Browsing by Author "Pati Tripathi, Chandra Dev"

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    Leishmania donovani secretory protein nucleoside diphosphate kinase b localizes in its nucleus and prevents ATP mediated cytolysis of macrophages
    (Academic Press, 2022-02-25T00:00:00) Kushawaha, Pramod K.; Pati Tripathi, Chandra Dev; Dube, Anuradha
    Leishmania donovani pathogenicity is closely linked to its ability to live and replicate in the hostile environment of macrophages. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDKs) are enzymes required to preserve the intracellular nucleoside phosphate equilibrium. For some pathogens, secretion of ATP-utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor mediated, ATP-induced death of infected macrophages. Here, Leishmanaia donovani nucleoside diphosphate kinase (LdNDKb) was cloned, expressed and purified by Ni2+-NTA affinity chromatography to elucidate its biological significance. The presence of secreted form of LdNDKb in the medium was confirmed by Western blot analysis. Interestingly, cellular localization by confocal microscopy showed that this protein was localized in the nucleus, inner leaflet of membrane and on the flagella of this parasite which indicates its multiple role in the life cycle of Leishmania donovani. Its possibility to bind with DNA was confirmed by gel retardation assay and electrophoretic mobility shift assay (EMSA) which show the binding with linear and supercoiled is not sequence specific. Further, treatment of J774 macrophages with recombinant LdNdKb and periodate oxidized ATP - a P2X7 receptor antagonist, inhibited ATP-induced cytolysis in vitro, as determined by lactate dehydrogenise release from J774 macrophages. Thus, LdNDKb prevents ATP-mediated host-cell plasma membrane permeabilization by hydrolyzing extracellular ATP, thereby, preserving the integrity of the host cells for the benefit of the parasite. This study indicates that LdNDKb could be explored for its potentiality as a drug/vaccine target against visceral leishmaniasis. � 2022
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    Leishmania donovani secretory protein nucleoside diphosphate kinase b localizes in its nucleus and prevents ATP mediated cytolysis of macrophages
    (Academic Press, 2022-02-25T00:00:00) Kushawaha, Pramod K.; Pati Tripathi, Chandra Dev; Dube, Anuradha
    Leishmania donovani pathogenicity is closely linked to its ability to live and replicate in the hostile environment of macrophages. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDKs) are enzymes required to preserve the intracellular nucleoside phosphate equilibrium. For some pathogens, secretion of ATP-utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor mediated, ATP-induced death of infected macrophages. Here, Leishmanaia donovani nucleoside diphosphate kinase (LdNDKb) was cloned, expressed and purified by Ni2+-NTA affinity chromatography to elucidate its biological significance. The presence of secreted form of LdNDKb in the medium was confirmed by Western blot analysis. Interestingly, cellular localization by confocal microscopy showed that this protein was localized in the nucleus, inner leaflet of membrane and on the flagella of this parasite which indicates its multiple role in the life cycle of Leishmania donovani. Its possibility to bind with DNA was confirmed by gel retardation assay and electrophoretic mobility shift assay (EMSA) which show the binding with linear and supercoiled is not sequence specific. Further, treatment of J774 macrophages with recombinant LdNdKb and periodate oxidized ATP - a P2X7 receptor antagonist, inhibited ATP-induced cytolysis in vitro, as determined by lactate dehydrogenise release from J774 macrophages. Thus, LdNDKb prevents ATP-mediated host-cell plasma membrane permeabilization by hydrolyzing extracellular ATP, thereby, preserving the integrity of the host cells for the benefit of the parasite. This study indicates that LdNDKb could be explored for its potentiality as a drug/vaccine target against visceral leishmaniasis. � 2022