Slusky Pilot Project Summary

Antibiotic resistant infections kill more people per year than HIV/AIDS or malaria. Clinical antibiotic resistance is correlated with the overexpression of particular efflux pumps. These efflux pumps shuttle out most classes of antibiotics so that the antibiotics can’t reach their targets. The most accessible part of the efflux pump is its outer membrane barrel. Plugging efflux pumps would make the antibiotics we already have work like new by stopping antibiotics from being removed from the cell and allowing them to reach their targets. Our long term goal is to develop peptides to combat antibiotic resistant infection. The objective of the proposed research is to determine what protein-protein interactions are necessary to create high affinity plugs for the TolC efflux pump. Prior efforts to disable efflux pumps have focused on inhibiting the pump’s periplasmic drug binding sites. However, those efforts yielded compounds that are cytotoxic. Because membrane barrels are not similar to mammalian proteins, we anticipate proteins that target the barrels will have fewer off target interactions and less cytotoxicity. Moreover, because the membrane beta barrel is on the surface of the cell it is an easily accessible target. Our central hypothesis is that we can plug the outer membrane beta barrel component of efflux pumps with peptides modeled on bacteriocin protein klebC which natively binds the efflux pumps to stabilize the bacteriocin interaction on the cell surface. The structure of this complex was recently solved and we have already found that peptide fragments of other klebC homologs make E. coli more susceptible to antibiotics. There have never before been peptides designed to plug outer membrane barrels and much of the critical information about those protein-protein interactions is unknown. In order to understand broadly how to make peptide plugs for beta barrels, we plan to 1) determine the strength of interactions between TolC and minimalist fragments of the bacteriocin klebC, and 2) computationally design efflux pump plugs modeled on native interactions between klebC and TolC. We will measure specificity and affinity of our interactions in vitro using isothermal titration. We will measure how the plugs interact with bacteria using measurements of minimum inhibitory concentration of antibiotics. The expected outcomes of these experiments are a) a
better understanding of how to plug large outer membrane β-barrels and b) creation of a set of peptide plugs that inhibit antibiotic efflux in E. coli. This work is poised to make a significant contribution because it will demonstrate the important protein-protein interactions necessary to create membrane protein plugs and can thereby enable a revival of existing antibiotics against resistant superbugs.