Diverse viscerotropic isolates of Leishmania all express a highly conserved secretory nuclease during human infections

• Published in: Molecular and cellular biochemistry 2012-02, Vol.361 (1-2), p.169-179
• Main Authors: Joshi, Manju B., Hernandez, Yunuen, Owings, Joshua P., Dwyer, Dennis M.
• Format: Article
• Language: English
• Subjects: Africa, Eastern; Amastigotes; Animals; Antibodies, Protozoan - blood; Antigens, Protozoan - blood; Antigens, Protozoan - immunology; Antigens, Protozoan - metabolism; Base Sequence; Biochemistry; Biomedical and Life Sciences; Blotting, Southern; Brazil; Cardiology; Cell Line; Chromatography, Affinity; Dogs; Enzymatic activity; Enzymes; Epidemiology; Esterases - blood; Esterases - immunology; Esterases - metabolism; Health aspects; Humans; Immunoprecipitation; India; Infection; Leishmania donovani; Leishmania donovani - enzymology; Leishmania donovani - genetics; Leishmania donovani - immunology; Leishmania donovani - isolation & purification; Leishmania infantum - enzymology; Leishmania infantum - genetics; Leishmaniasis, Visceral - immunology; Leishmaniasis, Visceral - parasitology; Life Sciences; Medical Biochemistry; Molecular Sequence Data; Oncology; Parasites; Parasitic diseases; Pathology; Peptides; Protozoa; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Vector-borne diseases
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1007/s11010-011-1101-1

Previously, we characterized a gene encoding the unique nuclease ( Ld Nuc s ) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite . Currently, we assessed whether an Ld Nuc s -like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc s gene homologs were present in each of the viscerotropic Leishmania tested (i.e . , L. donovani isolates from the Sudan , Ethiopia and India as well as L. infantum ). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc s -like nuclease activity. In Western blots, our anti- Ld Nuc s (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti- Ld Nuc s antibody above. In situ gel analyses showed that each of these immunoprecipitates had Ld Nuc s -like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an Ld Nuc s -HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc s homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.