DIFFICROSS

Clostridioides difficile incidence has tripled over the past 15 years and this bacterium has become the main causative agent of antibiotic-associated and health care-associated diarrhea. The impact of C. difficile infection (CDI) in hospitals is considerable in terms of mortality, morbidity and disease management as well as for its financial burden. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. However, the mechanism for toxin release is still poorly understood. CDI is difficult to prevent and treat, as the pathogen is resistant to many antimicrobial agents. Although resistance of C. difficile to ß-lactams, the most commonly prescribed class of antibiotics, is a leading contributor to the development of CDI, the underlying mechanisms of resistance are currently unknown. Due to the unsatisfactory nature of standard therapy for CDI, a current imperative is to better understand the molecular mechanisms involved in antibiotic resistance and controlling the key steps of CDI in the perspective of developing novel preventive and curative therapies. In C. difficile, the majority of the peptidoglycan (PG) cross-links is synthesized by non-classical transpeptidases, namely the L,D-transpeptidases (LDTs), insensitive to inhibition by most ß-lactam antibiotics. Our central hypothesis is that LDTs are key enzymes for PG biosynthesis that confer ß-lactam resistance and play a crucial role in toxin release in C. difficile. The overall aim of this project is therefore to characterize the three LDTs identified in C. difficile and the role of each of them. Our first objective will be to elucidate the functions of LDTs in the cell wall biology of C. difficile. We will study the relative roles of the unusual L,D-transpeptidation and the classical D,D-transpeptidation pathways in the formation of cross-links during the expansion and maturation of the C. difficile PG. We will also determine to which extent LDTs are important for cell morphology and ß-lactams resistance. Through the use of a genome-wide approach, our second objective will be to identify novel genes regulating the L,D-transpeptidation pathway. A subset of these genes will be selected for further characterization. Toxin release in C. difficile relies on PG-degrading enzymes, whose activity is controlled by structural alterations of the PG substrate. Our third objective will therefore be to evaluate the impact of LDTs on toxin release in-vitro and on virulence in-vivo. We will also identify molecular actors involved in toxin release in C. difficile. Overall, this work will provide fundamental knowledge on C. difficile PG biosynthesis, a field of research still neglected in this bacterium. Since cell wall synthesis is one of the most important antibiotic targets, a better understanding of the cell wall biology of C. difficile may ultimately help in developing procedures to overcome antibiotic resistance in this bacterium and identifying novel drug targets for the specific eradication of C. difficile. Ultimately, principles from this work will have wider implications beyond C. difficile research and will greatly enhance our current understanding of PG biosynthetic control and dynamics.