Studies on the Bacterial Potassium efflux system Kef in Shewanella denitrificans SdKef
<p>The glutathione-gated potassium efflux system (Kef) is a K<sup>+</sup>/H<sup>+</sup> 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 el...
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| Format: | Thesis |
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2015
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| _version_ | 1826316615915929600 |
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| author | Grayer, S |
| author2 | Conway, S |
| author_facet | Conway, S Grayer, S |
| author_sort | Grayer, S |
| collection | OXFORD |
| description | <p>The glutathione-gated potassium efflux system (Kef) is a K<sup>+</sup>/H<sup>+</sup> 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 (<b>GSH</b>), ɣ L-glu-L-cys-gly, and activated by glutathione S-conjugates (<b>GSX</b>). Healy <em>et al</em>. have quantified the affinities of <b>GSH</b> and a range of <b>GSX</b> for <em>Shewanella denitrificans</em> Kef (SdKef). <b>GSH</b> was found to have a weak affinity of 900 μM, whereas the strongest GSX, <b><sup><em>t</em></sup></b></p>BuSG, was 400 nM. <p>This dissertation looks to understand the potency shown by <b><sup><em>t</em></sup></b></p>BuSG for <em>Sd</em>Kef by exploring the binding contributions from each group of the tripeptide. Truncated analogues of <b><sup><em>t</em></sup></b>BuSG were synthesised and evaluated using a competition fluorescence assay and <sup>1</sup>H CPMG NMR. In summary, removal of the glycine unit caused a complete loss in affinity for <em>Sd</em>Kef, whereas removing the glutamate unit resulted in a negligible loss. Interactions made by the Glu-Cys amide carbonyl oxygen, the Cys-Gly amide N<em>H</em> and a directional interaction of the conjugated thiol were also found to be important contributors to affinity. <p>The information obtained during this work allowed the development of a membrane permeant, truncated analogue of <b><sup><em>t</em></sup></b></p>BuSG, which lacks the majority of the glutamate, for use in in vivo studies. The truncated analogue is able to activate <em>Sd</em>Kef to elicit K<sup>+</sup> efflux, demonstrating that the majority of the glutamate is not essential for activity. Furthermore, application of this truncated analogue to <em>Escherichia coli</em> cells expressing the <em>sdkef</em> gene in a Kirby Bauer disc diffusion assay has demonstrated for the first time that small molecules activating <em>Sd</em>Kef can elicit inhibition of growth / cell death. Kef thus shows promise as a target for the development of novel antibacterial agents. |
| first_indexed | 2024-03-06T21:56:55Z |
| format | Thesis |
| id | oxford-uuid:4d392a8a-d80f-49b7-b350-b5a1248e099f |
| institution | University of Oxford |
| last_indexed | 2024-12-09T03:48:14Z |
| publishDate | 2015 |
| record_format | dspace |
| spelling | oxford-uuid:4d392a8a-d80f-49b7-b350-b5a1248e099f2024-12-08T11:10:38ZStudies on the Bacterial Potassium efflux system Kef in Shewanella denitrificans SdKefThesishttp://purl.org/coar/resource_type/c_db06uuid:4d392a8a-d80f-49b7-b350-b5a1248e099fORA Deposit2015Grayer, SConway, SMorris, G<p>The glutathione-gated potassium efflux system (Kef) is a K<sup>+</sup>/H<sup>+</sup> 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 (<b>GSH</b>), ɣ L-glu-L-cys-gly, and activated by glutathione S-conjugates (<b>GSX</b>). Healy <em>et al</em>. have quantified the affinities of <b>GSH</b> and a range of <b>GSX</b> for <em>Shewanella denitrificans</em> Kef (SdKef). <b>GSH</b> was found to have a weak affinity of 900 μM, whereas the strongest GSX, <b><sup><em>t</em></sup></b></p>BuSG, was 400 nM. <p>This dissertation looks to understand the potency shown by <b><sup><em>t</em></sup></b></p>BuSG for <em>Sd</em>Kef by exploring the binding contributions from each group of the tripeptide. Truncated analogues of <b><sup><em>t</em></sup></b>BuSG were synthesised and evaluated using a competition fluorescence assay and <sup>1</sup>H CPMG NMR. In summary, removal of the glycine unit caused a complete loss in affinity for <em>Sd</em>Kef, whereas removing the glutamate unit resulted in a negligible loss. Interactions made by the Glu-Cys amide carbonyl oxygen, the Cys-Gly amide N<em>H</em> and a directional interaction of the conjugated thiol were also found to be important contributors to affinity. <p>The information obtained during this work allowed the development of a membrane permeant, truncated analogue of <b><sup><em>t</em></sup></b></p>BuSG, which lacks the majority of the glutamate, for use in in vivo studies. The truncated analogue is able to activate <em>Sd</em>Kef to elicit K<sup>+</sup> efflux, demonstrating that the majority of the glutamate is not essential for activity. Furthermore, application of this truncated analogue to <em>Escherichia coli</em> cells expressing the <em>sdkef</em> gene in a Kirby Bauer disc diffusion assay has demonstrated for the first time that small molecules activating <em>Sd</em>Kef can elicit inhibition of growth / cell death. Kef thus shows promise as a target for the development of novel antibacterial agents. |
| spellingShingle | Grayer, S Studies on the Bacterial Potassium efflux system Kef in Shewanella denitrificans SdKef |
| title | Studies on the Bacterial Potassium efflux system Kef in Shewanella denitrificans SdKef |
| title_full | Studies on the Bacterial Potassium efflux system Kef in Shewanella denitrificans SdKef |
| title_fullStr | Studies on the Bacterial Potassium efflux system Kef in Shewanella denitrificans SdKef |
| title_full_unstemmed | Studies on the Bacterial Potassium efflux system Kef in Shewanella denitrificans SdKef |
| title_short | Studies on the Bacterial Potassium efflux system Kef in Shewanella denitrificans SdKef |
| title_sort | studies on the bacterial potassium efflux system kef in shewanella denitrificans sdkef |
| work_keys_str_mv | AT grayers studiesonthebacterialpotassiumeffluxsystemkefinshewanelladenitrificanssdkef |