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 nitroreductasedeficient 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.Prodrugs are particularly appealing as therapeutic agents because of their selective toxicity for microorganisms possessing specific activating enzymes (7). Metronidazole (MTZ) is a nitroimidazole prodrug that is often used in combination therapies against Helicobacter pylori, the microaerobic bacterium that causes gastritis and peptic ulcer disease and that is an early risk factor for gastric cancer (6,19,30). However, resistance to MTZ is very common and reduces the efficacy of MTZ-containing regimes (10). MTZ is also highly mutagenic, and hence its use may accelerate development of resistance to other clinically important antibiotics, such as clarithromycin (24,29). These considerations have stimulated interest in alternative redox-active prodrugs in either first-line or salvage (failed eradication) therapies, especially those against which resistance may be uncommon in H. pylori. Alternative prodrugs include the nitrofurans and nitrothiazoles, which exhibit antimicrobial activity against H. pylori and also against facultative anaerobes, anaerobic bacterial and protozoan species, and helminths (12,19,21,30,32,33). For these drugs, the spectrum of bioactivity and the mechanisms of activation are largely a function of the redox potential (EЈ 7 ) of the 5-nitro groups. For example, nitrofurans (nitrofurazone, nitrofurantoin, and furazolidone) display relatively high redox potentials (Ϫ250 to Ϫ270 mV) and are reductively activated by a...

In vitro efficacy of nitro- and bromo-thiazolyl-salicylamide compounds (thiazolides) against Besnoitia besnoiti infection in Vero cells

“…However, solid experimental evidence for this hypothesis is missing. Studies in H. pylori suggested that, in contrast to metronidazole, NTZ was largely non-mutagenic (Sisson et al 2002). However, the prescribing information on NTZ (Alinia TM ) states that the drug was genotoxic in one tester strain (TA 100) in the Ames bacterial mutation assay.…”

“…In addition, NTZ exhibits a broad spectrum of activities against a wide variety of enteric bacteria, protozoa, and intestinal and tissue-dwelling helminth parasites, infecting animals and humans (Fox and Saravolatz, 2005 ;Pankuch and Appelbaum, 2006). Against Helicobacter pylori, Giardia intestinalis and Cryptosporidium parvum, the drug has been postulated to act via reduction of its nitro-group by nitroreductases, including pyruvate ferredoxin oxidoreductase (PFOR) (Coombs and Muller, 2002 ;Sisson et al 2002). In terms of veterinary use, the drug is marketed for the treatment of equine myeloencephalitis caused by Sarcocystis neurona (Navigator TM ).…”

Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis

“…The 5-nitroimidazoles, such as metronidazole, are activated only by anaerobic enzyme systems that generate very low redox potentials (Ϫ480 mV), such as pyruvate-ferrodoxin oxidoreductases and hydrogenases (14). However, neither redox potential nor hydrophobicity correlates with the activity of various nitroimidazoles against Trichomonas vaginalis, suggesting a specific protein-ligand interaction (15).…”

The fdxA Ferredoxin Gene Can Down-Regulate frxA Nitroreductase Gene Expression and Is Essential in Many Strains of Helicobacter pylori

“…Susceptibility results from the action of one or two related nitroreductases that each mediate conversion of Mtz from harmless prodrug to hydroxylamine, a bactericidal and mutagenic agent; RdxA, which is abundant in essentially all Mtz s clinical isolates; and FrxA, which is present at only very low levels in most isolates (designated type I strains) but at higher levels in others (type II strains) (27,28). RdxA and FrxA differ in substrate specificity (49), but their normal substrates, products, and roles (e.g., whether purely metabolic or protective against reactive nitrogen and oxygen metabolites that are produced in the host response to infection; see reference 40) are not known.…”