Antimicrobials and antimicrobial resistance

Richardson, William (2017) Antimicrobials and antimicrobial resistance. MRes thesis, University of Nottingham.

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Abstract

Project 1: Developing new anti-infective agents is essential in our fight to combat AMR. Pseudomonas aeruginosa, an opportunistic pathogen, causes severe lung infections in cystic fibrosis patients. P. aeruginosa uses a highly inter-linked network of cell-to-cell communication pathways to coordinate the expression of virulence machinery necessary to maintain an infection. The PQS quorum sensing pathway has been shown to regulate biofilm formation and virulence. This study focuses on the optimisation of a thioacetamide class of quorum sensing inhibitors using in vitro cell based assays, structural analysis and isothermal titration calorimetry. An in vitro biosensor identified 5 potential inhibitors of PqsR, the PQS receptor protein, with varying potency. The most potent was analysed using ITC to validate the binding between the inhibitor and PqsR receptor domain. The binding constant of the inhibitor closely matched the native PQS signal. To understand inhibitor – protein interaction crystals were grown with inhibitors in solution although the resolution was insufficient to discern individual residues.

Project 2: Understanding the interaction profile of key proteins within pathogenic Gram negative bacteria is imperative in developing new antimicrobials. This study designed a high throughput method of protein-protein interaction screening using transposon mediated biomolecular fluorescent protein reconstitution. A yellow fluorescence protein was optimised for Gram negative expression and split with the larger fragment chromosomally fused to a nitrogen stress protein Hcp and the smaller fragment integrated into a custom transposon. The optimised yellow fluorescent protein allowed for E. coli sub-populations to be identified according to fluorescence emission detected with a FITC filter. Construct cloning was successful with a test plasmid containing a suspected Hcp binding partner Hcr created as a positive control. Some evidence of interaction was detected using a fluorescent cell based assay in a 96 well plate. Poor expression was attributed to weak fluorescence detection as the proteins are regulated tightly by oxygen availability and nitrogen stress.

Item Type: Thesis (University of Nottingham only) (MRes)
Supervisors: Williams, P.
Keywords: QS inhibitors, PqsR (MvfR), Anti-virulence, Inhibitor optimisation, P. aeruginosa. Protein-protein interactions, TraDIS, Biomolecular Complementation (BiFC), Fluorescent cell sorting, Nitrogen stress proteins
Subjects: Q Science > QR Microbiology > QR171 Microorganisms in the animal body
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 48109
Depositing User: Richardson, William
Date Deposited: 12 Jan 2018 13:58
Last Modified: 18 Jan 2018 08:26
URI: http://eprints.nottingham.ac.uk/id/eprint/48109

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