Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole Drugs

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma− phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections

A human Na+/H+ antiporter sharing evolutionary origins with bacterial NhaA may be a candidate gene for essential hypertension

Phylogenetic analysis of the cation/proton antiporter superfamily has uncovered a previously unknown clade of genes in metazoan genomes, including two previously uncharacterized human isoforms, NHA1 and NHA2, found in tandem on human chromosome 4. The NHA (sodium hydrogen antiporter) family members share significant sequence similarity with Escherichia coli NhaA, including a conserved double aspartate motif in predicted transmembrane 5. We show that HsNHA2 (Homo sapiens NHA2) resides on the plasma membrane and, in polarized MDCK cells, localizes to the apical domain. Analysis of mouse tissues indicates that NHA2 is ubiquitous. When expressed in the yeast Saccharomyces cerevisiae lacking endogenous cation/proton antiporters and pumps, HsNHA2 can confer tolerance to Li+ and Na+ ions but not to K+. HsNHA2 transformants accumulated less Li+ than the salt-sensitive host; however, mutagenic replacement of the conserved aspartates abolished all observed phenotypes. Functional complementation by HsNHA2 was insensitive to amiloride, a characteristic inhibitor of plasma membrane sodium hydrogen exchanger isoforms, but was inhibited by phloretin. These are hallmarks of sodium–lithium countertransport activity, a highly heritable trait correlating with hypertension. Our findings raise the possibility that NHA genes may contribute to sodium–lithium countertransport activity and salt homeostasis in humans

A Phenomics Approach in Yeast Links Proton and Calcium Pump Function in the Golgi

The Golgi-localized Ca(2+)- and Mn(2+)-transporting ATPase Pmr1 is important for secretory pathway functions. Yeast mutants lacking Pmr1 show growth sensitivity to multiple drugs (amiodarone, wortmannin, sulfometuron methyl, and tunicamycin) and ions (Mn(2+) and Ca(2+)). To find components that function within the same or parallel cellular pathways as Pmr1, we identified genes that shared multiple pmr1 phenotypes. These genes were enriched in functional categories of cellular transport and interaction with cellular environment, and predominantly localize to the endomembrane system. The vacuolar-type H(+)-transporting ATPase (V-ATPase), rather than other Ca(2+) transporters, was found to most closely phenocopy pmr1Δ, including a shared sensitivity to Zn(2+) and calcofluor white. However, we show that pmr1Δ mutants maintain normal vacuolar and prevacuolar pH and that the two transporters do not directly influence each other's activity. Together with a synthetic fitness defect of pmr1ΔvmaΔ double mutants, this suggests that Pmr1 and V-ATPase work in parallel toward a common function. Overlaying data sets of growth sensitivities with functional screens (carboxypeptidase secretion and Alcian Blue binding) revealed a common set of genes relating to Golgi function. We conclude that overlapping phenotypes with Pmr1 reveal Golgi-localized functions of the V-ATPase and emphasize the importance of calcium and proton transport in secretory/prevacuolar traffic