The HilC and HilD proteins of Salmonella enterica serovar Typhimurium are members of the AraC/XylS family of transcription regulators. They are encoded on Salmonella pathogenicity island 1 (SPI1) and control expression of the hilA gene, which encodes the major transcriptional activator for many genes encoded on SPI1 and elsewhere that contribute to invasion of host cells. Gel electrophoretic shift and DNase footprinting assays revealed that purified HilC and HilD proteins can bind to multiple regions in the hilA and hilC promoters and to a single region in the hilD promoter. Although both HilC and -D proteins can bind to the same DNA regions, they showed different dependencies on the sequence and lengths of their DNA targets. To identify the binding-sequence specificity of HilC and HilD, a series of single base substitutions changing each position in a DNA fragment corresponding to positions ؊92 to ؊52 of the hilC promoter was tested for binding to HilC and HilD in a gel shift DNA-binding assay. This mutational analysis in combination with sequence alignments allowed deduction of consensus sequences for binding of both proteins. The consensus sequences overlap but differ so that HilC can bind to both types of sites but HilD only to one. The hilA and hilC promoters contain multiple binding sites of each type, whereas the hilD promoter contains a site that binds HilC but not HilD without additional binding elements. The HilC and HilD proteins had no major effect on transcription from the hilA or hilD promoters using purified proteins in vitro but changed the choice of promoter at hilC. These results are consistent with a model derived from analysis of lacZ fusions stating that HilC and HilD enhance hilA expression by counteracting a repressing activity.

Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ϳ250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05-to 2-g/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.

The coordinate expression of Salmonella enterica invasion genes on Salmonella pathogenicity island 1 is under the control of the complex circuits of regulation that involve the AraC/XylS family transcriptional activators HilD, HilC, and RtsA and nucleoid-associated proteins. Single-copy transcription fusions were used to assess the effects of nucleoid-associated proteins Hha and H-NS on hilD, hilC, and rtsA expression. The data show that all three genes, hilD, hilC, and rtsA, were repressed by H-NS and/or Hha. The repression of rtsA was the highest among tested genes. The level of rtsA-lac was equally elevated in hns and hha mutants and was further enhanced in the hns hha double mutant under low-osmolarity conditions. Electrophoretic mobility shift experiments showed that H-NS and Hha directly bind to the rtsA promoter. In addition to the negative control that was exerted by H-NS/Hha under low-osmolarity conditions, the homologous virulence activators HilD, HilC, and RtsA (Hil activators) induced rtsA-lac expression in a high-salt medium. A DNase footprinting assay of the rtsA promoter revealed one common DNA-binding site for all three Hil activators centered at position ؊54 relative to the transcriptional start site. In the absence of Hha and H-NS, however, osmoregulation of the rtsA promoter was lost, and Hil activators were not required for rtsA transcription. These results taken together suggest that the HilD, HilC, and RtsA proteins induce the transcription of the rtsA promoter by counteracting H-NS/Hhamediated repression.

Metronidazole[1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole] (MTZ) (Fig. 1) and related 5-nitroimidazoles are redox-active prodrugs commonly used to treat infections caused by anaerobic bacteria and intestinal parasites [1]. In these organisms, MTZ is reduced (nitroreduction) to mutagenic and DNA-damaging hydroxylamine adducts by ferredoxin electron carriers associated with the pyruvate ⁄ ketoacid:ferredoxin oxidoreductases [2]. Clinically significant MTZ resistance is rare in anaerobes because pyruvate ⁄ ketoacid:ferredoxin oxidoreductase enzymes are essential components of core metabolism [3]. By contrast, MTZ resistance is common among clinical isolates of the gastric microaerophile Helicobacter pylori, especially those from geographic regions where MTZ usage is high [4]. H. pylori establish life-long infections of the gastric mucosa of billions of people worldwide [5]. It is the major cause of peptic and duodenal ulcers and is a Metronidazole (MTZ) is widely used in combination therapies against the human gastric pathogen Helicobacter pylori. Resistance to this drug is common among clinical isolates and results from loss-of-function mutations in rdxA, which encodes an oxygen-insensitive nitroreductase. The RdxAassociated MTZ-reductase activity of H. pylori is lost upon cell disruption. Here we provide a mechanistic explanation for this phenomenon. Under aerobic conditions, His6-tagged RdxA protein (purified from Escherichia coli), catalyzed NAD(P)H-dependent reductions of nitroaromatic and quinone substrates including nitrofurazone, nitrofurantoin, furazolidone, CB1954 and 1,4-benzoquinone, but not MTZ. Unlike other nitroreductases, His6-RdxA exhibited potent NAD(P)H-oxidase activity (k cat = 2.8 s )1 ) which suggested two possible explanations for the role of oxygen in MTZ reduction: (a) NAD(P)H-oxidase activity promotes cellular hypoxia (nonspecific reduction of MTZ), and (b) molecular oxygen out-competes MTZ for reducing equivalents. The first hypothesis was eliminated upon finding that rdxA expression, although increasing MTZ toxicity in both E. coli and H. pylori constructs, did not increase paraquat toxicity, even though both are of similar redox potential. The second hypothesis was confirmed by demonstrating NAD(P)H-dependent MTZ-reductase activity (apparent K m = 122 ± 58 lm, k cat = 0.24 s )1 ) under strictly anaerobic conditions.The MTZ-reductase activity of RdxA was 60 times greater than for NfsB (E. coli NTR), but 10 times lower than the NADPH-oxidase activity. Whether molecular oxygen directly competes with MTZ or alters the redox state of the FMN cofactors is discussed.Abbreviations MTZ, metronidazole; NTR, nitroreductase; PQ, paraquat; SOD, superoxide dismutase.

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The RdxA oxygen insensitive nitroreductase of the human gastric pathogen Helicobacter pylori is responsible for the susceptibility of this organism to the redox active prodrug metronidazole (MTZ). Loss-of-function mutations in rdxA are primarily responsible for resistance to this therapeutic. RdxA exhibits potent NADPH oxidase activity under aerobic conditions and metronidazole reductase activity under strictly anaerobic conditions. Here we report the crystal structure of RdxA, which is a homodimer exhibiting domain swapping and containing two molecules of FMN bound at the dimer interface. We have found a gap between the side chain of Tyr47 and the isoalloxazine ring of FMN that seems appropriate for substrate binding. The structure does not include residues 97–128, which corresponds to a locally unstable part of the NTR from E. coli, and might be involved in cofactor binding. Comparison of H pylori RdxA to other oxidoreductases of known structure suggests RdxA may belong to a new subgroup of oxidoreductases in which a cysteine sidechain close to the FMN cofactor could be involved in the reductive activity. In this respect, mutation of C159 to A or S (C159A/S) has resulted in loss of MTZ reductase activity, but not NADPH oxidase activity. The RdxA structure allows interpretation of the many loss-of-function mutations previously described, including those affecting C159, a residue whose interaction with FMN is required for nitroreduction of MTZ. Our studies provide unique insights into the redox behavior of the flavin in this key enzyme for metronidazole activation, and with potential use in gene therapy.

A library composed of Nitazoxanide-based analogues was synthesized and assayed for increased antibacterial efficacy against the pyruvate:ferredoxin oxidoreductase (PFOR) utilizing microorganisms Helicobacter pylori, Campylobacter jejuni and Clostridium difficile. Derivatives were found to recapitulate and improve activity against these organisms and select analogues were tested for their ability to disrupt the PFOR enzyme directly. The library was also screened for activity against staphylococci and resulted in the identification of analogues capable of inhibiting both staphylococci and all PFOR organisms at low μM MIC concentrations with low toxicity to human foreskin cells.

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