Membrane-Active Peptides Derived from Natural Transmembrane Domains Function as Antibiotic Potentiators

Antimicrobial resistance continues to be a prevailing threat to human health worldwide, largely due to the arsenal of resistance mechanisms bacteria have evolved over years of exposure to traditional antibiotics. As an approach to devising therapeutics that can overcome this resistance, we hypothesized that peptides consisting of single transmembrane (TM) segments of membrane proteins may permeabilize bacterial membranes and thereby facilitate access of antimicrobials to their cytoplasmic targets. Using peptides derived from a natural TM helix from the AcrB component of the AcrAB-TolC efflux protein, we found that AcrB TM8 (wild type sequence: KKKK-FL(Abu)LAALYESWSI-NH₂) and a "scrambled" analog of identical composition, charge, and overall hydrophobicity (TM8-S: KKKK-FSLEALW(Abu)ISAYL-NH₂) (Abu = α-aminobutyric acid), resensitize <i>Escherichia coli</i> to sublethal doses of the antibiotics cloxacillin and nalidixic acid. These peptides induce 50-100% reduction of cell growth compared to bacteria treated with either peptide or antibiotic alone. The molecular basis for peptide-based permeabilization of the bacterial outer and inner membranes that contributes to the observed antibiotic potentiation was studied through several <i>in vitro</i> liposome-based assays and <i>in vivo</i> fluorescence-based assays. The overall results suggest that membrane proteins harbor a wealth of peptide sequences that may function as effective membrane-active peptides for the targeting of difficult-to-treat Gram-negative bacterial infections, and as such, provide valuable guidelines for the design of membrane-active peptides with synergistic antibacterial and antibiotic potentiation activity.