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J. Biol. Chem., Vol. 279, Issue 52, 54088-54096, December 24, 2004

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Genetic Disruption of the Plasmodium falciparum Digestive Vacuole Plasmepsins Demonstrates Their Functional Redundancy^*

A. Levi Omara-Opyene¶, Pedro A. Moura||, Carlos R. Sulsona, J. Alfredo Bonilla, Charles A. Yowell, Hisashi Fujioka**, David A. Fidock||, and John B. Dame

From the Department of Pathobiology, University of Florida, Gainesville, Florida 32611-0880, the ^||Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461, and ^**Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106

The digestive vacuole plasmepsins PfPM1, PfPM2, PfPM4, and PfHAP (a histoaspartic proteinase) are 4 aspartic proteinases among 10 encoded in the Plasmodium falciparum malarial genome. These have been hypothesized to initiate and contribute significantly to hemoglobin degradation, a catabolic function essential to the survival of this intraerythrocytic parasite. Because of their perceived significance, these plasmepsins have been proposed as potential targets for antimalarial drug development. To test their essentiality, knockout constructs were prepared for each corresponding gene such that homologous recombination would result in two partial, nonfunctional gene copies. Disruption of each gene was achieved, as confirmed by PCR, Southern, and Northern blot analyses. Western and two-dimensional gel analyses revealed the absence of mature or even truncated plasmepsins corresponding to the disrupted gene. Reduced growth rates were observed with PfPM1 and PfPM4 knockouts, indicating that although these plasmepsins are not essential, they are important for parasite development. Abnormal mitochondrial morphology also appeared to accompany loss of PfPM2, and an abundant accumulation of electron-dense vesicles in the digestive vacuole was observed upon disruption of PfPM4; however, those phenotypes only manifested in about a third of the disrupted cells. The ability to compensate for loss of individual plasmepsin function may be explained by close similarity in the structure and active site of these four vacuolar enzymes. Our data imply that drug discovery efforts focused on vacuolar plasmepsins must incorporate measures to develop compounds that can inhibit two or more of this enzyme family.

Received for publication, August 20, 2004 , and in revised form, October 13, 2004.

^* This work was supported by National Institutes of Health Grants AI39211 (to J. B. D.) and AI058186 (to H. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^¶ Present address: Dept. of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, P. O. Box 100275, Gainesville, FL 32610-0275.

To whom correspondence should be addressed. Tel.: 352-392-4700 (ext. 5818); Fax: 352-392-9704; E-mail: damej{at}mail.vetmed.ufl.edu.

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