FIRL potentiates antibiotic activity against multidrug-resistant Gram-negative bacteria: selective BamA-BamD...

The outer membrane (OM) of Gram-negative bacteria limits antibiotic entry and contributes substantially to multidrug resistance. OM integrity depends on β-barrel outer membrane protein (OMP) biogenesis mediated by the β-barrel assembly machinery (BAM) complex, making BAM an attractive target for antibiotic adjuvant development.

To define the molecular mechanism by which the previously described BamD-derived peptide FIRL functions as a BAM-targeting antibiotic adjuvant, with particular emphasis on its activity at the BamA-BamD interface.

Antibiotic potentiation was evaluated against susceptible and multidrug-resistant Gram-negative bacteria. Molecular dynamics simulations, mutagenesis, and binding analyses were used to characterize FIRL-mediated perturbation of BamA-BamD coupling. Effects on OM integrity, envelope stress responses, OMP abundance, intracellular antibiotic accumulation, and therapeutic efficacy were examined.

FIRL exhibited minimal effects on bacterial growth at the concentrations used in mechanistic studies but potentiated permeability-restricted antibiotics across multiple Gram-negative species and restored polymyxin B activity against mcr-1-positive isolates. FIRL bound the BamA POTRA2 region and perturbed a BamA-BamD interaction network involving BamD residue R97. This perturbation was associated with activation of the σ^E envelope stress response, reduced abundance of major OMPs, increased OM permeability, and enhanced intracellular erythromycin accumulation. FIRL also increased polymyxin-associated OM permeability in mcr-1-positive strains. In Galleria mellonella and murine wound infection models, FIRL significantly enhanced the therapeutic efficacy of polymyxin B.

FIRL promotes conditional OM vulnerability through perturbation of BamA-BamD coupling. These findings implicate the POTRA2-BamD R97 interaction network in OM homeostasis and support BAM-directed antibiotic adjuvant strategies against multidrug-resistant Gram-negative infections.