Open AccessGene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates onEscherichia coliandSalmonella entericaSerovar Typhimurium in Pure Culture and inTissue Culture
Author(s) -
Lucas D. Tilley,
Orion S. Hine,
J. Kellogg,
Jed N. Hassinger,
Dwight D. Weller,
Patrick L. Iversen,
Bruce L. Geller
Publication year - 2006
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.01286-05
Subject(s) - morpholino , salmonella enterica , escherichia coli , salmonella , serotype , microbiology and biotechnology , enterobacteriaceae , conjugate , biology , oligomer , peptide , oligopeptide , virology , gene , chemistry , bacteria , biochemistry , genetics , mathematical analysis , mathematics , organic chemistry , zebrafish
The objective was to improve efficacy of antisense phosphorodiamidatemorpholino oligomers (PMOs) by improving their uptake into bacterialcells. Four different bacterium-permeating peptides, RFFRFFRFFXB, RTRTRFLRRTXB,RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B isβ-alanine), were separately coupled to two different PMOs thatare complementary to regions near the start codons of a luciferasereporter gene (luc ) and a gene required for viability(acpP ). Luc peptide-PMOs targeted toluc inhibitedluciferase activity 23 to 80% in growing cultures ofEscherichiacoli . In cell-free translation reactions, LucRTRTRFLRRTXB-PMO inhibited luciferase synthesissignificantly more than the other Luc peptide-PMOs or the Luc PMO notcoupled to peptide. AcpP peptide-PMOs targeted toacpP inhibited growth ofE. coli orSalmonella enterica serovar Typhimurium to various extents, depending on the strain. Theconcentrations of AcpP RFFRFFRFFXB-PMO, AcpPRTRTRFLRRTXB-PMO, AcpPKFFKFFKFFKXB-PMO, and ampicillin that reducedCFU/ml by 50% after 8 h of growth (50% inhibitoryconcentration [IC50 ]) were 3.6, 10.8, 9.5, and 7.5μM, respectively, inE. coli W3110. Sequence-specificeffects of AcpP peptide-PMOs were shown by rescuing growth of amerodiploid strain that expressedacpP with silent mutationsin the region targeted by AcpP peptide-PMO. In Caco-2 cultures infectedwith enteropathogenicE. coli (EPEC), 10 μM AcpPRTRTRFLRRTXB-PMO or AcpPRFFRFFRFFXB-PMO essentially cleared theinfection. The IC50 of either AcpPRTRTRFLRRTXB-PMO or AcpPRFFRFFRFFXB-PMO in EPEC-infected Caco-2 culturewas 3 μM. In summary, RFFRFFRFFXB,RTRTRFLRRTXB, orKFFKFFKFFXB, when covalently bonded to PMO,significantly increased inhibition of expression of targeted genescompared to PMOs without attachedpeptide.