The role of transporters in antibiotic uptake into Escherichia coli

Abstract

The widespread use of antibiotics in human health and agriculture has driven the development of antimicrobial resistance, and there is an urgent need to develop new antibiotics. An important challenge in doing so is the lack of a granular understanding of how such compounds actually gain entry to bacterial cells, to reach their intracellular targets. The prevailing view is that drugs almost invariably cross the cytoplasmic membrane (CM) by diffusion directly across the lipid bilayer (“passive lipoidal diffusion”). However, the O’Neill laboratory has recently shown that a number of structurally-distinct antibacterial drug classes actually reach the cytoplasm of the Gram-positive pathogen, Staphylococcus aureus, by “hitch-hiking” on membrane transporters. The present study sought to establish whether this phenomenon also holds true in a Gram-negative bacterium, using a similar approach to that employed for S. aureus. A library of strains (“ECMKO”) independently deleted for most of the membrane transporters in E. coli, was screened in a competition-growth assay, on the basis that strains deleted for a transporter involved in antibiotic uptake would likely exhibit improved competitive fitness over the parent strain in the presence of that antibiotic. To facilitate this screening process, a fluorescence-based competition-growth assay was first established and validated. Initial hits from the screen were confirmed by more traditional (colony-counting) competition-growth assays, and antibiotic accumulation into these strains was then measured by LC-MS. Nine confirmed hits were identified from the ECMKO; six for ciprofloxacin, two for tetracycline and one for trimethoprim. These single gene deletion mutants showed around 10-15% reduction in accumulation of the corresponding antibiotic, suggesting that there may be multiple carriers of ciprofloxacin. The six hits for ciprofloxacin were therefore used to engineer poly mutant strains carrying up to four deletions for genes identified as being involved with uptake of this drug, on the basis that increased number of deletions should show a decreased accumulation, even allowing for redundancy in transporters. Quadruple mutants showed up to 50% reduction in accumulation and elevated MIC. The results from this study confirms that some antibiotics traverse the Gram-negative CM by transporters, and underscores the idea of designing new drugs that mimic the metabolites for these transporters.