Identifying Cell-Penetrating Peptides for Effectively Delivering Antimicrobial Molecules into .

Cell-penetrating peptides (CPPs) are promising carriers to effectively transport antisense oligonucleotides (ASOs), including peptide nucleic acids (PNAs), into bacterial cells to combat multidrug-resistant bacterial infections, demonstrating significant therapeutic potential. , a Gram-positive bacterium, is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. In this study, through the combination of super-resolution structured illumination microscopy (SR-SIM), flow cytometry analysis, and toxicity analysis assays, we investigated the suitability of four CPPs for delivering PNAs into cells: HIV-1 TAT efficiently penetrated cells with low toxicity against ; (RXR)XB had high penetration efficiency with inherent toxicity against ; (KFF)K showed lower penetration efficiency than HIV-1 TAT and (RXR)XB; K8 failed to penetrate cells. HIV-1 TAT-conjugated PNA specific for the essential gyrase A subunit gene (TAT-anti- PNA) effectively inhibited the growth of . TAT-anti- PNA exhibited a significant bactericidal effect on serotypes 2, 4, 5, 7, and 9 strains of , which are known to cause human infections. Our study demonstrates the potential of CPP-ASO conjugates as new antimicrobial compounds for combating infections. Furthermore, our findings demonstrate that applying SR-SIM and flow cytometry analysis provides a convenient, intuitive, and cost-effective approach to identifying suitable CPPs for delivering cargo molecules into bacterial cells.