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J. Biol. Chem., Vol. 282, Issue 43, 31789-31802, October 26, 2007

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Identification and Biochemical Characterization of Unique Secretory Nucleases of the Human Enteric Pathogen, Entamoeba histolytica^*

From the Cell Biology Section, Laboratory of Parasitic Diseases, Division of Intramural Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425

The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.

Received for publication, July 20, 2007

^* This work was supported in part by the intramural research program of the Division of Intramural Research, NIAID, National Institutes of Health. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by an intramural postdoctoral research fellowship from the NIAID, National Institutes of Health.

² Supported by an appointment to the Oak Ridge Institute for Science and Education, Research Specialist Program at the National Institutes of Health.

³ To whom correspondence should be addressed. Tel.: 301-496-5969; Fax: 301-402-0079; E-mail: ddwyer{at}niaid.nih.gov.

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