Abstract
The double membrane architecture of Gram-negative bacteria forms a barrier that is effectively impermeable to extracellular threats. Accordingly, researchers have shown increasing interest in developing antibiotics that target the accessible, surface-exposed proteins embedded in the outer membrane. TolC forms the outer membrane channel of an antibiotic efflux pump in Escherichia coli. Drawing from prior observations that colicin E1, a toxin produced by and lethal to E. coli, can bind to the TolC channel, we investigate the capacity of colicin E1 fragments to ‘plug’ TolC and inhibit its efflux function. First, using single-molecule fluorescence, we show that colicin E1 fragments that do not include the cytotoxic domain localize at the cell surface. Next, using real-time efflux measurements and minimum inhibitory concentration assays, we show that exposure of wild-type E. coli to fragments of colicin E1 indeed disrupts TolC efflux and heightens bacterial susceptibility to four common classes of antibiotics. This work demonstrates that extracellular plugging of outer membrane transporters can serve as a novel method to increase antibiotic susceptibility. In addition to the utility of these protein fragments as starting points for the development of novel antibiotic potentiators, the variety of outer membrane protein colicin binding partners provides an array of options that would allow our method to be used to inhibit other outer membrane protein functions.
Significance We find that fragments of a protein natively involved in intraspecies bacterial warfare can be exploited to plug the E. coli outer membrane antibiotic efflux machinery. This plugging disables a primary form of antibiotic resistance. Given the diversity of bacterial species of similar bacterial warfare protein targets, we anticipate that this method of plugging is generalizable to disabling the antibiotic efflux of other proteobacteria. Moreover, given the diversity of the targets of bacterial warfare proteins, this method could be used for disabling the function of a wide variety of other bacterial outer membrane proteins.
