The global problem of antibiotic resistance makes identification of new antibiotic targets essential. Potassium ion (K+) transport is crucial for bacterial growth and survival through its role in regulating cytoplasmic pH and cell turgor. We have studied the potassium (K+) efflux system (Kef), a ligand-gated K+/H+ antiporter in Gram-negative bacteria, which protects them from toxic electrophiles. The rate and extent of Kef activation are critical factors for bacterial survival after exposure to electrophiles. Kef is therefore a promising new target for antibiotics. However, Kef-targeting drugs can only be developed with an understanding of Kef at the molecular level. We aimed to identify Kef systems in bacterial pathogens, synthesise and test chemical probes to determine if they activate diverse Kef systems. Genome-wide bioinformatic searches revealed Kef systems in the bacterial pathogens, YpKef in Yersinia pestis (plague) and BpKef1 and -2 in Burkholderia pseudomallei (melioidosis). YpKef gave good homology scores to EcKefC (43% identities, 63% positives) while BpKef1 and -2 gave lower scores (BpKef1: 33% identities, 51% positives; BpKef2: 25% identities, 44% positives). It was hypothesised that our dipeptide tool compounds, previously shown to activate EcKefC, were likely to activate YpKef but not BpKef1 and -2. The newly-identified kef genes were cloned by recombinant DNA technology. The resulting plasmids were used in Kirby-Bauer disc diffusion assays in E. coli to screen for ligands that can kill and/or inhibit bacteria. Hits were found for YpKef but not for BpKef1 or -2, consistent with our initial hypothesis. In a minimum inhibitory concentration (MIC) assay in E. coli expressing YpKef, analogue (3) was bacteriostatic with an MIC of 16 μg mL-1. The expression level of YpKef in the two E. coli strains used in the disc assay will be determined using SDS-PAGE/western blotting. These findings demonstrate the potential of Kef as a novel antibiotic target in pathogenic bacteria.

Analysis of potassium (K+) efflux systems (Kef) as an antibiotic target in pathogenic bacteria

256th National Meeting (2018)

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