Designed to penetrate: Time-resolved interaction of single antibiotic molecules with bacterial pores

Abstract

Membrane permeability barriers are among the factors contributing to the intrinsic resistance of bacteria to antibiotics. We have been able to resolve single ampicillin molecules moving through a channel of the general bacterial porin, OmpF (outer membrane protein F), believed to be the principal pathway for the ␤-lactam antibiotics. With ion channel reconstitution and high-resolution conductance recording, we find that ampicillin and several other efficient penicillins and cephalosporins strongly interact with the residues of the constriction zone of the OmpF channel. Therefore, we hypothesize that, in analogy to substrate-specific channels that evolved to bind certain metabolite molecules, antibiotics have ''evolved'' to be channel-specific. Molecular modeling suggests that the charge distribution of the ampicillin molecule complements the charge distribution at the narrowest part of the bacterial porin. Interaction of these charges creates a region of attraction inside the channel that facilitates drug translocation through the constriction zone and results in higher permeability rates.

The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria

“…Inspection of the porin structure suggests that the permeation of molecules through the channel is driven by molecular interactions with the surface rather than by free diffusion 68,69 . The first evidence for facilitated diffusion through a binding site was found in maltoporin, a maltose-specific channel from the outer membrane in E. coli [70][71][72][73] .…”

“…In addition, the phenyl group is attracted to a hydrophobic pocket 66,68 . This observation has led to the development of molecular modelling approaches to elucidate the possible pathways of antibiotics through the OmpF channel 69,74,75 . Here, we describe the biophysical methods that can be used to estimate translocation rates through porins.…”

“…This dialogue provides the molecular specificity and it is chemical structure, which exert the dominant influence on permeation pathways (and not the molecules size). The current understanding of the molecular mechanism of antibiotic permeation is based on the available high-resolution crystal structure of OmpF 66,68,69 . This structural knowledge, together with an extensive collection of relevant mutants, now offers a unique opportunity to model and modify the surface of the OmpF channel and to compare the theoretical transport pathways with flux experiments.…”

“…Addition of antibiotics or sugar molecules on one (or both) side of the bilayer causes them to diffuse towards the channel. High-resolution ion-current fluctuation analysis allows detection of the passage of single molecules and thus characterization of facilitated permeation of sugars and antibiotics [68][69][70] . Addition of millimolar concentrations of ampicillin to the system can cause fluctuations in the ion current.…”

Investigating reaction pathways in rare events simulations of antibiotics diffusion through protein channels

“…In principle, standard MD simulations would have the required microscopic accuracy to link the structure and dynamics (of the drug and porin) to the rate of permeation. However, standard simulations are limited to hundred of nanoseconds at most and they do not allow the study of the reactive pathway that antibiotics follow during passive diffusion, which is on the order of hundreds of microseconds [4].…”