Antisense peptide nucleic acids targeting ribosomal protein genes: A novel strategy against carbapenem-resistant...

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a nosocomial pathogen associated with substantial morbidity and mortality globally. Due to its high resistance to most antimicrobial agents, including carbapenems, novel treatment strategies are urgently required for CRAB infection. We explored peptide nucleic acids (PNAs) targeting essential ribosomal proteins against CRAB. Potential targets were identified using the Database of Essential Genes, and a cell-penetrating peptide (CPP)-PNA conjugate library was synthesized. The PNAs were screened using colony-forming unit counts and minimum inhibitory concentration assays. Inhibition of mRNA and protein expression was confirmed using real-time PCR (real-time PCR) and western blotting, respectively. Cytotoxicity and efficacy were evaluated using human cell lines, in vivo micronucleus tests, and insect infection models. Two specific CPP-PNAs targeting the 50S ribosomal proteins, L11 (rplK) and L13 (rplM), effectively inhibited bacterial growth in a dose-dependent manner. Furthermore, rplK and rplM mRNA and protein levels were significantly reduced in CPP-PNA-treated bacteria. The CPP-PNAs showed minimal cytotoxicity in human cell lines and no genotoxic effects in mice, while maintaining robust in vivo efficacy in infected insect models and activity against clinically relevant CRAB isolates. These findings suggest that CPP-PNAs targeting rplK and rplM could serve as effective antisense therapies against CRAB infections.