Clostridioides difficile, an anaerobic Gram-positive spore-forming bacterium, is responsible for a wide spectrum of infections ranging from diarrhea to life-threatening pseudomembranous colitis. The use of antibiotic therapy raises concerns about the selection of antibiotic-resistant bacteria at hospitals. New therapeutic targets should be investigated as alternatives to antibiotic treatments. Polysaccharides (PS) biosynthesis enzymes are encoded in a PS locus where most genes are essential for bacterial viability. We propose therefore the enzymes involved in PS biosynthesis as new therapeutic targets. Moreover, vaccines currently under development target the toxins and may not prevent C. difficile colonization and dissemination. We also propose in this project to evaluate PSII and/or LTA as vaccine component(s) of a vaccine that may prevent C. difficile colonization and dissemination. To that aim, the project will (i) define if either one or both PSII and LTA are essential for bacterial viability, ii) identify specific enzymes involved in PSII or LTA biosynthesis, (iii) target them with inhibitors and (iv) test PSII and LTA as vaccine candidates using an innovative approach. We recently developed a new genetic conditional lethal mutant system and have already obtained antibodies directed against PSII. Using these tools, we showed that the PSII seems to be essential for bacterial viability. The project will be divided into three tasks. The first will determine whether PSII, its anchoring and/or LTA are essential and define at least two enzymes as new therapeutic targets, one of each involved in each PS biosynthesis. The second task will look for inhibitors able to target these specific enzymes, using in silico models and a chemistry approach. The last task will test PSII and LTA as vaccine candidates. This project aims to combat C. difficile infections and prevent them using vaccination.

Clostridioides difficile (C. difficile) is a toxigenic bacterium responsible for severe nosocomial intestinal infections. About 500.000 patients are infected every year in the USA, causing 13.000 deaths, according to the American CDC. To treat such intestinal infections, only 2 antibiotics are recommended as first line oral treatments, exposing to a 15-25% risk of resistance and infection recurrences. Regarding phase III clinical trials, only 1 molecule is being studied against C. difficile, worrying situation which explains why this pathogen was listed among the 5 urgent bacterial threats by the CDC. To develop new orally-active antibiotics against C. difficile, cationic amphiphilic antimicrobial peptides (AMP) are quite inspiring natural antibiotics which present several advantages: i) their mechanism of action is different from the ones of commercial anti-C. difficile antibiotics, ii) their capacity to disrupt the membrane/cell wall exposes to a lower risk of resistance and iii) their peptidic structure allows a rapid degradation in the environment, limiting the risk of selection of resistant bacteria. Nevertheless, these molecules cannot be administered orally and often exert severe systemic toxicity when administered IV. Then, based on preliminary in vitro results, OBTAC is a drug-design project to develop novel amphiphilic peptoïdic polymers, as simplified orally-active AMP analogues, to treat C. difficile infections (CDI) at low industrial production costs. A consortium of 4 complementary partners, proposes a “hit-to-lead” pharmacochemistry approach involving i) structure-activity relationship studies, ii) optimization of the synthesis of monomers and of the polymerization process to decrease production cost and environmental impact, iii) exhaustive microbiological studies including in vivo evaluation on a mouse infectious model and iv) tritium labeling to allow biodistribution and pharmacokinetic evaluation in mouse, in order to identify an innovative orally-active pre-clinical antibiotic candidate against CDI with easy and low cost scale-up synthesis.

Clostridium difficile is the leading cause of intestinal nosocomial post-antibiotic infections. The impact of C. difficile infection (CDI) in hospitals is considerable in terms of mortality, morbidity and disease management as well as for its social economic burden. Importantly, relapse of CDI, which occurs in more than 20% of patients after the first episode, is one of the most significant clinical issues of this disease. There is therefore an urgent need to better understand the molecular mechanisms controlling key steps of CDI including persistence of C. difficile in intestinal tract. For many bacterial pathogens, biofilm formation is linked to survival persistence, resistance to stress and antibiotics, and colonization that cause disease. Moreover, chronic bacterial infections are also often correlated to the ability of bacteria to form biofilms. Although C. difficile was shown to form single or mixed biofilms in vitro, little is known on factors involved in C. difficile biofilm formation in vivo and its consequences. By combining biofilm studies, global and targeted functional genetics and animal assays, the DifBioRel project aims to explore mechanisms involved in the C. difficile biofilm formation in response to inducers encountered in dysbiosis gut environment and to assess the impact of biofilm in the persistence of this important pathogen. Based on strong preliminary results and in vitro and in vivo approaches developed and currently used by the 3-partner consortium we propose to i) identify and select the main stresses and/or stimuli present in the gut dysbiosis environment that induce C. difficile biofilm formation, ii) characterize the molecular mechanisms involved and iii) evaluate the contribution of the biofilm in the persistence of C. difficile in the gut and relapse of CDI. In addition to inducers of single and mixed C. difficile biofilm formation already identified by the consortium, the three Partners P1 (Bruno Dupuy, Institut Pasteur), P2 (Claire Janoir, Université Paris Sud) and P3 (Jean-Marc Ghigo, Institut Pasteur) will cooperate to identify other potential factors varying gut dysbiosis and/or resilience of the microbiota, and triggering C. difficile biofilm formation. These will include nutritional compounds such as amino acids promoting C. difficile growth, antimicrobial peptides, sub-inhibitory concentrations of antibiotics or osmotic stresses. The molecular mechanisms involved in C. difficile biofilm formation in response to the major gut inducers will then be characterized. C. difficile biofilm-deficient mutants with no other phenotypic difference compared to the wild type will be used as controls to evaluate the role of biofilms in gut C. difficile persistence and CDI relapse. Partners 1 and 3 will investigate the cooperative interactions between C. difficile and several prominent commensal bacteria favoring C. difficile (mixed) biofilms to study the impact of the microbial community on C. difficile biofilm formation. In addition, Partners 1 and 2 will explore C. difficile biofilm formation in mono-associated mouse model and then study the in vivo dynamic of mixed-biofilm formation between C. difficile and tested intestinal commensal bacteria. Finally, the role of C. difficile biofilm formation in the relapse of CDI after antibiotherapy will be assessed. For this purpose, we will use adapted C. difficile-infected animal models mimicking dysbiosis and relapse developed by Partner 2 to test appropriate C. difficile mutants unable to form biofilm when exposed to the studied inducers. The ambition of the DifBioRel project is to provide new insights on key C. difficile biofilm factors involved in in vivo persistence and relapse processes. Moreover, DifBioRel should lead to innovative strategies based on the use of specific inhibitors of biofilm formation and/or inducers of biofilm dispersion to limit bacterial survival, and therefore the spread and recurrence of CDI.