Antisense phosphorodiamidate morpholino oligomer inhibits viability of Escherichia coli in pure culture and in mouse peritonitis

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Abstract
Objectives: Antisense phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA mimics that specifically inhibit gene expression in pure cultures of Escherichia coli. Previously, an 11 base PMO targeted to an essential gene (acpP) for phospholipid biosynthesis was shown to inhibit growth of a pure culture of E. coli AS19, which has an abnormally permeable outer membrane. The objectives of experiments in this report are to show that the AcpP PMO significantly inhibits growth of strain SM105, which has a normal, intact outer membrane, both in pure culture and in infected mice. Methods: In pure culture, SM105 was grown in rich broth supplemented with 20 μM AcpP PMO, and growth was monitored by optical density and viable cell count. Mice were infected by intraperitoneal injection with a non-lethal inoculum of either E. coli AS19 or SM105. Following infection, mice were treated intraperitoneally with 300 μg of the 11 base antisense PMO targeted to acpP, a scrambled sequence PMO or PBS. Results: Growth of SM105 was slower and viable cells were significantly reduced by up to 61% in pure cultures supplemented with AcpP PMO compared with untreated cultures or cultures supplemented with a scrambled sequence PMO. A single dose of AcpP PMO reduced peritoneal cfu of E. coli AS19 about 39- to 600-fold compared with controls at 2, 7, 13 and 23 h after treatment. The same PMO significantly reduced cfu of E. coli SM105 75% compared with controls at 12 h after treatment. However, there was no difference in cfu at 2, 7 or 24 h. A second dose at 24 h again reduced SM105 cfu about 10-fold by 48 h post-infection. In other experiments with infected mice, multiple doses of AcpP PMO sustained the ∼10-fold reduction in SM105 cfu at 6, 12 and 24 h post-infection. Compared with equivalent (micromolar) doses of ampicillin, AcpP PMO was significantly more effective at all time points. Specificity of PMO inhibition was shown in other experiments by treating infected mice with a PMO targeted to a non-essential reporter gene for luciferase. A luciferase-specific PMO reduced both the amount and activity of luciferase to the same extent, whereas scrambled PMO had no effect. Conclusions: An 11 base antisense PMO targeted to acpP significantly inhibited viability of a strain of E. coli with a normal, intact outer membrane both in pure culture and in infected mice. Inhibition by PMOs was sequence-specific.