Transporter excess and clustering facilitate adaptor protein shuttling for bacterial efflux

Multidrug efflux pumps confer not only antibiotic resistance to bacteria but also cell proliferation. In gram-negative bacteria, the ATP-binding cassette (ABC)-family transporter MacB, the adaptor protein MacA, and the outer membrane protein TolC form the MacA₆:MacB₂:TolC₃ assembly to extrude antibiotics and virulence factors. Here, using quantitative single-molecule single-cell imaging, we uncover that, in <i>E. coli</i> cells, there is a large excess of MacB (and TolC) driving the limiting adaptor protein MacA mostly into the MacAB-TolC assembly. Moreover, the excess MacB transporters can dynamically cluster around the assembly, and MacA can dynamically disassemble from the MacAB-TolC assembly, leading to an adaptor protein shuttling mechanism for efficient substrate sequestration from the periplasm toward efflux. We further show that both MacB clustering and MacAB-TolC assembly can be perturbed chemically or physically via microfluidics-based extrusion loading for compromised antibiotic tolerance. These insights may provide opportunities for countering the activities of multidrug efflux systems for antimicrobial treatments.