Candida albicans VMA3 Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation

Abstract

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 VATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. Candida albicans is a major opportunistic human fungal pathogen and is responsible for 6.8% of hospital-acquired infections in the United States (1). Despite the availability of several classes of antifungal drugs, attributable mortality, cost of care, and length of stay due to invasive candidiasis remain unacceptably high (2, 3). In addition, resistance to currently available antifungal drugs is emerging (see reference 4 for a review). Therefore, development of new antifungal drug targets remains a critical need. A diverse set of factors contributing to C. albicans virulence have been identified, including the secretion of aspartyl proteases and lipases, filamentation, and biofilm formation (5-8). Understanding the biology and regulation of these processes and pathways may illuminate new candidates for antifungal therapy.The vacuole is a dynamic acidic organelle found in yeast and plants that is analogous to the mammalian lysosome. It functions in an array of cellular homeostasis processes and thereby plays an important role in stress response, adaptation to novel environments, and cell differentiation (9-13). Furthermore, in C. albicans, intact vacuolar function is important for filamentation and virulence (12)(13)(14)(15). Vacuolar function depends on the maintenance of acidic pH by the vacuolar H-ATPase (V-ATPase), an enzyme complex that functions in organelle acidification across eukaryotes (16,17). The V-ATPase utilizes hydrolysis of ATP to transport protons from the cytosol into a variety of organelles. V-ATPase-mediated acidification and membrane energization are necessary for important vacuolar functions, including calcium and metal homeostasis (18), cargo sorting and membrane trafficking in endocytic and secretory pathways (19), and drug resistance (20). In Saccharomyces cerevisiae, the V-ATPase is expressed at the vacuolar membrane and the membrane of prevacuolar compartments and the Golgi compartment.The V-ATPase complex consists of the V 1 and V o subcomplexes (16). The V 1 subcomplex is composed of peripherally associated subunits that form the sites of ATP binding and hydrolysis on the cytosolic side of the membrane. The V o su...

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

“…Our initial attempt to disrupt C. albicans VMA2 by generating a vma2⌬ homozygous null mutant on acidic media was unsuccessful; after screening over 150 transformants from three separate lineages of heterozygous null mutants, a homozygous null mutant was not recovered. These results are consistent with previous attempts to disrupt V-ATPase genes in C. albicans (4). Therefore, we constructed the strain tetR-VMA2, a tetracycline-regulatable VMA2 mutant in which the VMA2 gene is repressed upon the addition of DOX (18).…”

“…Vacuolar acidification assays. Quinacrine staining was performed to visualize acidified vacuoles as described previously (27), with modifications as indicated previously (4). Briefly, cells were grown for 24 h in unbuffered YPD with or without DOX and then reset in fresh unbuffered YPD with or without DOX and grown to early log phase.…”

“…In addition to its requirement for intraorganellar pH homeostasis, V-ATPase function has been implicated in cytosolic and extracellular pH homeostasis (1,(8)(9)(10)(11), underscoring the far-reaching importance of this pump to overall cellular homeostasis. V-ATPase inhibition also interferes with virulence-related traits in C. albicans, including the secretion of degradative enzymes and yeast-to-hypha transitioning (3,4,12,13). Notably, V-ATPase previously has been shown to be important for in vivo virulence in C. albicans (3, 13); however, the requirement of acidic environmental pH for the growth of VATPase mutants complicates the interpretation of these studies due to the growth-limiting alkaline pH in the bloodstream of the murine host.…”

“…The acidification of these organelles has several important functions: first, the protons pumped into the compartment by V-ATPase energize multiple secondary transporter systems, such as those involved in metal ion homeostasis, and second, the acidic pH created by V-ATPase is necessary for the activity of degradative enzymes. Accordingly, in both Saccharomyces cerevisiae and C. albicans, mutations in VATPase lead to the Vma Ϫ phenotype, which is characterized by poor growth at alkaline pH as well as defective metal sequestration, glycerol metabolism, and other responses to environmental stress conditions (1,(3)(4)(5)(6). V-ATPase mutants are also impaired in vacuolar biogenesis due to defects in both vacuolar membrane fusion and fission (7).…”

“…Studies of S. cerevisiae have shown that disruption of VMA2 completely inhibits both ATPase activity and proton transport by the V-ATPase (14). We have previously investigated the contribution of several subunits of C. albicans VATPase to cell biology and virulence-related traits (4,12). This is the first study analyzing a subunit of the catalytic hexamer in the V 1 domain of V-ATPase in C. albicans.…”