Pseudomonas aeruginosa is a Gram-negative species of bacteria that is of high clinical importance and is on the ESKAPE list of pathogens, causing the most serious nosocomial infections World-wide. Current antibiotics in clinical use against P. aeruginosa include carbenicillin, cefepime, ceftazidime, and the fluoroquinoline ciprofloxacin. The multi-drug resistant (MDR) phenotype exhibits widespread resistance to β-lactams and antibiotics from multiple classes often need to be administered in conjunction with β-lactamase inhibitors. Penicillin binding proteins (PBPs) are essential for cell viability and are important antibiotic targets, with the Class A bifunctional PBPs synthesising the principle component of the bacterial cell wall, peptidoglycan. The focus of this project was to investigate the kinetics of transglycosylation and transpeptidation mechanisms with a view to improving our understanding of bacterial cell wall synthesis, maintenance and regeneration. In this project, the function of Class A PBPs 1A and 1B from Escherichia coli and P. aeruginosa, with particular focus on PBP1A has been investigated. Transglycosylation and transpeptidation have been probed from multiple angles using orthogonal assays and fluorescently-labelled and native lipid substrates, as well as Lipid II structural variants. P. aeruginosa PBP1A transglycosylase and transpeptidase activities are demonstrated continuously, and compared to the model organism equivalent, E. coli PBP1A. The continuous monitoring of activity gave insights into the possibility of two catalytic sites for transglycosylation: a donor and an acceptor site. Data was fitted to different kinetic models to elucidate the best fit for extracting meaningful kinetic parameters. The data acquired over the course of this project suggests that P. aeruginosa PBP1A and E. coli PBP1A exhibit similar functional behaviour to each other, with E. coli PBP1B showing distinct activity from PBP1A. PaPBP1B activity was not detected at a level required to kinetically characterise the enzyme and it is possible that this PBP has an as yet undetermined stimulatory cofactor. The assays developed and optimised in this thesis will pave the way for further in-depth studies of P. aeruginosa PBP1A, which could be used to screen for putative inhibitor compounds and potentially identify certain characteristics required for antimicrobial efficacy. Over the past 4 years, major leaps in our knowledge of assay development, the transglycosylase and transpeptidase activities of Class A PBPs and substrate features and requirements for transglycosylase and transpeptidase donors and acceptors have thus far and will continue to unravel the unique and fascinating mechanistic intricacies of the bifunctionality of PBPs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:725293 |
| Date | January 2017 |
| Creators | O'Reilly, Amy |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/93414/ |
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