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.