title: Cytotoxic activity of nucleoside diphosphate kinase secreted from Mycobacterium tuberculosis.
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.
authors.affiliation: Institute of Genomics and Integrative Biology, Mall Road, Delhi, India.
authors.forename: Puneet
authors.initials: P
authors.lastname: Chopra
authors.forename: Anubha
authors.initials: A
authors.lastname: Singh
authors.forename: Anil
authors.initials: A
authors.lastname: Koul
authors.forename: S
authors.initials: S
authors.lastname: Ramachandran
authors.forename: Karl
authors.initials: K
authors.lastname: Drlica
authors.forename: Anil K
authors.initials: AK
authors.lastname: Tyagi
authors.forename: Yogendra
authors.initials: Y
authors.lastname: Singh
mesh_terms: D000225:Adenine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D015153:Blotting, Western; D016923:Cell Death; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D058919:Purinergic P2 Receptor Antagonists; D058486:Receptors, Purinergic P2X7
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D058919:Purinergic P2 Receptor Antagonists; D058486:Receptors, Purinergic P2X7; C004282:periodate-oxidized adenosine 5'-triphosphate; D005973:Glutamine; D006639:Histidine; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D020558:GTP Phosphohydrolases; D000225:Adenine