CPP-PNA Conjugate-Mediated Inhibition of pdxA Gene Impairs Vitamin B6 Biosynthesis and Growth in Acinetobacter baumannii

<i>Acinetobacter baumannii</i> represents a critical-priority organism due to its multidrug resistance. The emergence of carbapenem-resistant strains poses a major clinical challenge, underscoring the urgent need for novel antibacterial agents with alternative mechanisms. As peptide nucleic acids (PNAs) have recently gained attention as antisense therapeutics, we aimed to validate their potential as novel antimicrobial strategies against multidrug-resistant <i>A. baumannii</i>. We synthesized a cell-penetrating peptide (CPP)-PNA conjugate targeting <i>pdxA</i>, an essential gene involved in vitamin B6 biosynthesis. Among several candidate genes tested, the <i>pdxA</i>-targeting PNA exhibited the strongest inhibitory activity, achieving complete growth suppression of <i>A. baumannii</i> at 1.56 μM. Although quantitative real-time polymerase chain reaction did not reveal significant reductions in <i>pdxA</i> transcript levels, ELISA quantification revealed an approximately 80% reduction in intracellular vitamin B6, indicating translational inhibition rather than mRNA degradation. The <i>pdxA</i>-targeting CPP-PNA showed negligible activity against other Gram-negative or Gram-positive species, indicating high target specificity; no detectable cytotoxicity in human cells was observed even at relatively high concentrations. CPP-PNA conjugates targeting <i>pdxA</i> interfere with vitamin B6 biosynthesis, leading to growth inhibition of <i>A. baumannii</i>. These findings support PNA as a promising antisense antimicrobial platform that inhibits multidrug-resistant <i>A. baumannii</i> by blocking vitamin B6 biosynthesis.