph1Δ/ΔR). V-ATPase activity, vacuolar physiology, and in vitro virulence-related phenotypes were unaffected in the stv1Δ/Δ mutant. The vph1Δ/Δ 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 vph1Δ/Δ cells also had abnormal endocytosis and vacuolar morphology and an alkalinized vacuolar lumen (pHvph1Δ/Δ = 6.8 versus pHvph1Δ/ΔR = 5.8) in both yeast cells and hyphae. Secreted protease and lipase activities were significantly reduced, and M199-induced filamentation was impaired in the vph1Δ/Δ mutant. However, the vph1Δ/Δ 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.
Highlights
V-ATPase regulates pH, and Candida albicans virulence is pH-dependent
To study the importance of the individual Vo domain (Voa) subunit isoforms of V-ATPase in C. albicans physiology, we generated both stv1/stv1 and vph1/vph1 homozygous null mutants via PCR-mediated gene disruption
Loss of V-ATPase Voa Isoforms Vph1p and Stv1p Separately Does Not Reduce Growth of C. albicans—The S. cerevisiae vph1 ⌬ mutant displays a mild vma phenotype characterized by normal growth at acidic pH and reduced growth at neutral pH [41], whereas the stv1⌬ strain does not exhibit vma mutant growth
Summary
Results: Deletion of V-ATPase Voa subunit Vph1p, but not Stv1p, alkalinizes vacuoles; several virulence-related traits remain unaffected. The vph1⌬/⌬ 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 nonvacuolar (Stv1p) V-ATPase activity is necessary to affect in. A. L.) and a Biomedical Research Institute of New Mexico grant Voa is the only fungal V-ATPase subunit encoded by two functional homologs: STV1 and VPH1 These isoforms allow for organelle targeting of V-ATPase; in S. cerevisiae, complexes containing Stv1p localize to the Golgi and pre-vacuolar compartments, whereas Vph1p targets V-ATPase to the vacuole [6, 7]. The S. cerevisiae isoforms differ in their regulation and kinetics, as Vph1p-containing complexes uniquely dissociate in
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