An antisense peptide-conjugated peptide nucleic acid (PPNA) for peptidoglycan recycling inhibition reduces AmpC hyperproduction and β–lactam resistance in Pseudomonas aeruginosa

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. IMPORTANCE In 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.