Novel Synthetic Tools as Bacterial Diagnostics and Therapeutics

  • Jenny L Samphire

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)

Abstract

The issue of antimicrobial resistance is of increasing concern and so is the ability to develop probes that can specifically recognise and target bacteria with alternative antibacterial mechanisms which will be of the utmost importance in countering its growth. The aim of this project is to develop a new class of diagnostic and antibiotic tools that can offer improvements on current protocols and alternative mechanisms of action. This thesis investigates the use of a bench stable, non-toxic, carbon-based nanomaterial, termed carbon dots (CDs), as bacterial labelling and antibacterial therapeutic tools. Previously reported green-fluorescent CDs (GCDs), synthesised via a three-minute microwave reaction, are shown to fluorescently label four different species of bacteria with incubation times as low as one minute. Also discussed is the antimicrobial effect exhibited by the GCDs in the presence of blue-LED irradiation, in both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Specific targeting of E. coli through the exploitation of the interaction between the membrane-bound carbohydrate binding protein (lectin) FimH and mannose is also targeted. Different nanoparticle platforms and a variety of mannose-derived ligands are also explored. A successful nanoprobe was developed, a blue fluorescent CD functionalised with a seven carbon mannoside ligand, which was seen to interact with FimH expressing E. coli in an agglutination-based assay.
Additionally, to the nanoparticle-based systems developed, a novel class of G4-binding antimicrobial compounds were investigated. G4s are nucleic acid secondary structures found in guanine rich sequences and have been identified in the genomes of multiple organisms, with particular research interest in their involvement cancer. However, little is known about their role and effect in bacteria. A variety of multidrug resistant, clinically relevant bacterial strains were screened against a previously synthesised library of G-quadruplex (G4) stabilising ligands. Several G4 stabilising ligands were found to exhibit antibacterial activity against the drug-resistant strains, one stiff-stilbene based ligand, L6, showing potency in both E. coli and S. aureus with minimum inhibitory concentrations as low as 1 μg/mL. An RNA G4 sequence from E. coli is also assessed and the stabilisation induced from G4 binding ligands measured with circular dichroism and FRET melting assays. The potent L6 ligand exhibited better thermal stabilisation than the G4 benchmark ligands BRCO19 and TMPyP4. Nineteen compounds from the G4 ligand library were also shown to exert an antibacterial effect on M. tuberculosis model strain M. smegmatis. The results found in nanoparticle systems and G4 targeting ligands offer alternative strategies for bacterial identification and antibacterial therapeutics, where rapid diagnostics and novel antibiotic mechanisms are vital in the fight against antimicrobial resistance.
Date of Award28 Sept 2021
Original languageEnglish
Awarding Institution
  • University of Bristol
SupervisorM C Galan (Supervisor)

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