Rane Hallie S, Bernardo Stella M, Raines Summer M, Binder Jessica L, Parra Karlett J, Lee Samuel A
Section of Infectious Diseases, New Mexico Veterans Healthcare System, Albuquerque, New Mexico, USA.
Eukaryot Cell. 2013 Oct;12(10):1369-82. doi: 10.1128/EC.00118-13. Epub 2013 Aug 2.
The 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 in Saccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen Candida albicans, we created a conditional mutant in which VMA3 was placed under the control of a tetracycline-regulated promoter (tetR-VMA3 strain). Repression of VMA3 in the tetR-VMA3 strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of C. albicans V-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-VMA3 strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-VMA3 strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in an in vitro macrophage killing model. Repression of VMA3 suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression of RIM8, MDS3, EFG1, CST20, or UME6, 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 in C. albicans.
液泡膜ATP酶（V-ATP酶）是一种蛋白质复合体，它利用ATP水解将质子从细胞质溶胶转运到液泡腔中，使液泡酸化，并调节酿酒酵母中的几个关键细胞反应系统。为了研究V-ATP酶对机会性真菌病原体白色念珠菌的生物学特性和毒力属性的贡献，我们构建了一个条件突变体，其中VMA3置于四环素调控启动子的控制之下（tetR-VMA3菌株）。在tetR-VMA3菌株中抑制VMA3可阻止V-ATP酶在液泡膜上组装，并使制霉菌素A敏感的ATP酶特异性活性和质子转运降低90%以上。白色念珠菌V-ATP酶活性的丧失使液泡腔碱化，并具有多效性作用，包括pH依赖性生长、钙敏感性和冷敏感性。tetR-VMA3菌株还表现出异常的液泡形态，表明液泡膜分裂存在缺陷。tetR-VMA3菌株的天冬氨酸蛋白酶和脂肪酶分泌受损，并且在体外巨噬细胞杀伤模型中毒力减弱。抑制VMA3可抑制丝状化，并且编码丝状化正调控因子的RIM8、MDS3、EFG1、CST20或UME6的过表达不能挽救V-ATP酶依赖性丝状化缺陷。对Vma3p功能的特异性化学抑制也导致丝状化缺陷。这些发现表明，要么V-ATP酶在这些转录调节因子的下游起作用，要么V-ATP酶在丝状化过程中的功能涉及独立的机制和替代信号通路。综上所述，这些数据表明V-ATP酶活性是白色念珠菌几个关键毒力相关性状的基本要求。
