title: Enzymes associated with reductive activation and action of nitazoxanide, nitrofurans, and metronidazole in Helicobacter pylori.
abstract: Nitazoxanide (NTZ) is a redox-active nitrothiazolyl-salicylamide prodrug that kills Helicobacter pylori and also many anaerobic bacterial, protozoan, and helminthic species. Here we describe development and use of a spectrophotometric assay, based on nitroreduction of NTZ at 412 nm, to identify H. pylori enzymes responsible for its activation and mode of action. Three enzymes that reduce NTZ were identified: two related NADPH nitroreductases, which also mediate susceptibility to metronidazole (MTZ) (RdxA and FrxA), and pyruvate oxidoreductase (POR). Recombinant His-tagged RdxA, FrxA, and POR, overexpressed in nitroreductase-deficient Escherichia coli, each rapidly reduced NTZ, whereas only FrxA and to a lesser extent POR reduced nitrofuran substrates (furazolidone, nitrofurantoin, and nitrofurazone). POR exhibited no MTZ reductase activity either in extracts of H. pylori or following overexpression in E. coli; RdxA exhibited no nitrofuran reductase activity, and FrxA exhibited no MTZ reductase activity. Analysis of mutation to rifampin resistance (Rif(r)) indicated that NTZ was not mutagenic and that nitrofurans were only weakly mutagenic. Alkaline gel DNA electrophoresis indicated that none of these prodrugs caused DNA breakage. In contrast, MTZ caused DNA damage and was strongly mutagenic. We conclude that POR, an essential enzyme, is responsible for most or all of the bactericidal effects of NTZ against H. pylori. While loss-of-function mutations in rdxA and frxA produce a Mtz(r) phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.
authors.affiliation: Department of Microbiology and ImmunologyFaculty of Medicine, Dalhousie University, HaliFax, Nova Scotia B3H 4H7, Canada.
authors.forename: Gary
authors.initials: G
authors.lastname: Sisson
authors.forename: Avery
authors.initials: A
authors.lastname: Goodwin
authors.forename: Ausra
authors.initials: A
authors.lastname: Raudonikiene
authors.forename: Nicky J
authors.initials: NJ
authors.lastname: Hughes
authors.forename: Asish K
authors.initials: AK
authors.lastname: Mukhopadhyay
authors.forename: Douglas E
authors.initials: DE
authors.lastname: Berg
authors.forename: Paul S
authors.initials: PS
authors.lastname: Hoffman
mesh_terms: D001711:Biotransformation; D003001:Cloning, Molecular; D004249:DNA Damage; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D016480:Helicobacter pylori / Q000187:drug effects / Q000201:enzymology*; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D008795:Metronidazole / Q000378:metabolism* / Q000494:pharmacology; D009154:Mutation; D009574:Nitro Compounds; D009581:Nitrofurans / Q000378:metabolism* / Q000494:pharmacology; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D050743:Pyruvate Synthase; D013379:Substrate Specificity; D013844:Thiazoles / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D009574:Nitro Compounds; D009581:Nitrofurans; D013844:Thiazoles; D008795:Metronidazole; C029492:aromatic NADH-dependent nitroreductase; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase; D009601:Nitroreductases; C413454:flavin nitroreductase; C041747:nitazoxanide