Antimicrobial peptide-induced inner membrane hyperpolarization is associated with antibiotic sensitization and attenuated MIC escalation in multidrug-resistant Gram-negative pathogens.

Antimicrobial resistance and dysregulated inflammation drive mortality in multidrug-resistant (MDR) sepsis. We evaluated the cationic peptide TP2-5 as a low-dose antibiotic adjuvant. At sub-MIC concentrations, TP2-5 enhanced antibiotic susceptibility of MDR E. coli in broth and 50% human serum, and in combination with antibiotics was associated with attenuated MIC escalation during 21-day serial passage. Membrane potential assays and cryo-electron tomography showed envelope perturbation characterized by inner-membrane hyperpolarization. This biophysical state was temporally associated with preferential interactions with lipopolysaccharide (LPS) and anionic phospholipids rather than nonspecific permeabilization. TP2-5 neutralized LPS and reduced TLR4-dependent cytokine production. In our murine polymicrobial CLP sepsis model, TP2-5 alone or with meropenem achieved 100% survival, accompanied by reduced bacterial burden and systemic inflammatory cytokines, consistent with combined antibacterial and host-directed effects, supporting a multifunctional adjuvant profile. This study did not measure bacterial membrane potential in vivo, and the causal role of hyperpolarization in protection or attenuated MIC escalation remains to be determined.