Peptide-Based Approach to Inhibition of the Multidrug Resistance Efflux Pump AcrB

Abstract

Clinically relevant multidrug-resistant bacteria often arise due to overproduction of membrane-embedded efflux proteins that are capable of pumping antibiotics out of the bacterial cell before the drugs can exert their intended toxic effect. The Escherichia coli membrane protein AcrB is the archetypal protein utilized for bacterial efflux study because it can extrude a diverse range of antibiotic substrates and has close homologues in many Gram-negative pathogens. Three AcrB subunits, each of which contains 12 transmembrane (TM) helices, are known to trimerize to form the minimal functional unit, stabilized noncovalently by helix−helix interactions between TM1 and TM8. To inhibit the efflux activity of AcrB, we have rationally designed synthetic peptides aimed at destabilizing the AcrB trimerization interface by outcompeting the subunit interaction sites within the membrane. Here we report that peptides mimicking TM1 or TM8, with flanking N-terminal peptoid tags, and C-terminal lysine tags that aid in directing the peptides to their membrane-embedded target, decrease the AcrBmediated efflux of the fluorescent substrate Nile red and potentiate the effect of the antimicrobials chloramphenicol and ethidium bromide. To further characterize the motif encompassing the interaction between TM1 and TM8, we used Forster resonance energy transfer to demonstrate dimerization. Using the TM1 and TM8 peptides, in conjunction with several selected mutant peptides, we highlight residues that may increase the potency and specificity of the peptide drug candidates. In targeting membrane-embedded protein−protein interactions, this work represents a novel approach to AcrB inhibition and, more broadly, a potential route to a new category of efflux pump inhibitors.

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

“…Peptide-based approaches have also been explored as a means to inhibit tripartite efflux systems. By exploiting the transmembrane AcrB TM1–TM8 protein–protein interface, Jesin et al 910 designed synthetic peptides to mimic TM1 or TM8 of AcrB to interfere with AcrB trimerization. Using Nile Red efflux assays and antibiotic susceptibility testing, TM1 and TM8 peptides were shown to significantly reduce efflux activity and potentiate the activity of antimicrobials.…”

Lipid-mediated antimicrobial resistance: a phantom menace or a new hope?

“…2C ). This strategy has been effective against the RND efflux pump AcrB in E. coli (Jesin et al 2020 ), and against an SMR efflux protein of Pseudomonas aeruginosa, PAsmr, inhibiting antimicrobial efflux (Mitchell et al 2019 ). These and other efflux pump inhibitors (EPIs), including PAβN, pyridopyrimidines, quinoline derivatives, arylpiperidines and arylpiperazines have been identified as having the potential to abrogate resistance in a number of problematic pathogens (Kabra et al 2019 ).…”

Update on the Discovery of Efflux Pump Inhibitors against Critical Priority Gram-Negative Bacteria

“…The value of RND efflux pumps as molecular targets is underscored by their propensity to support the acquisition of additional resistance mechanisms [29,30], their overexpression in dormant bacteria [31], and their role in cell-to-cell communication [32,33], biofilm formation [34], pathogenicity, and virulence [35]. Strategies against multidrug efflux pumps have been targeting their (a) expression [36], (b) complex assembly [37], or (c) the fully assembled active transporter.…”