Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types

Abstract

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we characterised two possible motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free swimming bacteria and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+ motors comparable to phenamil at matching concentrations, and could also stop rotation of tethered cells driven by H+ motors. Bead measurements in presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+ and H+ stator types, and in pathogenic and non-pathogenic strains. ImportanceHere we characterised two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. Our two compounds were shown to inhibit flagellar motility at 10 M across multiple strains, from non-pathogenic E. coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (EHEC/UPEC) and lastly in sodium-powered Vibrio alginolyticus. Broad anti-motility compounds such as these are important tools in our efforts control virulence of pathogens in health and agricultural settings.