Analysis of the L,D transpeptidation pathway in Clostridium difficile: contribution to peptidoglycan synthesis and antibiotic resistance

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Clostridium difficile is a nosocomial pathogen causing diarrhoea associated with high morbidity/mortality and a high healthcare cost. The microbial imbalance in the gut caused by antibiotic treatment is a key factor for the germination of spores and the production of toxins causing the disease. C. difficile infections are therefore underpinned by the intrinsic resistance of this bacterium to many antibiotics including beta-lactams (such as cephalosporins) that target the synthesis of peptidoglycan, the essential component of the bacterial cell envelope. In C. difficile, peptidoglycan synthesis essentially relies on a family of enzymes called L,D-transpeptidases (Ldts) that have been associated with beta-lactam resistance in other pathogens. We therefore hypothesise that the widespread resistance of C. difficile to beta-lactams can, in large part, be attributed to this group of enzymes. Our aim is to explore the contribution of the L,D transpeptidation pathway to bacterial cell growth and antibiotic resistance in C. difficile using a multidisciplinary approach. We propose to construct mutants by gene replacement to investigate the role of these enzymes during cell growth and division and elucidate the impact of Ldts activities to peptidoglycan structure and beta-lactam resistance. To gain further insights into the individual role of C. difficile Ldts, we will study their enzymatic activity in vitro and explore their structural properties by X-ray crystallography. Finally, building upon our detailed analysis of Ldt functionality in a model C. difficile strain, we will explore the natural variation in PG structure in circulating clinical strains and determine how this relates to the spectrum of resistance to a range of beta-lactam antibiotics. This project will further our understanding of the mechanism underpinning C. difficile infections and generate knowledge to develop new treatments against this key hospital pathogen.