Kushawaha Pramod K, Pati Tripathi Chandra Dev, Dube Anuradha
Department of Microbiology, Central University of Punjab, Bathinda, India.
Division of Parasitology, CSIR- Central Drug Research Institute, Lucknow, India.
Microb Pathog. 2022 May;166:105457. doi: 10.1016/j.micpath.2022.105457. Epub 2022 Feb 25.
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 Ni-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 P2X 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.
杜氏利什曼原虫的致病性与其在巨噬细胞的恶劣环境中生存和繁殖的能力密切相关。所有原生动物寄生虫，包括利什曼原虫，都无法从头合成嘌呤，而核苷二磷酸激酶（NDK）是维持细胞内核苷磷酸平衡所需的酶。对于一些病原体来说，将利用ATP的酶分泌到细胞外环境中，通过P2Z受体介导的、ATP诱导的受感染巨噬细胞死亡，有助于病原体的存活。在此，通过镍-氮三乙酸亲和层析克隆、表达并纯化了杜氏利什曼原虫核苷二磷酸激酶（LdNDKb），以阐明其生物学意义。通过蛋白质免疫印迹分析证实了培养基中存在分泌形式的LdNDKb。有趣的是，共聚焦显微镜下的细胞定位显示，这种蛋白质定位于该寄生虫的细胞核、膜的内小叶和鞭毛上，这表明它在杜氏利什曼原虫的生命周期中具有多种作用。凝胶阻滞试验和电泳迁移率变动分析（EMSA）证实了它与DNA结合的可能性，结果表明它与线性和超螺旋DNA的结合不具有序列特异性。此外，用重组LdNdKb和高碘酸盐氧化ATP（一种P2X受体拮抗剂）处理J774巨噬细胞，抑制了体外ATP诱导的细胞溶解，这是通过检测J774巨噬细胞释放的乳酸脱氢酶来确定的。因此，LdNDKb通过水解细胞外ATP来防止ATP介导的宿主细胞质膜通透性增加，从而为了寄生虫的利益维持宿主细胞的完整性。这项研究表明，LdNDKb作为抗内脏利什曼病的药物/疫苗靶点的潜力值得探索。
