Molecular Characterization of the Plasma Membrane H⁺-ATPase, an Antifungal Target inCryptococcus neoformans

Abstract

The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H ؉ -ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H ؉ -ATPase was shown by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H ؉ -ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, K m and V max values of 0.5 mM and 3.1 mol mg ؊1 min ؊1, respectively, and an apparent K i for vanadate inhibition of 1.6 M. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H ؉ -ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the Cryptococcus H ؉ -ATPase as a viable target for antifungal drug discovery.The opportunistic pathogen Cryptococcus neoformans causes pulmonary and central nervous system disease in immunocompromised individuals and is known to produce life-threatening meningoencephalitis in 5 to 10% of AIDS patients (1). Treatment of cryptococossis typically involves combined and sequential therapy with the polyene antibiotic amphotericin and azole-based drugs such as fluconazole. However, such therapy presents clinical problems, since amphotericin treatment results in a high degree of nephrotoxicity and azole resistance has emerged as a complicating factor, especially in AIDS patients with repeated or chronic exposure to fluconazole (4). As is the case with other fungal pathogens, the development of newer antifungal drugs with alternative sites of action that can reduce toxicity and be used in combination to minimize resistance is an important goal.The plasma membrane H ϩ -ATPase is a high-capacity proton pump that plays a critical role in fungal cell physiology by helping to regulate intracellular pH and maintain transmembrane electrochemical proton gradients necessary for nutrient uptake (26). The H ϩ -ATPase has been characterized biochemically from various fungi in which it is known to be a predominant membrane protein comprised of a single 100-kDa subunit that contains both a membrane-spanning transport domain and a cytoplasmically located catalytic ATP hydrolysis domain. The gene encoding this enzyme, PMA1, has been cloned from diverse fungi and has been shown to be highly conserved (31). Gene-disruption experiments in Saccharomyces cerevisiae have confirmed the essential nature of this gene product (27). The H ϩ -ATPase has recently been proposed as a target for antifungal drug development, largely because of its well-characterized biochemical and genetic properties (20) and the ava...

Molecular Evaluation of the Plasma Membrane Proton Pump fromAspergillus fumigatus

“…The A. fumigatus enzyme in plasma membranes was found to have an optimal pH of 6.5 and displayed K m and V max values of 0.5 mM and 0.2 mol of P i released per mg of protein per min, respectively, which are typical values for fungal H ϩ -ATPases (21,26). An IC 50 of 1.8 M was found for the mechanism-specific inhibitor vanadate (5), which compares favorably with VO 4 IC 50 s of 0.8, 1.6, and 9 M for H ϩ -ATPases from S. cerevisiae (15), C. neoformans (40), and C. albicans (23), respectively.…”

The Cryptococcal Enzyme Inositol Phosphosphingolipid-Phospholipase C Confers Resistance to the Antifungal Effects of Macrophages and Promotes Fungal Dissemination to the Central Nervous System

“…A possible explanation for the ⌬isc1 strain's sensitivity to acidic stress could be the altered function of the proton pump Pma1, which is critical for maintaining the intracellular pH of C. neoformans within the physiological ranges necessary for optimal enzymatic activities and crucial metabolic processes (22). Indeed, Pma1's physiological importance is reflected by the inviability of Pma1 deletion mutants of S. cerevisiae or C. neoformans (1,41). Several reports have shed light on the role that sphingolipids have on the correct oligomerization and localization of Pma1 during its synthesis and transport to the plasma membrane (reviewed in reference 14).…”

“…Pma1 is important for the regulation of intracellular pH (22,41,42) and for the response to oxidative (40) and nitrosative (50) …”

A Putative P-Type ATPase, Apt1, Is Involved in Stress Tolerance and Virulence in Cryptococcus neoformans

“…Phylogenetic analysis revealed that Apt1 from C. neoformans is closely related to P-type ATPase of the Drs2 family of APTs (P4 type) but clearly distinct from the other main classes of P-type ATPases identified so far in C. neoformans. These include the P2 types, such as the sarcoplasmic/ER Ca 2ϩ ATPase EcaI (14), the sodium/ potassium P-type ATPase Ena1 (30), the H ϩ ATPases Pma1 and Pma2 (21,53), the Ca 2ϩ /Mg 2ϩ -transporting ATPase Pmr1, and a P1-type copper-exporting ATPase, Atu2/Ccc2 (57) (see Fig. S1 in the supplemental material).…”