title: Role of SbmA in the uptake of peptide nucleic acid (PNA)-peptide conjugates in E. coli.
abstract: Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli 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 sbmA, 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 sbmA. Furthermore, deletion of sbmA in E. coli AS19, a strain that is sensitive to unmodified PNA, resulted in resistance to PNA. Finally, PNA conjugated with the corresponding non-biological H-D((KFF)(3)K) peptide retained antibacterial activity in sbmA 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-D((KFF)(3)K), 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.
authors.affiliation: Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark.
authors.forename: Anubrata
authors.initials: A
authors.lastname: Ghosal
authors.forename: Ally
authors.initials: A
authors.lastname: Vitali
authors.forename: James E M
authors.initials: JE
authors.lastname: Stach
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen
mesh_terms: D001692:Biological Transport; D004926:Escherichia coli / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D004676:Myelin Basic Protein / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D004676:Myelin Basic Protein; D010446:Peptide Fragments; D020135:Peptide Nucleic Acids; C529573:SbmA protein, E coli; C018357:peptide L