Targeting carA Using Optimized Antisense Peptide Nucleic Acid–Cell-Penetrating Peptide Conjugates in Acinetobacter baumannii: A Novel Antibacterial Approach

<i>Acinetobacter baumannii</i> is an opportunistic pathogen associated with severe bloodstream infections. It exhibits a high level of multidrug resistance, posing major clinical challenges. Antisense peptide nucleic acids (PNAs) represent a promising alternative to conventional antibiotics; however, their therapeutic efficacy depends on optimal delivery and molecular design. In this study, we aimed to enhance the antibacterial activity of PNAs against <i>A. baumannii</i> by systematically optimizing cell-penetrating peptides (CPPs), PNA length, target region, and chemical modifications. The efficacy and safety of CPP-PNA constructs were evaluated using a comprehensive set of approaches, including determination of minimum bactericidal and minimum inhibitory concentrations, quantitative reverse transcription polymerase chain reaction, Western blotting, and cytotoxicity assays. Three CPP-PNA constructs targeting <i>carA</i> were synthesized. Among these, the KFFK (FFK)₂-PNA conjugate showed the strongest bacterial growth-inhibitory effect, while the addition of extra lysine residues reduced its efficacy. Further analyses showed that a 10-mer alpha (α)-PNA modification targeting the ribosomal binding site of <i>carA</i> had the greatest inhibitory effect. These results underscore the importance of rational CPP design and PNA optimization in developing effective antisense antimicrobials against <i>A. baumannii</i>.