Abstract
Membrane efflux pumps are a leading cause of increasing multidrug resistant bacterial infections, which pose a significant threat to global human health. Understanding the mechanisms that underpin their function is critical for the development of inhibitors targeting these systems, with the aim being to ‘revive’ the activities of pre-existing antibiotics known to suffer bacterial resistance. AcrAB-TolC is a membrane spanning, tripartite multidrug efflux pump native to Escherichia coli and prototypical of homologous systems across other ESKAPE bacteria. The work in this thesis investigates the role of structural dynamics in the function, assembly, and inhibition of AcrAB-TolC, with a focus on the membrane fusion protein (MFP) AcrA, to reveal critical information on how these efflux systems work, which could be essential for developing novel methods of inhibition to combat multidrug resistance. Throughout this work, structural mass spectrometry (MS) techniques such as hydrogen deuterium exchange mass spectrometry (HDX-MS) and native MS were used alongside a range of complementary biophysical/biochemical techniques to investigate AcrAB-TolC.This work reveals that AcrA lipidation promotes the propensity of AcrA to form oligomers, whereas a non-lipidated, water soluble AcrA construct (AcrAS) is still monomeric. Moreover, HDX-MS showed AcrAS exhibits increased backbone structural dynamics at pH 6.0 compared to pH 7.4, yet this was largely tempered by the presence of magnesium. In the periplasm, the pH can often be ∼1.7 pH units lower than in the cytosol, and there is a significantly higher concentration of magnesium ions (7.56 times). This suggests a regulatory role of magnesium to help AcrA function within the periplasmic environment. To expand the investigations on AcrAS further, a soluble pseudo-dimer construct (AcrASD) was used to infer biological information on the AcrA functional dimer. It was found the pseudo-dimer has unique structural dynamics compared to AcrA, with extensive protection in the α-helices and in regions of the αβ-barrel and MP domains. Furthermore, whilst AcrAS and AcrASD appeared to bind peptidoglycan similarly, AcrASD had a higher propensity to form higher order complexes with AcrB. This suggests dimerization may help prime the AcrA protomers for interactions with its binding partners.
Traditionally, efflux pumps inhibitors (EPIs) have been targeted against AcrB, but none have made it past clinical trials, often due to toxicity issues. This has led to a switch in focus for the next generation of EPIs, with AcrA becoming a promising target. In this work, HDX-MS and native MS were used in combination with molecular dynamics (MD) simulations, to investigate the effect of a recently identified EPI, NSC 60339, on the structural dynamics of AcrAS. The data showed NSC 60339 likely binds to AcrA in a cleft bridging the lipoyl and αβ-barrel domains, stabilising these areas as well as the MP domain which usually exhibits intrinsic disorder; NSC 60339 inhibition of AcrASD presented the same. This work proposes the first mechanism of action regarding an AcrA inhibitor and reveals a promising new way to target the AcrAB-TolC complex.
Due to the hydrophobic nature of membrane proteins, a suitable membrane mimetic is required for in vitro investigations. As the AcrAB-TolC multidrug efflux pump spans the entire Gram-negative cell envelope and are therefore membrane proteins, studying them in lipid environments rather than detergents is essential as they provide a more representative environment. In this work, HDX-MS was used to show MBX-3756 stabilises the hydrophobic trap of AcrB in membrane scaffold protein (MSP) nanodiscs. Furthermore, a novel SMALP-liposome-SMALP assay was utilised to show that previously designed AcrB antimicrobial peptides did not make the AcrB trimer, purified in styrene maleic acid lipid particles (SMALPs), dissociate into monomers. Lastly, assembly of the AcrAB-TolC complex was probed using two different pull-down assays and native polyacrylamide gel electrophoresis (PAGE), however the heterogeneity and hydrophobicity of SMALPs complicated these investigations, combined with the slow energetics of this assembly in vitro.
| Date of Award | 1 Apr 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Eamonn Reading (Supervisor) & Paula Booth (Supervisor) |