title: Antisense PNA effects in Escherichia coli are limited by the outer-membrane LPS layer.
abstract: Antisense peptide nucleic acids (PNAs) can inhibit Escherichia coli gene expression and cell growth through sequence-specific RNA binding, and this opens possibilities for novel anti-infective agents and tools for microbial functional genomics. However, the cellular effects of PNAs are limited relative to effects in cell extracts, presumably because of cell barrier components such as the outer-membrane lipopolysaccharide (LPS) layer or drug efflux pumps, both of which function to exclude antibiotics and other foreign molecules. To evaluate the importance of such cellular factors on PNA effects, the authors developed a positive assay for antisense inhibition by targeting the lac operon repressor and compared PNA susceptibilities in mutant and wild-type E. coli by assessing lacZ induction. Strains with defective LPS (AS19 and D22) were more permeable to the antibiotic nitrocefin and more susceptible to PNA than the wild-type. Also, PNA potency was improved in wild-type cells grown in the presence of certain cell-wall-permeabilizing agents. In contrast, the activities of the Acr and Emr drug efflux pumps were not found to affect PNA susceptibility. The results show that the LPS layer is a major barrier against cell entry, but PNAs that can enter E. coli are likely to remain active inside cells.
authors.affiliation: Center for Genomics Research, Karolinska Institute, Berzelius väg 37, 171 77, Stockholm, Sweden1.
authors.forename: Liam
authors.initials: L
authors.lastname: Good
authors.affiliation: Center for Genomics Research, Karolinska Institute, Berzelius väg 37, 171 77, Stockholm, Sweden1.
authors.forename: Rickard
authors.initials: R
authors.lastname: Sandberg
authors.affiliation: Center for Genomics Research, Karolinska Institute, Berzelius väg 37, 171 77, Stockholm, Sweden1.
authors.forename: Ola
authors.initials: O
authors.lastname: Larsson
authors.affiliation: Center for Biomolecular Recognition, Panum Institute, Blegdamsvej 3c, DK 2200 Copenhagen N., and Pantheco A/S, Fruebjergvej 3, DK 2100 Copenhagen Ø., Denmark2.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Nielsen
authors.affiliation: Center for Genomics Research, Karolinska Institute, Berzelius väg 37, 171 77, Stockholm, Sweden1.
authors.forename: Claes
authors.initials: C
authors.lastname: Wahlestedt
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004352:Drug Resistance, Microbial / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007763:Lac Operon / Q000235:genetics; D008070:Lipopolysaccharides / Q000378:metabolism*; D008074:Lipoproteins / Q000235:genetics; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D020320:Oligoribonucleotides, Antisense / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D020135:Peptide Nucleic Acids / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015854:Up-Regulation
chemical_list: C083497:AcrA protein, E coli; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D008074:Lipoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D020320:Oligoribonucleotides, Antisense; D020135:Peptide Nucleic Acids; C077169:VceB protein, Vibrio cholerae