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Cytotoxic activity of nucleoside diphosphate kinase secreted from Mycobacterium tuberculosis
Puneet Chopra1, Anubha Singh, Anil Koul
1Institute of Genomics and Integrative Biology, Mall Road, Delhi, India.
Abstract:
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.
Insights
Mycobacterium tuberculosis secretes nucleoside diphosphate kinase (Ndk), an enzyme that promotes macrophage death. This bacterial cytotoxic factor aids in tuberculosis dissemination and immune evasion.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Mycobacterium tuberculosis (M. tuberculosis) pathogenesis involves survival within macrophages.
- Extracellular ATP can induce death in infected macrophages, a mechanism exploited by some pathogens.
- Nucleoside diphosphate kinase (Ndk) is implicated in this bacterial survival strategy.
Purpose of the Study:
- To investigate the role of M. tuberculosis Ndk in macrophage cytotoxicity.
- To determine if secreted Ndk contributes to M. tuberculosis pathogenicity.
Main Methods:
- Cloning and expression of the M. tuberculosis ndk gene in E. coli.
- Assessing Ndk secretion via enzymatic activity and Western blotting.
- Evaluating Ndk's effect on ATP-induced macrophage cell death using [14C]adenine release.
- Testing catalytic mutants and P2Z receptor inhibitor (oATP).
- Characterizing Ndk's enzymatic properties, including autophosphorylation and GTPase activity.
Main Results:
- M. tuberculosis secretes Ndk into the culture medium.
- Purified Ndk enhances ATP-induced macrophage cell death.
- A catalytically inactive Ndk mutant and the P2Z receptor inhibitor oATP blocked this cell death.
- Ndk exhibits broad nucleotide specificity in autophosphorylation, dependent on its nucleotide-binding site.
- Ndk possesses GTPase activity.
Conclusions:
- Secreted M. tuberculosis Ndk functions as a cytotoxic factor against macrophages.
- This Ndk-mediated cytotoxicity likely facilitates M. tuberculosis dissemination.
- The enzyme may play a role in immune system evasion by the pathogen.