Efflux pumps control intracellular drug-target kinetics by limiting rebinding in bacteria

Bacterial efflux pumps are major contributors to multidrug resistance, classically described as "gatekeepers" that reduce drug entry. Here, we uncover a post-entry mechanism of efflux pumps, revealing their function deep into intracellular drug-target interactions. Using quantitative live-cell imaging, we monitored the activity of major efflux systems in Escherichia coli and Pseudomonas aeruginosa with Hoechst 33342 (HCT), a DNA binding inhibitor. We found that inactivation of efflux (ΔtolC in E. coli and Δ6 in P. aeruginosa) increased the apparent HCT-DNA affinity, mediated by a decreased apparent unbinding rate, whereas the intrinsic rate remained unchanged. Statistical physics modeling and experimental testing show that, unlike under dilute in vitro conditions, drug molecules that unbind from their targets in intracellular environments undergo successive rebinding, prolonging the total lifetime of the drug bound to the target. However, efflux pumps counteract this effect by suppressing rebinding, thereby kinetically destabilizing drug-target interactions. This biophysical mechanism acts multiplicatively with the canonical gatekeeping effect to broaden and amplify drug resistance.