Functional Analysis of the RdxA and RdxB Nitroreductases of Campylobacter jejuni Reveals that Mutations in rdxA Confer Metronidazole Resistance

Campylobacter jejuni is a leading cause of gastroenteritis in humans and a commensal bacterium of the intestinal tracts of many wild and agriculturally significant animals. We identified and characterized a locus, which we annotated as rdxAB, encoding two nitroreductases. RdxA was found to be responsible for sensitivity to metronidazole (Mtz), a common therapeutic agent for another epsilonproteobacterium, Helicobacter pylori. Multiple, independently derived mutations in rdxA but not rdxB resulted in resistance to Mtz (Mtz^r), suggesting that, unlike the case in H. pylori, Mtz^r might not be a polygenic trait. Similarly, Mtz^r C. jejuni was isolated after both in vitro and in vivo growth in the absence of selection that contained frameshift, point, insertion, or deletion mutations within rdxA, possibly revealing genetic variability of this trait in C. jejuni due to spontaneous DNA replication errors occurring during normal growth of the bacterium. Similar to previous findings with H. pylori RdxA, biochemical analysis of C. jejuni RdxA showed strong oxidase activity, with reduction of Mtz occurring only under anaerobic conditions. RdxB showed similar characteristics but at levels lower than those for RdxA. Genetic analysis confirmed that rdxA and rdxB are cotranscribed and induced during in vivo growth in the chick intestinal tract, but an absence of these genes did not strongly impair C. jejuni for commensal colonization. Further studies indicate that rdxA is a convenient locus for complementation of mutants in cis. Our work contributes to the growing knowledge of determinants contributing to susceptibility to Mtz (Mtz^s) and supports previous observations of the fundamental differences in the activities of nitroreductases from epsilonproteobacteria.Nitroreductases form a large family of enzymes whose physiological roles have been implicated or proposed to function in diverse processes, such as the generation of nitrogen sources for metabolism, degradation of potentially toxic nitro compounds, vitamin and bioluminescence production, redox balancing, and oxidative stress responses (20, 31, 32, 35, 41, 43, 58). These enzymes can been subdivided into two main categories based on characteristics of their reductive processes, including the mechanism of electron transfer and sensitivity to oxygen. Type I (O₂-insensitive) nitroreductases catalyze a sequential two- or three-step reduction of the nitro group on heterocyclic compounds via paired-electron transfer to produce either hydroxylamine or amino derivatives. Type II (O₂-sensitive) nitroreductases catalyze a single-electron reduction of heterocyclic nitro compounds that is reversible in the presence of oxygen (40). Nitroreductases are common in bacteria, with a given bacterial species often containing multiple paralogs that presumably reduce different substrates. The genes encoding nitroreductases have received intense study due to their unusual nature in degrading or transforming xenobiotic chemicals. Consequently, these enzymes have become attractive candidates for bioremediation processes, and some are utilized in cancer chemotherapies (27, 49). However, the nitroreductases are also a puzzling class of enzymes, because the natural substrates for most remain unknown and these proteins likely did not evolve to exclusively manipulate xenobiotic compounds.Metronidazole (Mtz) has been used in multidrug therapy for Helicobacter pylori infections due to production of factors that convert this 5-nitroimidazole product to a toxic form (1, 45, 53). Therapeutic failure with Mtz has been predominantly associated with mutations occurring in one of two genes of H. pylori encoding the nitroreductases RdxA and FrxA. A previous biochemical study characterized the Mtz reductase activity of RdxA (37). Even though RdxA was capable of reducing other nitro compounds under aerobic conditions, the enzyme was unable to reduce Mtz. However, under anaerobic conditions, RdxA was shown to catalyze the reduction of Mtz, and its specific activity for this reaction was 60-fold greater than that of the NfsB nitroreductase of Escherichia coli under similar conditions. In addition, this work revealed that RdxA exhibited a potent NADPH oxidase activity not appreciated in other nitroreductases. Not only did this study demonstrate a direct reduction of Mtz by a nitroreductase, but results from this work implied that RdxA of H. pylori possessed novel biochemical properties relative to other nitroreductases.Like H. pylori, Campylobacter jejuni is a Gram-negative bacterium belonging to the epsilonproteobacteria class. C. jejuni is a common commensal bacterium of the intestinal tracts of wild and agriculturally significant animals, especially poultry. In contrast, C. jejuni causes acute diarrhea in humans, ranging from a mild enteritis to a bloody diarrheal syndrome, and is one of the most prevalent causes of food-borne gastritis (4, 5, 34, 38). Additionally, postinfectious sequelae can develop in a small percentage of patients following a C. jejuni infection. One major complication is Guillan-Barré syndrome, a temporary and partial paralysis of the peripheral nervous system (21).Many individuals with C. jejuni enteritis resolve the infection without therapeutic treatment. If antibiotics are administered, fluoroquinolones or macrolides, such as ciprofloxacin or erythromycin, are common drugs of choice, with therapeutic use of Mtz for C. jejuni infections being unconventional. However, Mtz-resistant (Mtz^r) C. jejuni isolates have been recovered from humans and animals. In agriculture, 19 to 92% of C. jejuni isolates from avian species (including chickens and turkeys) and 6 to 20% of isolates from lambs, sheep, and cows were Mtz^r (13, 47). One study also demonstrated that 62% of C. jejuni clinical isolates from humans were Mtz^r (47). These data are curious, since these C. jejuni isolates would have likely developed Mtz^r during infections in the absence of selection.Because susceptibility to Mtz (Mtz^s) was also found in isolates associated with each host in studies described above and since C. jejuni is closely related to H. pylori, we hypothesized that C. jejuni may produce a nitroreductase to reduce Mtz to its toxic form, leading to Mtz^s. In this report, we identify and characterize the gene required for Mtz^s in C. jejuni. Mutations in this gene, encoding a putative nitroreductase, but not in a downstream paralog were linked to the development of Mtz^r, indicating that Mtz^r in C. jejuni appears to be linked to mutation of only one nitroreductase. Supporting our findings, we provide evidence that a proportion of Mtz^r isolates of C. jejuni are due to spontaneous errors during DNA replication in the absence of Mtz exposure, resulting in a variety of mutations. Biochemical analysis of these C. jejuni nitroreductases demonstrated that these proteins had potent NADPH oxidase activity and could reduce Mtz under anaerobic conditions. These results, along with previous biochemical analysis of H. pylori RdxA (37), demonstrate that the nitroreductases of epsilonproteobacteria have unique characteristics in comparison to other bacterial counterparts.