Molecular and functional analyses of a novel class I secretory nuclease from the human pathogen, Leishmania donovani.
The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc(s), a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc(s) chimeric construct was used in conjunction with an anti-LdNuc(s) peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single- and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen.
Joshi
Manju B
MB
Cell Biology Section, Laboratory of Parasitic Diseases, Division of Intramural Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425.
Dwyer
Dennis M
DM
Cell Biology Section, Laboratory of Parasitic Diseases, Division of Intramural Research, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0425. Electronic address: ddwyer@niaid.nih.gov.
D000595:Amino Acid Sequence; D000818:Animals; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007893:Leishmania donovani / Q000201:enzymology*; D008969:Molecular Sequence Data; D012260:Ribonucleases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism / Q000502:physiology*
D003851:Deoxyribonucleases; D012260:Ribonucleases