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ABSTRACTThe gene encoding the plasma membrane proton pump (H+-ATPase) of Aspergillus fumigatus, PMA1, was characterized from A. fumigatusstrain NIH 5233 and clinical isolate H11-20. An open reading frame of 3,109 nucleotides with two introns near the N terminus predicts a protein consisting of 989 amino acids with a molecular mass of approximately 108 kDa. The predicted A. fumigatusenzyme is 89 and 51% identical to H+- ATPases of Aspergillus nidulansand Saccharomyces cerevisiae, respectively. The A. fumigatus PMA1is a typical member of the P-type ATPase family that contains 10 predicted transmembrane segments and conserved sequence motifs TGES, CSDKTGT, MLTGD, and GDGVN within the catalytic region. The enzyme represents 2% of the total plasma membrane protein, and it is characteristically inhibited by orthovanadate, with a 50% inhibitory concentration of ∼1.8 μM. H+-ATPases from Aspergillusspp. contain a highly acidic insertion region of 60 amino acids between transmembrane segments 2 and 3, which was confirmed for the membrane-assembled enzyme with a peptide-derived antibody. An increasing A. fumigatus PMA1copy number confers enhanced growth in low-pH medium, consistent with its role as a proton pump. These results provide support for the development of the A. fumigatusH+-ATPase as a potential drug discovery target.

ABSTRACTThe Cryptococcus neoformans PMA1gene, 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 sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiaeH+-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, Kmand Vmaxvalues of 0.5 mM and 3.1 μmol mg−1min−1, respectively, and an apparent Kifor vanadate inhibition of 1.6 μM. 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. neoformansprotein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1genes from plants. Collectively, this study provides the basis for establishing the CryptococcusH+-ATPase as a viable target for antifungal drug discovery.

We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosisthat is caused by a reversible frameshift mutation in the M. tuberculosisorngene. A frameshift mutation in ornproduces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis. However, the same ornmutation in a low-level EMB-resistant double embB-aftAmutant (MIC = 8 μg/ml) produces an SCV with an EMB MIC of 32 μg/ml.