Inhibition of the outer membrane channel TolC with phage-displayed peptides
Anderson, Matthew S.
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Gram negative bacteria have evolved Multi-Drug Resistance (MDR) efflux pumps to extrude harmful substances from the bacterial cell. In Escherichia coli, the outer membrane channel TolC is a vital component of several MDR efflux pumps. In addition to its contribution to drug efflux, TolC is necessary for the secretion of several virulence factors. In this study, we seek to inhibit the drug efflux activity of TolC through the use of phage-displayed peptides. We hypothesize that phage-displayed peptides bound to specific sites in the TolC molecule will be able to inhibit drug efflux through either 1) blocking of the TolC pore, 2) prevention of open-channel formation, or 3) prevention of TolC interaction with other efflux pump components. A series of insertion mutants, in which a Tobacco Etch Virus (TEV) peptide sequence is randomly inserted into tolC (tolC::tev) (28), was employed to identify sites in the TolC protein that are accessible from the extracellular environment and important to its function. Display Phage 14, isolated against the TEV peptide, was shown to bind to E. coli cells containing the TolC::TEV insertions located throughout TolC. The displayed peptide, TB14, was shown to inhibit TolC-mediated efflux in two different tolC::tev insertion mutants. Unfortunately, attempts to isolate display phage which could bind with high affinity to wild-type TolC peptide sequences located at these two sites failed. We have recently isolated a collection of display phage that bind native TolC with high affinity. Future studies would involve their characterization and their ability to bind and inhibit TolC function.