title: Gene-specific effects of antisense phosphorodiamidate morpholino oligomer-peptide conjugates on Escherichia coli and Salmonella enterica serovar typhimurium in pure culture and in tissue culture.
abstract: The objective was to improve efficacy of antisense phosphorodiamidate morpholino oligomers (PMOs) by improving their uptake into bacterial cells. Four different bacterium-permeating peptides, RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B is beta-alanine), were separately coupled to two different PMOs that are complementary to regions near the start codons of a luciferase reporter gene (luc) and a gene required for viability (acpP). Luc peptide-PMOs targeted to luc inhibited luciferase activity 23 to 80% in growing cultures of Escherichia coli. In cell-free translation reactions, Luc RTRTRFLRRTXB-PMO inhibited luciferase synthesis significantly more than the other Luc peptide-PMOs or the Luc PMO not coupled to peptide. AcpP peptide-PMOs targeted to acpP inhibited growth of E. coli or Salmonella enterica serovar Typhimurium to various extents, depending on the strain. The concentrations of AcpP RFFRFFRFFXB-PMO, AcpP RTRTRFLRRTXB-PMO, AcpP KFFKFFKFFKXB-PMO, and ampicillin that reduced CFU/ml by 50% after 8 h of growth (50% inhibitory concentration [IC(50)]) were 3.6, 10.8, 9.5, and 7.5 microM, respectively, in E. coli W3110. Sequence-specific effects of AcpP peptide-PMOs were shown by rescuing growth of a merodiploid strain that expressed acpP with silent mutations in the region targeted by AcpP peptide-PMO. In Caco-2 cultures infected with enteropathogenic E. coli (EPEC), 10 microM AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO essentially cleared the infection. The IC(50) of either AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO in EPEC-infected Caco-2 culture was 3 microM. In summary, RFFRFFRFFXB, RTRTRFLRRTXB, or KFFKFFKFFXB, when covalently bonded to PMO, significantly increased inhibition of expression of targeted genes compared to PMOs without attached peptide.
authors.affiliation: AVI BioPharma, Inc., Corvallis, Oregon, USA.
authors.forename: Lucas D
authors.initials: LD
authors.lastname: Tilley
authors.forename: Orion S
authors.initials: OS
authors.lastname: Hine
authors.forename: Jill A
authors.initials: JA
authors.lastname: Kellogg
authors.forename: Jed N
authors.initials: JN
authors.lastname: Hassinger
authors.forename: Dwight D
authors.initials: DD
authors.lastname: Weller
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Iversen
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Geller
mesh_terms: D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D015169:Colony Count, Microbial; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000188:drug therapy; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D005798:Genes, Bacterial; D017930:Genes, Reporter; D006801:Humans; D066298:In Vitro Techniques; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008156:Luciferases / Q000037:antagonists & inhibitors; D009025:Morpholines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D060172:Morpholinos; D009154:Mutation; D010455:Peptides / Q000037:antagonists & inhibitors* / Q000235:genetics; D012486:Salmonella typhimurium / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D012703:Serotyping
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009025:Morpholines; D060172:Morpholinos; D010455:Peptides; D008156:Luciferases