Escobar-Salom M; Barceló IM; Sansó-Sastre J; Torrens G; Jordana-Lluch E; Moyà B; Oliver A; Juan C

Abstract: We performed a proof-of-concept study to validate a peptide-conjugated peptide nucleic acid (PPNA) directed to inhibit peptidoglycan recycling as strategy to reduce AmpC hyperproduction and β-lactam resistance in Pseudomonas aeruginosa. Our nagZ-targeting PPNA at 2 µM decreased mRNA levels of nagZ and ampC to about a quarter in the AmpC high-level hyperproducer mutant PAdacBΔD and a previously characterized clinical strain with similar features, causing low cytotoxicity on human A549 cells. Ceftazidime minimum inhibitory concentration decreased from 64 to 8 mg/L in both strains after combination with 2 µM PPNA (which showed significant synergy in checkerboard assays), suggesting that nagZ-targeting PPNAs can be explored as weapons to sensitize P. aeruginosa against β-lactams and return therapeutic value to these essential drugs.IMPORTANCEIn the current scenario of threatening antibiotic resistance rates in Pseudomonas aeruginosa, the quest for alternative therapeutic weapons must consider all options, including the use of antisense oligonucleotides (e.g., peptide-conjugated peptide nucleic acids [PPNAs]) to silence the production of key target proteins. In this regard, we designed a proof-of-concept study to validate a PPNA directed to inhibit peptidoglycan recycling as a strategy to impair AmpC β-lactamase hyperproduction and derived resistance in P. aeruginosa. Our results indicate that the designed PPNA (targeting the N-acetyl-glucosaminidase NagZ) at low concentrations significantly decreased AmpC production and ceftazidime resistance in clinically relevant high-level hyperproducer P. aeruginosa strains, suggesting interesting therapeutic potentials. Microbiology spectrum: 2025 doi: 10.1128/spectrum.02622-24 https://pubmed.ncbi.nlm.nih.gov/40736236