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Title: Studies on the bacterial potassium efflux system Kef in Shewanella denitrificans SdKef
Author: Grayer, Samuel Charles
ISNI:       0000 0004 6062 6531
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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The glutathione-gated potassium efflux system (Kef) is a K+/H+ antiporter found in the majority of Gram negative pathogens, which shows potential as a novel antibiotic target. Kef plays a vital role in the protection of bacteria against toxic electrophiles through the regulation of cytoplasmic pH. Kef is inhibited by glutathione (GSH), γ L-glu-L-cys-gly, and activated by glutathione S-conjugates (GSX). Healy et al. have quantified the affinities of GSH and a range of GSX for Shewanella denitrificans Kef (SdKef). GSH was found to have a weak affinity of 900 μM, whereas the strongest GSX, tBuSG, was 400 nM. This dissertation looks to understand the potency shown by tBuSG for SdKef by exploring the binding contributions from each group of the tripeptide. Truncated analogues of tBuSG were synthesised and evaluated using a competition fluorescence assay and 1H CPMG NMR. In summary, removal of the glycine unit caused a complete loss in affinity for SdKef, whereas removing the glutamate unit resulted in a negligible loss. Interactions made by the Glu-Cys amide carbonyl oxygen, the Cys-Gly amide NH and a directional interaction of the conjugated thiol were also found to be important contributors to affinity. The information obtained during this work allowed the development of a membrane permeant, truncated analogue of tBuSG, which lacks the majority of the glutamate, for use in in vivo studies. The truncated analogue is able to activate SdKef to elicit K+ efflux, demonstrating that the majority of the glutamate is not essential for activity. Furthermore, application of this truncated analogue to Escherichia coli cells expressing the sdkef gene in a Kirby Bauer disc diffusion assay has demonstrated for the first time that small molecules activating SdKef can elicit inhibition of growth / cell death. Kef thus shows promise as a target for the development of novel antibacterial agents.
Supervisor: Conway, Stuart ; Morris, Garrett Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available