title: Identification and biochemical characterization of unique secretory nucleases of the human enteric pathogen, Entamoeba histolytica.
abstract: 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.
authors.affiliation: Cell Biology Section, Laboratory of Parasitic Diseases, Division of Intramural Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425, USA.
authors.forename: Glen C
authors.initials: GC
authors.lastname: McGugan
authors.forename: Manju B
authors.initials: MB
authors.lastname: Joshi
authors.forename: Dennis M
authors.initials: DM
authors.lastname: Dwyer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002474:Cell-Free System / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000472:pathogenicity; D000939:Epitopes; D017125:Genes, Protozoan; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D011233:Precipitin Tests; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D012260:Ribonucleases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D004220:Disulfides; D000939:Epitopes; D021382:Protein Sorting Signals; D006639:Histidine; D012260:Ribonucleases; D003545:Cysteine