Gene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates onEscherichia coliandSalmonella entericaSerovar Typhimurium in Pure Culture and inTissue Culture
- 1 August 2006
- journal article
- research article
- Published by American Society for Microbiology in Antimicrobial Agents and Chemotherapy
- Vol. 50 (8) , 2789-2796
- https://doi.org/10.1128/aac.01286-05
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.Keywords
This publication has 16 references indexed in Scilit:
- Peptide Nucleic Acid Antisense Oligomer as a Therapeutic Strategy against Bacterial Infection: Proof of Principle Using Mouse Intraperitoneal InfectionAntimicrobial Agents and Chemotherapy, 2005
- Arginine-Rich Peptide Conjugation to Morpholino Oligomers: Effects on Antisense Activity and SpecificityBioconjugate Chemistry, 2005
- Antisense phosphorodiamidate morpholino oligomer inhibits viability of Escherichia coli in pure culture and in mouse peritonitisJournal of Antimicrobial Chemotherapy, 2005
- Antibacterial antisense.2005
- Antisense Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition inEscherichia coliAntimicrobial Agents and Chemotherapy, 2005
- Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acidsPublished by Elsevier ,2004
- PNA TechnologyMolecular Biotechnology, 2004
- Inhibition of Gene Expression inEscherichia coliby Antisense Phosphorodiamidate Morpholino OligomersAntimicrobial Agents and Chemotherapy, 2003
- Bactericidal antisense effects of peptide–PNA conjugatesNature Biotechnology, 2001
- Morpholino Antisense Oligomers: Design, Preparation, and PropertiesAntisense and Nucleic Acid Drug Development, 1997