characterisation of penicillin binding proteins and ß-lactamases to investigate ß-lactam action and resistance in klebsiella pneumoniae.

Context: Antimicrobial resistance (AMR) is a prevalent and increasing global health threat; 1.3 million deaths were directly attributed to AMR in 2019, with 4.95 million deaths associated with resistance. Klebsiella pneumoniae (Kp) is a Gram-negative pathogen classed by the World Health Organisation as critical priority, responsible for severe global infections and a top 3 pathogen for AMR-related deaths worldwide. ß-Lactams, the most prescribed antibiotic class worldwide, kill bacteria by binding and inhibiting Penicillin Binding Proteins (PBPs). ß-Lactam resistance can be conferred by ß-lactamases (BLAs), enzymes evolutionarily related to PBPs that inactivate the antibiotics, threatening their continued clinical efficacy. Challenge: We require new antibiotics and BLA inhibitors. Discovery, development and optimisation of new antimicrobial therapies is guided through understanding the complex ß-lactam action and resistance landscape. ß-Lactam:BLA inhibitor combinations are validated routes to overcome AMR through successful inhibition of PBPs and BLAs. However, old drug combinations are failing in clinic due to the rise in AMR, requiring informed new approaches to outsmart resistance. PBPs are a large enzyme family with myriad roles in cell wall biosynthesis to include glycan chain polymerisation and cross-linking. They differ in affinity, structure and function across and within bacterial species. Kp PBPs are poorly characterised and of clear importance to understand at a fundamental biochemical level to develop efficacious Kp treatments (antimicrobial developments). Aims and objectives: I will utilise an interdisciplinary suite of approaches in microbiology, biochemistry, structure, and computational simulation on Kp PBPs, BLAs (and variants) to: Identify key factors governing ß-lactam antibiotic affinity, selectivity, and resistance. I will visualise protein structures of PBPs bound to antibiotics (X-ray crystallography and cryo-EM) and link this to activity (enzyme kinetic) data to highlight important structural features required for potent binding, guiding strategies to develop and improve current drugs. Understand how PBPs interact with their natural substrates. I will produce soluble fragments of natural substrates to understand, using structure and kinetics, how PBPs catalyse peptidoglycan synthesis. Data will extend our understanding of essential cell wall synthesis and remodelling in Kp and other bacteria. Test novel compounds as dual PBP and BLA inhibitors. I will evaluate the efficacy and potency of new inhibitors) and antibiotics (collaborations across Oxford, Ljubljana and SMU) against target Kp PBPs and BLAs. Utilise the latest developments in dynamic structural biology and computational simulation to further explore PBP and BLA reactions. New, powerful, X-ray free electron lasers (XFELs) seek to visualise a moving picture of enzyme activity by capturing time-resolved snapshots along the reaction pathway. These dynamic data will feed into high-level quantum simulations to explore and model chemistry inaccessible in the laboratory. Together, these methods can create molecular movies of PBP and BLA reactions, predict impacts of sequence variants and novel antibiotics on enzyme activity, providing mechanistic insights into antibiotic efficacy and resistance. Optimise current and newly-developed antibacterials to treat Kp. Microbiological assays with current/new inhibitors alongside antibiotic partners will be tested against a range of clinical Kp strains. These minimal inhibitory concentration data will inform antibiotic synergy (combinations) in Kp, identifying new routes to effective treatments. Applications and benefits: I will advance the understanding of PBP/BLA mechanisms and inhibition in Kp.  This work will identify new candidate inhibitors, elucidate trends in antibiotic resistance and guide developments of more potent antibiotics or synergistic drug combinations which improve the clinical treatment of multi-drug resistant Kp.