Chopra Puneet, Singh Anubha, Koul Anil, Ramachandran S, Drlica Karl, Tyagi Anil K, Singh Yogendra
Institute of Genomics and Integrative Biology, Mall Road, Delhi, India.
Eur J Biochem. 2003 Feb;270(4):625-34. doi: 10.1046/j.1432-1033.2003.03402.x.
Pathogenicity of Mycobacterium tuberculosis is closely related to its ability to survive and replicate in the hostile environment of macrophages. For some pathogenic bacteria, secretion of ATP-utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor-mediated, ATP-induced death of infected macrophages. A component of these enzymes is nucleoside diphosphate kinase (Ndk). The ndk gene was cloned from M. tuberculosis H37Rv and expressed in Escherichia coli. Ndk was secreted into the culture medium by M. tuberculosis, as determined by enzymatic activity and Western blotting. Purified Ndk enhanced ATP-induced macrophage cell death, as assayed by the release of [14C]adenine. A catalytic mutant of Ndk failed to enhance ATP-induced macrophage cell death, and periodate-oxidized ATP (oATP), an irreversible inhibitor of P2Z receptor, blocked ATP/Ndk-induced cell death. Purified Ndk was also found to be autophosphorylated with broad specificity for all nucleotides. Conversion of His117-->Gln, which is part of the nucleotide-binding site, abolished autophosphorylation. Purified Ndk also showed GTPase activity. Collectively, these results indicate that secreted Ndk of M. tuberculosis acts as a cytotoxic factor for macrophages, which may help in dissemination of the bacilli and evasion of the immune system.
结核分枝杆菌的致病性与其在巨噬细胞的恶劣环境中存活和复制的能力密切相关。对于一些病原菌来说，向细胞外环境分泌利用ATP的酶有助于病原菌通过P2Z受体介导的、ATP诱导的被感染巨噬细胞死亡而存活。这些酶的一个成分是核苷二磷酸激酶（Ndk）。从结核分枝杆菌H37Rv中克隆出ndk基因并在大肠杆菌中表达。通过酶活性和蛋白质印迹法确定，Ndk由结核分枝杆菌分泌到培养基中。通过[14C]腺嘌呤的释放测定，纯化的Ndk增强了ATP诱导的巨噬细胞死亡。Ndk的催化突变体未能增强ATP诱导的巨噬细胞死亡，而高碘酸盐氧化的ATP（oATP），一种P2Z受体的不可逆抑制剂，阻断了ATP/Ndk诱导的细胞死亡。还发现纯化的Ndk对所有核苷酸具有广泛特异性的自身磷酸化作用。核苷酸结合位点的一部分His117向Gln的转化消除了自身磷酸化。纯化的Ndk还表现出GTP酶活性。总体而言，这些结果表明，结核分枝杆菌分泌的Ndk作为巨噬细胞的细胞毒性因子发挥作用，这可能有助于杆菌的传播和免疫系统的逃避。
