Role of SbmA in the Uptake of Peptide Nucleic Acid (PNA)-Peptide Conjugates in <i>E. coli</i>

Antisense PNA oligomers targeting essential genes (<i>acp</i>P or <i>fts</i>Z) and conjugated to the delivery peptide <i>L</i>((KFF)₃K) show complete growth inhibition of wild type <i>E. coli</i> strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA-peptide conjugates had disruptions in the region of <i>sbm</i>A, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with <i>sbm</i>A. Furthermore, deletion of <i>sbm</i>A in <i>E. coli</i> AS19, a strain that is sensitive to unmodified PNA, resulted in resistance to PNA. Finally, PNA conjugated with the corresponding non-biological H-<i>D</i>((KFF)₃K) peptide retained antibacterial activity in <i>sbm</i>A deletion strains, whereas the same conjugate with a protease-sensitive linker did not. These results clearly identify SbmA as a carrier of naked PNA over the inner bacterial membrane and thereby infer that the peptide is transporting the PNA conjugates over the outer membrane. Strains lacking SbmA were used to screen novel peptide-PNA carriers that were SbmA-independent. Four such PNA-peptide conjugates, H-<i>D</i>((KFF)₃K), H-(RFR)₄-Ahx-βAla, H-(R-Ahx-R)₄-Ahx-βAla, and H-(R-Ahx)₆-βAla, were identified that utilize an alternative uptake mechanism but retain their antimicrobial potency. In addition SbmA is the first protein identified to recognize PNA