MexXY-OprM efflux pump mediates resistance to odilorhabdins in <i>Pseudomonas aeruginosa</i>

ABSTRACT The urgent need for new antibiotic classes to treat infections caused by multidrug-resistant gram-negative pathogens has driven interest in odilorhabdins (ODLs), a novel class of cationic antimicrobial peptides that target the bacterial ribosome. NOSO-502, the first clinical candidate in this family, exhibits potent activity against multidrug-resistant Enterobacteriaceae but is inactive against Pseudomonas aeruginosa isolates. This study investigates the intrinsic resistance of P. aeruginosa to NOSO-502 using the reference strain PAO1. Our data indicate that NOSO-502 induces overexpression of the MexXY-OprM efflux pump, which mediates drug extrusion from the periplasm and limits antibacterial activity. To further explore the role of MexXY-OprM, we evaluated NOSO-95C, the first purified ODL from natural origin. Unlike NOSO-502, NOSO-95C does not induce overexpression of MexXY-OprM at subinhibitory concentrations, resulting in measurable antibacterial activity against PAO1. However, NOSO-95C remains susceptible to MexXY-OprM-mediated efflux, as evidenced by its limited efficacy against strains that constitutively overexpress this pump. To better understand the structural basis of these differences, we investigated the relationship between ODL structures and the ability to induce mexXY expression. Our results demonstrate that both the amino acid at position 7 and the C -terminal region of the ODL scaffold are critical determinants of MexXY induction and efflux susceptibility. These findings offer valuable insights for the design of next-generation ODLs with improved efficacy against P. aeruginosa . IMPORTANCE Pseudomonas aeruginosa is a major cause of hospital-acquired infections. Odilorhabdins (ODLs) are a novel class of ribosome-targeting antibiotics with promising activity against multidrug-resistant gram-negative bacteria. Unfortunately, their efficacy against P. aeruginosa remains limited. In this study, we identify the MexXY-OprM efflux pump as the primary determinant of intrinsic resistance of P. aeruginosa to ODLs. We show that the clinical candidate NOSO-502 strongly induces mexXY expression, resulting in active self-efflux and loss of antibacterial activity, whereas the natural ODL NOSO-95C displays reduced induction and partial activity. By combining microbiological, transcriptional, uptake, and structure-activity analyses, we demonstrate that specific structural patterns of ODLs govern both efflux susceptibility and efflux pump induction. These findings pave the way for the design of the next ODL generation with improved activity against P. aeruginosa .