Rationally Designed Self-Derived Peptides Kill <i>Escherichia coli</i> 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 <i>Escherichia coli</i> 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-permeabilized<i>E. coli</i> 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 intact<i>E. coli</i> cells. Further, both of them significantly change the cell membrane integrity of<i>E. coli</i>, 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 living<i>E. coli</i> 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.