Rationally Designed Self-Derived Peptides Kill Escherichia coli by Targeting BamA and BamD Essential for Outer Membrane Protein Biogenesis

There is an urgent need to develop antibiotics with new mechanisms of action for combating antibiotic-resistant bacteria, particularly against Gram-negative pathogens that severely threaten human health. Here, we introduce the rational design and comprehensive characterization of self-derived antibacterial peptides that specifically target Escherichia coli BamA and BamD, vital components of the β-barrel assembly machine (BAM) for the folding and membrane integration of outer membrane proteins (OMPs) in Gram-negative bacteria. Among the three BamA-targeted peptides, BamA_543-551, which corresponds to an extracellular loop of BamA, exhibits remarkable bactericidal activity against OM-permeabilizedE. coli cells. Similarly, among four BamD-targeted peptides, BamD_163-187 corresponding to a BamA-interacting α-helix exhibits potent bactericidal activity. Notably, both BamA_543-551 and BamD_163-187 are able to kill other OM-permeabilized Gram-negative pathogens but not Gram-positive ones, and fusion with a cell membrane-penetrating peptide enabled them to directly kill intactE. coli cells. Further, both of them significantly change the cell membrane integrity ofE. coli, induce the accumulation of misfolded OmpF, and reduce the level of folded OmpF. In particular, in vivo photo-cross-linking analysis indicates that BamA_543-551 disrupts the direct interaction between BamA and periplasmic chaperone SurA in livingE. coli cells, thus offering insights into their mode of action. Collectively, our findings confirm the potential of BamA and BamD as promising antibiotic targets and suggest that BamA- and BamD-derived peptides can be candidates for antibiotic development.