Department of Biochemistry and Molecular Biology, School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
J Biol Chem. 2013 Mar 1;288(9):6190-201. doi: 10.1074/jbc.M112.426197. Epub 2013 Jan 11.
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.
液泡质子转运 ATP 酶（V-ATPase）是细胞 pH 动态平衡的核心调节剂，所有 V-ATPase 功能的失活已被证明可防止白色念珠菌的感染性。Vo 结构域的液泡质子转运 ATP 酶亚基 a（Voa）存在两种真菌同工型：Stv1p（高尔基体）和 Vph1p（液泡）。为了阐明 Stv1p 和 Vph1p 对白色念珠菌生理学的个体贡献，我们创建了 stv1Δ/Δ 和 vph1Δ/Δ 突变体，并将它们与相应的重组菌株（stv1Δ/ΔR 和 vph1Δ/ΔR）进行了比较。Stv1Δ/Δ 突变体的 V-ATPase 活性、液泡生理学和体外与毒力相关的表型均不受影响。Vph1Δ/Δ 突变体表现出 V1Vo 组装缺陷和在用康纳霉素 A 敏感的 ATP 酶活性和纯化液泡膜中的质子转运降低 90%，表明 Vph1p 同工型是白色念珠菌液泡 V-ATPase 活性所必需的。Vph1Δ/Δ 细胞还具有异常的内吞作用和液泡形态以及碱性化的液泡腔（pHvph1Δ/Δ = 6.8，而 pHvph1Δ/ΔR = 5.8），无论是在酵母细胞还是菌丝体中。分泌的蛋白酶和脂肪酶活性显著降低，并且 Vph1Δ/Δ 突变体中 M199 诱导的丝状形成受损。然而，Vph1Δ/Δ 细胞仍然能够被 Spider 培养基和 YPD、10% FCS 诱导丝状化，并且体外生物膜形成和巨噬细胞杀伤不受影响。这些研究表明，不同的毒力机制不同程度地依赖于酸化的液泡，并且液泡（Vph1p）和非液泡（Stv1p）V-ATPase 活性的丧失对于影响体外与毒力相关的表型是必需的。作为白色念珠菌发病机制的决定因素，液泡 pH 值可能证明不如最初假设的那么关键。
