Studies on the Bacterial Potassium efflux system Kef in Shewanella denitrificans SdKef

Thesis

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), ɣ 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, ^t

BuSG, was 400 nM.

This dissertation looks to understand the potency shown by ^t

BuSG 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 ^t

BuSG, 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.

Authors: Grayer, S

• DOI: 10.5287/ora-xoy228jom
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Keywords: efflux; bacteria; antibiotic; antiporter; potassium; glutathione; chemical probes; novel mechanism; glutathione S-conjugate; Kef; ion channel