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

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.

C. difficile causes gut infections that vary in severity from mild diarrhea to life threatening, inflammatory complications. The bacterium is intrinsically resistant to many antibiotics, including beta-lactams such as cephalosporins, and infection commonly follows antibiotic-induced intestinal dysbiosis that gives C. difficile a competitive advantage. The molecular basis of resistance is not understood but the cell wall peptidoglycan contains an unusually high degree of 3-3 crosslinks, catalysed by a family of L,D transpeptidases (Ldts), the activity of which has been associated with beta-lactam resistance in other species.
Most C. difficile strains encode three Ldts; two of these appear to have only a minor contribution to the formation of 3-3 crosslinks, while the third has yet to be characterised in any detail. We aim to fully characterise the L,D transpeptidation pathway in C. difficile: elucidating the role of each Ldt in peptidoglycan synthesis and beta-lactam resistance, and exploring the structural basis of these functions. We will also analyse Ldt diversity in a panel of clinically relevant C. difficile strains and determine how this impacts peptidoglycan structure and beta-lactam resistance. We will adopt a highly multidisciplinary approach, combining expertise in peptidoglycan biochemistry, C. difficile molecular biology, structural biology and clinical microbiology. The insights gained from our analysis of this important biochemical pathway will shed light on C. difficile multidrug resistance and pave the way for the development of Ldt inhibitors to directly kill C. difficile or induce susceptibility to traditional beta-lactams.

The proposed project deals with the characterisation of the crucial Clostridium difficile L,D transpeptidation pathway, determining how these enzymes contribute to the synthesis of peptidoglycan and resistance to beta-lactam antibiotics. This research will provide a number of benefits to the general public, the NHS and industry in the UK and further afield.

(i) The local community in Sheffield (short term):
In recent years the problems of AMR and hospital-acquired infections have entered the public consciousness. The local community will gain a broader knowledge of this important scientific area, and have an opportunity to engage with and contribute to the research happening within their city during our outreach activities.

(ii) Society (medium to long term):
In the longer term, our work has the potential to lead to development of new therapeutics that target L,D transpeptidases directly or that restore the utility of existing beta-lactams through circumvention of resistance. The development of a new treatment for C. difficile would have a major impact on society more widely. There are approximately 2,000 deaths involving C. difficile in UK every year, in addition to considerable morbidity. Treatment of C. difficile infections and the resulting extended hospital stays also place a huge burden on the NHS budget.

(iii) National Health Service (medium to long term):
Management of C. difficile infection is a current NHS priority and is a major financial drain on the health service. Development of novel therapies to treat C. difficile associated disease, particularly those that reduce the rate of relapse, will have a major impact on the NHS.

(iv) Industry (medium to long term):
In the context of the current antimicrobial resistance (AMR) crisis, the development of new antimicrobials is an absolute priority. Biotech and pharmaceutical companies with an interest in the development of antimicrobials will benefit both directly and indirectly from this project. The C. difficile L,D transpeptidation pathway is an attractive target for new targeted antimicrobials and anti-resistance therapeutics. This project will provide crucial information about the biochemical basis of C. difficile peptidoglycan synthesis and beta-lactam resistance. Ultimately this will enable the rational design small molecule inhibitors that either directly kill C. difficile by compromising cell wall synthesis or restore susceptibility to traditional beta-lactam antibiotics.

(v) Students and Staff at the University of Sheffield (Short to medium term):
Undergraduate and postgraduate students at the University of Sheffield will benefit from their involvement in the project, developing knowledge and skills that will contribute to successful future careers. The postdoctoral research associate employed on the project will also develop crucial research and career development skills, with opportunities for involvement in outreach, science communication and industry collaborations.