Journal
FEMS MICROBIOLOGY REVIEWS
Volume 44, Issue 6, Pages 782-792Publisher
OXFORD UNIV PRESS
DOI: 10.1093/femsre/fuaa031
Keywords
microscopy in live bacterial cells; drug-efflux; drug-resistance; TetA efflux pump protein
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Funding
- ATIP-Avenir program
- Schlumberger Foundation for Education and Research [FSER 2019]
- FINOVI [AO-2014]
- ANR PRC program (PlasMed)
- FRM (Foundation pour la Recherche Medicale) [ECO201806006855]
- University of Lyon [ARF20150934201]
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Drug-efflux by pump proteins is one of the major mechanisms of antibiotic resistance in bacteria. Here, we use quantitative fluorescence microscopy to investigate the real-time dynamics of drug accumulation and efflux in live E. coli cells. We visualize simultaneously the intrinsically fluorescent protein-synthesis inhibitor tetracycline (Tc) and the fluorescently labelled Tc-specific efflux pump, TetA. We show that Tc penetrates the cells within minutes and accumulates to stable intracellular concentration after similar to 20 min. The final level of drug accumulation reflects the balance between Tc-uptake by the cells and Tc-efflux by pump proteins. In wild-type Tc-sensitive cells, drug accumulation is significantly limited by the activity of the multidrug efflux pump, AcrAB-TolC. Tc-resistance wild-type cells carrying a plasmid-borne Tn10 transposon contain variable amounts of TetA protein, produced under steady-state repression by the TetR repressor. TetA content heterogeneity determines the cells' initial ability to efflux Tc. Yet, efflux remains partial until the synthesis of additional TetA pumps allows for Tc-efflux activity to surpass Tc-uptake. Cells overproducing TetA no longer accumulate Tc and become resistant to high concentrations of the drug. This work uncovers the dynamic balance between drug entry, protein-synthesis inhibition, efflux-pump production, drug-efflux activity and drug-resistance levels.
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