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Towards a mechanistic understanding of PBP-mediated beta-lactam resistance in Streptococcus pneumoniae
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Rowland, Catherine Emily (2019) Towards a mechanistic understanding of PBP-mediated beta-lactam resistance in Streptococcus pneumoniae. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3494272~S15
Abstract
Cell wall biosynthesis is a crucial process for bacterial growth and a well-validated source of targets for existing antimicrobials. Among such targets are the penicillin-binding proteins (PBPs), which act within multiprotein complexes to assemble peptidoglycan from Lipid II, catalysing both glycosyltransferase (GT) and transpeptidase (TP) activities. This work focussed on the PBPs of Streptococcus pneumoniae, a key pathogen in terms of its healthcare burden and the prevalence of pneumococcal resistance to b-lactams.
The requirement for both PBP modification and branched muropeptides in the pneumococcal b-lactam resistance mechanism has been well documented. Progress in demonstrating the mechanistic link between these two factors has historically been limited by availability of the native substrates. We aimed to address the hypothesis that branching of precursors is beneficial for competition of PG substrates against blactams, and also to explore the impact of lipid environment on PBP1a activity, based upon previous observations with the MurM protein that generates branched Lipid II precursors.
We used chemoenzymatic methods to demonstrate the first synthesis of potential donor- and acceptor-only amidated substrates, in addition to branched precursors, for pneumococcal PBPs. These substrates will provide valuable tools for study of pneumococcal PBP function with natural substrates, which is key for addressing pneumococcal b-lactam resistance. A novel spectrophotometric assay for transpeptidation was used to attempt observation of in vitro transpeptidase activity by PBP1a. Mass spectrometry revealed that D,D-carboxypeptidase activity alone occurred under the current reaction conditions. We discuss possible further work to establish in vitro transpeptidase activity.
Established assays for GT activity were used to identified a novel link between pneumococcal membrane phospholipids and the activity of PBP1a. This is the first observation of the impact of cardiolipin on the activity of a PBP, and corresponds with growing evidence in support of a role for cardiolipin in regulation of cell division proteins.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QK Botany Q Science > QP Physiology Q Science > QR Microbiology R Medicine > RS Pharmacy and materia medica |
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Library of Congress Subject Headings (LCSH): | Streptococcus pneumoniae, Beta lactam antibiotics., Penicillium, Protein binding, Bacterial cell walls -- Synthesis, Drug resistance in microorganisms, Gram-negative bacteria, Gram-positive bacteria | ||||
Official Date: | August 2019 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Dowson, Chris ; Lloyd, Adrian J. | ||||
Sponsors: | Micropathology Ltd. | ||||
Extent: | xviii, 315 leaves : illustrations, charts | ||||
Language: | eng |
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