Molecular characterization of the plasma membrane H(+)-ATPase, an antifungal target in Cryptococcus neoformans.
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 sulfate-polyacrylamide 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 micromol mg(-1) min(-1), respectively, and an apparent K(i) for vanadate inhibition of 1.6 microM. 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.
Soteropoulos
P
P
Public Health Research Institute, New York, NY 10016, USA.
Vaz
T
T
Santangelo
R
R
Paderu
P
P
Huang
D Y
DY
Tamás
M J
MJ
Perlin
D S
DS
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D001483:Base Sequence; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D003001:Cloning, Molecular; D003455:Cryptococcus neoformans / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004271:DNA, Fungal / Q000032:analysis; D006801:Humans; D054833:Isoindoles; D008969:Molecular Sequence Data; D016566:Organoselenium Compounds / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D012703:Serotyping
D000935:Antifungal Agents; D001393:Azoles; D016861:Cyclooxygenase Inhibitors; D004271:DNA, Fungal; D054833:Isoindoles; D016566:Organoselenium Compounds; C434364:PMA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C042986:ebselen; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases