The Esc(1-21)-1c Antimicrobial Peptide Inhibits a Specific Transcriptional Activator of the MexAB–OprM Efflux Pump in P. aeruginosa

The emergence of multidrug-resistant <i>Pseudomonas aeruginosa</i> strains is increasingly becoming a critical threat to global health. Among the resistance mechanisms, the MexAB-OprM efflux pump confers <i>P. aeruginosa</i> with an efficient method to export a broad spectrum of antibiotics. The antimicrobial peptide Esc (1-21)-1c was shown to downregulate this efflux system, though its mechanism of action has not been unveiled thus far. Here, we employed a combination of molecular modeling and experimental methods to investigate the precise peptide inhibitory mechanism. Functional proteomic experiments revealed the <i>P. aeruginosa</i> protein Q9I5H3, homologous to <i>E. coli</i> QseB, as a putative key target of Esc(1-21)-1c. Molecular docking predicted stable peptide-protein interactions, which were experimentally validated through fluorescence spectroscopy. Furthermore, electrophoretic mobility shift assays demonstrated that Q9I5H3 specifically binds the MexAB-OprM promoter and that Esc(1-21)-1c competitively inhibits this interaction in a dose-dependent manner. These findings reveal a previously uncharacterized regulatory pathway for efflux pump control and highlight Q9I5H3 as a promising therapeutic target against multidrug-resistant <i>P. aeruginosa</i>.