Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori . Here we show that NTZ is a noncompetitive inhibitor ( K _i , 2 to 10 μM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis , Entamoeba histolytica , Giardia intestinalis , Clostridium difficile , Clostridium perfringens , H. pylori , and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli . To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli , and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO ₂ ) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and ¹ H nuclear magnetic resonance analyses determined that the spectral shift ( A ₄₁₈ to A ₃₅₁ ) of NTZ was due to protonation of the anion (NTZ ⁻ ) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pK _a = 6.18). We propose that NTZ ⁻ intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the “activated cofactor” of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.

The location of Helicobacter pylori in the gastric mucosa of mammals is defined by natural pH gradients within the gastric mucus, which are more alkaline proximal to the mucosal epithelial cells and more acidic toward the lumen. We have used a microscope slide-based pH gradient assay and video data collection system to document pH-tactic behavior. In response to hydrochloric acid (HCl), H. pylori changes its swimming pattern from straight-line random swimming to arcing or circular patterns that move the motile population away from the strong acid. Bacteria in more-alkaline regions did not swim toward the acid, suggesting the pH taxis is a form of negative chemotaxis. To identify the chemoreceptor(s) responsible for the transduction of pH-tactic signals, a vector-free allelic replacement strategy was used to construct mutations in each of the four annotated chemoreceptor genes ( tlpA , tlpB , tlpC , and tlpD ) in H. pylori strain SS1 and a motile variant of strain KE26695. All deletion mutants were motile and displayed normal chemotaxis in brucella soft agar, but only tlpB mutants were defective for pH taxis. tlpD mutants exhibited more tumbling and arcing swimming, while tlpC mutants were hypermotile and responsive to acid. While tlpA , tlpC , and tlpD mutants colonized mice to near wild-type levels, tlpB mutants were defective for colonization of highly permissive C57BL/6 interleukin-12 (IL-12) (p40 ^−/− )-deficient mice. Complementation of the tlpB mutant ( tlpB expressed from the rdxA locus) restored pH taxis and infectivity for mice. pH taxis, like motility and urease activity, is essential for colonization and persistence in the gastric mucosa, and thus TlpB function might represent a novel target in the development of therapeutics that blind tactic behavior.

Importance of the field: Antibacterial drug discovery and development has slowed considerably in recent years, with novel classes discovered decades ago and regulatory approvals tougher to get. Tra...

It has been nearly 50 years since the golden age of antibiotic discovery (1945–1975) ended; yet, we still struggle to identify novel drug targets and to deliver new chemical classes of antibiotics to replace those rendered obsolete by drug resistance. Despite herculean efforts utilizing a wide range of antibiotic discovery platform strategies, including genomics, bioinformatics, systems biology and postgenomic approaches, success has been at best incremental. Obviously, finding new classes of antibiotics is really hard, so repeating the old strategies, while expecting different outcomes, seems to boarder on insanity. The key questions dealt with in this review include: (1) If mutation based drug resistance is the major challenge to any new antibiotic, is it possible to find drug targets and new chemical entities that can escape this outcome; (2) Is the number of novel chemical classes of antibacterials limited by the number of broad spectrum drug targets; and (3) If true, then should we focus efforts on subgroups of pathogens like Gram negative or positive bacteria only, anaerobic bacteria or other group where the range of common essential genes is likely greater?. This review also provides some examples of existing drug targets that appear to escape the specter of mutation based drug resistance, and provides examples of some intermediate spectrum strategies as well as modern molecular and genomic approaches likely to improve the odds of delivering 21st century medicines to combat multidrug resistant pathogens.

Pyruvate-dependent reduction of NADP has been demonstrated in cell extracts of the human gastric pathogen Helicobacter pylori. However, NADP is not a substrate of purified pyruvate:ferredoxin oxidoreductase (PFOR), suggesting that other redox active enzymes mediate this reaction. Here we show that fqrB (HP1164), which is essential and highly conserved among the epsilonproteobacteria, exhibits NADPH oxidoreductase activity. FqrB was purified by nickel interaction chromatography following overexpression in Escherichia coli . The protein contained flavin adenine dinucleotide and exhibited NADPH quinone reductase activity with menadione or benzoquinone and weak activity with cytochrome c , molecular oxygen, and 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB). FqrB exhibited a ping-pong catalytic mechanism, a k _cat of 122 s ⁻¹ , and an apparent K _m of 14 μM for menadione and 26 μM for NADPH. FqrB also reduced flavodoxin (FldA), the electron carrier of PFOR. In coupled enzyme assays with purified PFOR and FldA, FqrB reduced NADP in a pyruvate- and reduced coenzyme A (CoA)-dependent manner. Moreover, in the presence of NADPH, CO ₂ , and acetyl-CoA, the PFOR:FldA:FqrB complex generated pyruvate via CO ₂ fixation. PFOR was the rate-limiting enzyme in the complex, and nitazoxanide, a specific inhibitor of PFOR of H. pylori and Campylobacter jejuni , also inhibited NADP reduction in cell-free lysates. These capnophilic (CO ₂ -requiring) organisms contain gaps in pathways of central metabolism that would benefit substantially from pyruvate formation via CO ₂ fixation. Thus, FqrB provides a novel function in pyruvate metabolism and, together with production of superoxide anions via quinone reduction under high oxygen tensions, contributes to the unique microaerobic lifestyle that defines the epsilonproteobacterial group.

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 ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-μg/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.