Elucidating TolC Protein Dynamics: Structural Shifts Facilitate Efflux Mediated β-lactam Resistance

Abstract

ABSTRACT Efflux-mediated β-lactam resistance represents a significant public health challenge, limiting the efficacy of various β-lactam antibiotics against numerous clinically relevant pathogenic bacteria. Structural and functional analyses have revealed that the efflux protein TolC in several Gram-negative bacteria serves as a conduit for antibiotics, bacteriocins, and phages, affecting bacterial susceptibility and virulence. In this study, we conducted a comprehensive examination of the efflux of β-lactam drugs mediated by TolC, employing extensive experimental and computational analyses. Our computational investigations into the molecular dynamics of drug-free TolC revealed critical unidirectional movements of the trimeric TolC and identified residues significantly involved in TolC opening. To corroborate these findings, we performed a whole-gene-saturation mutagenesis assay, systematically mutating each residue of TolC to 19 other amino acids and measuring the fitness effects of these mutations under β-lactam-induced selection. The β-lactams oxacillin, piperacillin, and carbenicillin were selected for this study because they are effluxed by the AcrAB-TolC complex with varying efficiencies. This approach clarified the similarities and differences in the efflux processes of the three β-lactam antibiotics through the trimeric TolC. Further analysis of TolC’s efflux mechanism for these β-lactam antibiotics via steered molecular dynamics simulations revealed the existence of general and drug-specific mechanisms employed by TolC. We identified key positions at the periplasmic entry of TolC whose altered dynamics influence long-range efflux motions as allosteric modulators. Our findings provide valuable insights into the structural dynamics of TolC, establishing a foundation for understanding the key mechanisms behind multidrug resistance and principles for designing new antibiotics and antibiotic derivatives capable of circumventing the bacterial efflux mechanism.