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Molecular and Functional Characterization of a Unique Secretory Nuclease [ Ld Nuc s ] from the Human Protozoan Pathogen, Leishmania donovani
Author(s) -
Dwyer Dennis M.,
Owings Joshua P.,
Joshi Manju B.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1012.5
Subject(s) - biology , amastigote , nuclease , leishmania , leishmania donovani , nucleic acid , microbiology and biotechnology , enzyme , biochemistry , parasite hosting , visceral leishmaniasis , leishmaniasis , immunology , world wide web , computer science
Leishmania are parasites of humans that reside/multiply as promastigotes within the gut of their sandfly vectors and as obligate intracellular amastigotes in the phago‐lysosomal system of macrophages. These organisms are strict purine auxotrophs and must salvage such essential nutrients from their hosts. Here we identified, characterized and episomally‐expressed a gene (LdNuc s ) encoding a unique 35‐kDa, DTT‐sensitive, Class‐I “secretory nuclease” from L. donovani. Our results showed that LdNuc s mRNA and its encoded secretory nuclease activity were constitutively expressed by both parasite developmental forms. Western blot and immunoprecipitation (IP) assays showed that an anti‐ LdNuc s ‐peptide antibody recognized both the native and LdNuc s ‐expressed enzymes. In addition, coupled IP‐enzyme activity assays demonstrated that this enzyme could hydrolyze various synthetic polynucleotides as well as, both ss‐ and ds‐DNAs and RNA. Further, IP analyses showed that sera of visceral leishmaniasis patients from multiple endemic foci all recognized and immunoprecipitated the LdNuc s enzyme. The latter indicated that LdNuc s was in fact expressed by amastigotes during the course of human infections. Cumulatively, our observations suggest that this “secretory” nuclease functions to hydrolyze host‐derived nucleic acids to satisfy the essential purine requirements of these parasites. Thus, the LdNuc s must play critical role(s) in the survival, growth and development of these important human pathogens. This study was supported by the Intramural Research Program of the DIR, NIAID, NIH.