Structural shifts in TolC facilitate efflux-mediated β-lactam resistance

Efflux-mediated beta-lactam resistance is a major public health concern, reducing the effectiveness of beta-lactam antibiotics against many bacteria. Structural analyses show the efflux protein TolC in Gram-negative bacteria acts as a channel for antibiotics, impacting bacterial susceptibility and virulence. This study examines beta-lactam drug efflux mediated by TolC using experimental and computational methods. Molecular dynamics simulations of drug-free TolC reveal essential movements and key residues involved in TolC opening. A whole-gene-saturation mutagenesis assay, mutating each TolC residue and measuring fitness effects under beta-lactam selection, is performed. Here we show the TolC-mediated efflux of three antibiotics: oxacillin, piperacillin, and carbenicillin. Steered molecular dynamics simulations identify general and drug-specific efflux mechanisms, revealing key positions at TolC's periplasmic entry affecting efflux motions. Our findings provide insights into TolC's structural dynamics, aiding the design of new antibiotics to overcome bacterial efflux mechanisms. Modelling and deep mutational scanning of TolC provide insights for designing beta-lactams to evade resistance, by outlining how efflux is fine-tuned for efficiency via transient hydrogen bonds with antibiotics and how allostery impacts exit site motions