HT61 is a novel, quinoline-derived antimicrobial that has shown potent activity against both multiplying and non-multiplying Gram positive bacteria, specifically MRSA. It has been previously suggested, but not investigated, that HT61 damages the cytoplasmic membrane of susceptible bacteria. In this study, the putative membrane-activity of HT61 in both Gram positive and Gram negative bacteria was investigated using various techniques, including the BacLight, membrane potential, ATP release and protoplast assays. It was shown that HT61 disrupts both Gram positive and Gram negative membranes resulting in increased permeability and depolarisation of the membrane, leading to leakage of intracellular components and lysis of Staphylococcus aureus protoplasts. Biomimetic bacterial cytoplasmic membrane models were developed using synthetic lipids to investigate the putative disruptive effects of HT61 on the lipid portion of bacterial membranes. Langmuir monolayer studies showed that HT61 primarily interacts electrostatically with model systems c6ntaining anionic lipids, followed by penetration into the monolayer. In the presence of salt, HT61 was also able to penetrate into zwitterionic lipid monolayers due to increased hydrophobicity. HT61 causes enough disruption to bilayers containing anionic lipids to elicit release of dye from liposomes, whilst also allowing access of a quencher to the aqueous core of the liposome. Monitoring the size and number of liposomes using nanoparticle tracking analysis indicated that following HT61 exposure the number of liposomes decreased and their size distribution became more polydisperse. The use of neutron reflectivity confirmed that HT61 interacts with the lipid bilayer, without penetrating through the membrane, by non-specifically targeting anionic lipids, eventually causing loss of material from the bilayer. It is therefore proposed that HT61 interacts with the headgroups of anionic lipids, inserting itself within the bilayer, causing lipid packing disruption, and at concentrations above a threshold, result in lysis of the bilayer and mixed micelle formation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:676099 |
| Date | January 2015 |
| Creators | Hubbard, Alasdair Thomas Macadam |
| Publisher | St George's, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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