Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase) for anti-fungal therapy

Abstract

Vacuolar proton-translocating ATPase (V-ATPase) is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

Keywords: C. albicans virulence; anti-fungal target; fungal V-ATPase; pH homeostasis; vacuolar acidification; vacuolar proton pump.

FIGURE 1

V-ATPase subunit composition and mechanism of catalysis. The V-ATPase proton pump acidifies the lumen of organelles in the endomembrane system of all eukaryotic cells. V-ATPase has 14 subunits that form two domains, V₁ and V_o. V₁ (clear and gray subunits, A₃B₃CDE₃FG₃H) hydrolyzes ATP at the cytosolic side of the membrane, and V_o (blue subunits, ac_3-4c’c”de) translocates protons. Transport of protons against a concentration gradient entails a rotational mechanism. Hydrolysis of 3 ATP in the V₁A catalytic subunits drives rotation of a shaft (subunits D, F) that penetrates V₁ and is bound to a rotating proteolipid ring structure in V_o formed by subunits c_(3-4)c’c” (c-ring) that together with subunit V_oa forms the path for proton transport. With the exception of subunit V_oa, which exists in two isoforms (Vph1p and Stv1p), each V-ATPase subunit is encoded by a single gene in fungi. In contrast, multiple isoforms (two to four) exist for most subunits of the mammalian V-ATPase. Subunit V_oc′ is absent in mammals.

FIGURE 2

V-ATPase in non-vacuolar organelles (Stv1p-containing complexes) plays a yet unknown role in C. albicans virulence. Wild-type: When functional, V-ATPase-mediated acidification of vacuoles, Golgi, and secretory vesicles maintains organelle pH and supports traffic of Pma1p to the cell surface for proton efflux and maintenance of an alkaline cytosol. VPH1 deficient: C. albicans grows normally at neutral pH when only Vph1p-containing V-ATPase complexes (vacuolar membrane) are missing. Only modest filamentation defects are obvious, despite the concomitant vacuolar alkalinization and defective Pma1p activity (Raines et al., 2013 and unpublished results). VMA3 deficient: Vacuolar alkalinization and defective Pma1p activity occur at levels equal to that of VPH1 deficient cells when all V-ATPase function is missing (Stv1p- and Vph1-containing V-ATPase complexes; Rane et al., 2013 and unpublished results). However, VMA3 deficient C. albicans exhibits growth defects at neutral pH and severely reduced filamentation under these conditions. We therefore hypothesize that the presence of Stv1p-containing V-ATPase in non-vacuolar organelles maintains virulence in the face of defective vacuolar and cytoplasmic pH homeostasis. Pink = acidic/acidified, blue = alkaline/alkalinized.