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Antimicrobial Agents and Chemotherapy, July 2002, p. 2116-2123, Vol. 46, No. 7
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.7.2116-2123.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Enzymes Associated with Reductive Activation and Action of Nitazoxanide, Nitrofurans, and Metronidazole in Helicobacter pylori

Gary Sisson,1 Avery Goodwin,1,2 Ausra Raudonikiene,1 Nicky J. Hughes,2 Asish K. Mukhopadhyay,3 Douglas E. Berg,3 and Paul S. Hoffman1,2,4*

Department of Microbiology and Immunology,1 Division of Infectious Diseases, Department of Medicine, Faculty of Medicine, Dalhousie University, HaliFax, Nova Scotia B3H 4H7,4 GlaxoSmithKline, Collegeville, Pennsylvania,2 Department of Molecular Microbiology and Department of Genetics, Washington University Medical School, St. Louis, Missouri3

Received 17 December 2001/ Returned for modification 27 February 2002/ Accepted 10 April 2002

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 (Rifr) 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 Mtzr phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Sir Charles Tupper Medical Bldg., Room 7P, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. Phone: (902) 494-3889. Fax: (902) 494-5125. E-mail: phoffman{at}tupdean2.med.dal.ca.

Antimicrobial Agents and Chemotherapy, July 2002, p. 2116-2123, Vol. 46, No. 7
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.7.2116-2123.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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