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Toxins TcdA and TcdB are the main virulence factors of Clostridioides difficile, a leading cause of hospital-acquired diarrhea. Despite their importance, there is a significant knowledge gap of druggable targets for inhibiting toxin production. To address this, we screened nonantibiotic phytochemicals to identify potential chemical genetic probes to discover antivirulence drug targets. This led to the identification of 18β-glycyrrhetinic acid (enoxolone), a licorice metabolite, as an inhibitor of TcdA and TcdB biosynthesis. Using affinity-based proteomics, potential targets were identified as ATP synthase subunit alpha (AtpA) and adenine deaminase (Ade, which catalyzes conversion of adenine to hypoxanthine in the purine salvage pathway). To validate these targets, a multifaceted approach was adopted. Gene silencing of ade and atpA inhibited toxin biosynthesis, while surface plasmon resonance and isothermal titration calorimetry molecular interaction analyses revealed direct binding of enoxolone to Ade. Metabolomics demonstrated enoxolone induced the accumulation of adenosine, while depleting hypoxanthine and ATP in C. difficile. Transcriptomics further revealed enoxolone dysregulated phosphate uptake genes, which correlated with reduced cellular phosphate levels. These findings suggest that enoxolone's cellular action is multitargeted. Accordingly, supplementation with both hypoxanthine and triethyl phosphate, a phosphate source, was required to fully restore toxin production in the presence of enoxolone. In conclusion, through the characterization of enoxolone, we identified promising antivirulence targets that interfere with nucleotide salvage and ATP synthesis, which may also block toxin biosynthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Metronidazole, a mainstay treatment for Clostridium difficile infection (CDI), is often ineffective for severe CDI. Whilst this is thought to arise from suboptimal levels of metronidazole in the colon due to rapid absorption, empirical validation is lacking. In contrast, reutericyclin, an antibacterial tetramic acid from Lactobacillus reuteri, concentrates in the gastrointestinal tract. In this study, we modified metronidazole with reutericyclin's tetramic acid motif to obtain non-absorbed compounds, enabling assessment of the impact of pharmacokinetics on treatment outcomes., Methods: A series of metronidazole-bearing tetramic acid substituents were synthesized and evaluated in terms of anti-C. difficile activities, gastric permeability, in vivo pharmacokinetics, efficacy in the hamster model of CDI and mode of action., Results: Most compounds were absorbed less than metronidazole in cell-based Caco-2 permeability assays. In hamsters, lead compounds compartmentalized in the colon rather than the bloodstream with negligible levels detected in the blood, in direct contrast with metronidazole, which was rapidly absorbed into the blood and was undetectable in caecum. Accordingly, four leads were more efficacious (P < 0.05) than metronidazole in C. difficile-infected animals. Improved efficacy was not due to an alternative mode of action, as the leads retained the mode of action of metronidazole., Conclusions: This study provides the clearest empirical evidence that the high absorption of metronidazole lowers treatment outcomes for CDI and suggests a role for the tetramic acid motif for colon-specific drug delivery. This approach also has the potential to lower systemic toxicity and drug interactions of nitroheterocyclic drugs for treating gastrointestine-specific diseases., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The stationary phase of Clostridium difficile, which is primarily responsible for diarrhoeal symptoms, is refractory to antibiotic killing. We investigated whether disrupting the functions of the clostridial membrane is an approach to control C. difficile infections by promptly removing growing and non-growing cells., Methods: The bactericidal activities of various membrane-active agents were determined against C. difficile logarithmic-phase and stationary-phase cultures and compared with known antibiotics. Their effects on the synthesis of ATP, toxins A/B and sporulation were also determined. The effect of rodent caecal contents on anti-difficile activities was examined using two reutericyclin lead compounds, clofazimine, daptomycin and other comparator antibiotics., Results: Most membrane-active agents and partially daptomycin showed concentration-dependent killing of both logarithmic-phase and stationary-phase cultures. The exposure of cells to compounds at their MBC resulted in a rapid loss of viability with concomitant reductions in cellular ATP, toxins A/B and spore numbers. With the exception of nisin, these effects were not due to membrane pore formation. Interestingly, the activity of the proton ionophore nigericin significantly increased as the growth of C. difficile decreased, suggesting the importance of the proton gradient to the survival of non-growing cells. The activities of the lipophilic antimicrobials reutericyclins and clofazimine were reduced by caecal contents., Conclusions: These findings indicate that C. difficile is uniquely susceptible to killing by molecules affecting its membrane function and bioenergetics, indicating that the clostridial membrane is a novel antimicrobial target for agents to alleviate the burden of C. difficile infections.