Investigating the structure and function of metallo-β-lactamases with directed evolution

Metallo-β-lactamases (MBLs) are powerful enzymes capable of conferring pathogenic bacteria with effective resistance against all major classes of β-lactam antibiotics. Their continuing global dissemination, paired with a lack of therapeutic inhibitors, has combined to pose a significant threat to human health. This thesis aims to use an evolutionary perspective to better understand the structure, function, and behaviour of the MBLs. The comprehensive characterization of eight MBLs in three different host organisms, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, revealed that host specific constraints can limit the effective periplasmic expression of the enzymes, and as a result, might restrict the dissemination of MBLs to certain bacteria. The directed evolution of NDM-1, VIM-2, and IMP-1 for the provision of greater ampicillin resistance in Escherichia coli exposed the mechanisms by which MBLs may adapt to overcome these expression barriers, while revealing the critical role that the signal peptide plays in host adaptation. The subsequent directed evolution of the same three MBLs with two other β-lactam antibiotics, cefotaxime and meropenem, demonstrated the relative robustness of the family’s broad substrate specificity, as only two of seven complete trajectories featured a narrowing of specificity and changing the selection pressure on one of these trajectories swiftly restored broad specificity. The long-term genetic drift of VIM-2 under purifying selection at different thresholds revealed the plasticity of the MBL’s sequence and structure, but also the robustness of its activity and function. Overall, the results presented in this thesis contribute to our understanding of the MBL family and will help to develop better treatment strategies in the future.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat