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Antimicrobial Agents and Chemotherapy, September 2000, p. 2349-2355, Vol. 44, No. 9
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Patricia Soteropoulos, Tanya Vaz, Rosaria Santangelo, Padmaja Paderu, David Y. Huang, Markus J. Tamás,dagger and David S. Perlin*

Public Health Research Institute, New York, New York 10016

Received 1 February 2000/Returned for modification 4 May 2000/Accepted 8 June 2000

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, Km and Vmax values of 0.5 mM and 3.1 µmol mg-1 min-1, respectively, and an apparent Ki for 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. 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.

* Corresponding author. Mailing address: Public Health Research Institute, 455 First Ave., New York, NY 10016. Phone: (212) 578-0820. Fax: (212) 578-0804. E-mail: perlin{at}phri.nyu.edu.

dagger Present address: Laboratory of Molecular Cell Biology, K. U. Leuven, Leuven, Belgium.

Antimicrobial Agents and Chemotherapy, September 2000, p. 2349-2355, Vol. 44, No. 9
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

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