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.
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.
Tilley
Lucas D
LD
AVI BioPharma, Inc., Corvallis, Oregon, USA.
Hine
Orion S
OS
Kellogg
Jill A
JA
Hassinger
Jed N
JN
Weller
Dwight D
DD
Iversen
Patrick L
PL
Geller
Bruce L
BL
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
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009025:Morpholines; D060172:Morpholinos; D010455:Peptides; D008156:Luciferases