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.

FIG. 1.

POR assay and competition with benzyl viologen. (A) Competitive inhibition of POR activity (benzyl viologen reduction) in H. pylori extracts as a function of NTZ concentration was monitored spectrophotometrically at 546 nm. The specific activity at each concentration of nitazoxanide was recorded. (B) POR was assayed in cell extracts of H. pylori by monitoring the pyruvate-dependent reduction of nitazoxanide at 412 nm (A) as described in the text.

FIG. 2.

Lack of NTZ- or nitrofuran-induced DNA fragmentation in H. pylori. The MTZ^s strain H. pylori (Hp) 26695 was challenged with various concentrations of NTZ or nitrofurazone for 30 min as described in the text. The bacteria were suspended and lysed in agarose plugs, and agarose gels were run under alkaline conditions to display the extent of DNA fragmentation of denatured genomic DNA. Bacteria were treated with hydrogen peroxide (20 mM) for 15 min (positive controls). wt, wild type.

FIG. 3.

Lack of drug-induced DNA fragmentation of E. coli strain CC104 carrying pBSK. The bacteria were grown in the presence of the nitrofuran drugs as described in the text. The preparation of agarose plugs is as described in Fig. 2. Hydrogen peroxide was added at a 20 mM concentration as a positive control. The distinct bands noted in each of the lanes are pBSK plasmid DNA.

FIG. 4.

Lack of nitazoxanide-induced DNA fragmentation of E. coli strains carrying rdxA of H. pylori. E. coli strain CC104 containing either pBSK (control) or pGS950 (rdxA⁺) was grown in the presence of NTZ, and bacteria were suspended and lysed in agarose plugs and electrophoresed as described for Fig. 2 and detailed in the text. Hydrogen peroxide was added at a 20 mM concentration as a positive control. The distinct bands noted in the various lanes are plasmid DNA.