Inhibition of Pseudomonas aeruginosa by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers

Abstract

Pseudomonas aeruginosa is a highly virulent, multidrug-resistant pathogen that causes significant morbidity and mortality in hospitalized patients and is particularly devastating in patients with cystic fibrosis. Increasing antibiotic resistance coupled with decreasing numbers of antibiotics in the developmental pipeline demands novel antibacterial approaches. Here, we tested peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which inhibit translation of complementary mRNA from specific, essential genes in P. aeruginosa PPMOs targeted to acpP, lpxC, and rpsJ, inhibited P. aeruginosa growth in many clinical strains and activity of PPMOs could be enhanced 2- to 8-fold by the addition of polymyxin B nonapeptide at subinhibitory concentrations. The PPMO targeting acpP was also effective at preventing P. aeruginosa PAO1 biofilm formation and at reducing existing biofilms. Importantly, treatment with various combinations of a PPMO and a traditional antibiotic demonstrated synergistic growth inhibition, the most effective of which was the PPMO targeting rpsJ with tobramycin. Furthermore, treatment of P. aeruginosa PA103-infected mice with PPMOs targeting acpP, lpxC, or rpsJ significantly reduced the bacterial burden in the lungs at 24 h by almost 3 logs. Altogether, this study demonstrates that PPMOs targeting the essential genes acpP, lpxC, or rpsJ in P. aeruginosa are highly effective at inhibiting growth in vitro and in vivo These data suggest that PPMOs alone or in combination with antibiotics represent a novel approach to addressing the problems associated with rapidly increasing antibiotic resistance in P. aeruginosa.

Keywords: PPMO; Pseudomonas aeruginosa; antibiotic resistance; antimicrobial agents; antisense; experimental therapeutics; phosphorodiamidate morpholino oligomer.

FIG 1

Peptide-conjugate (RXR)₄XB was effective at delivering PMO into P. aeruginosa. (a) Heat map comparison of MICs for PPMOs with various peptide-conjugates against P. aeruginosa strains grown in MHII with PMBN at 2 μg/ml. (b) Heat map comparison of MICs for 5′ versus 3′ (RXR)₄XB-conjugated PPMOs against P. aeruginosa strains grown in MHII with PMBN at 2 μg/ml. (c) Heat map comparison of MICs for lead sequence PPMOs against P. aeruginosa strains grown in MHII with PMBN at 2 μg/ml, MOPS, and MHII alone.

FIG 2

PPMO treatment reduces existing P. aeruginosa biofilm. Biofilm was grown for 24 h and then treated at 24, 32, and 40 h with the indicated PPMO in MHII (a) or in MHII with PMBN at 2 μg/ml (b) and analyzed at 48 h by crystal violet. The dashed line represents the lower limit of detection. These data are combined from three independent experiments, the error bars represent standard deviations, and statistics were determined with a one-way analysis of variance (ANOVA) and a Holm-Ŝidák's multiple-comparison test, where P < 0.0001 (**) and P < 0.05 (*) are indicated compared to no PPMO. Spinning disk confocal microscopy images of MBEC pegs are depicted from above and the side incubated as in panel b with 10 μM Scr-0078 (c), 10 μM AcpP-0445 (d), or 2.5 μM AcpP-0445 (e). Green indicates PAO1-GFP, and red indicates biofilm stained with concanavalin A-Alexa Fluor 647.

FIG 3

PPMOs display synergy with traditional antibiotics. Three representative strains of P. aeruginosa (PAO1, M57-15, and W43532) were utilized to examine if PPMOs enhanced activity when combined with current traditional antibiotics. (a) Synergy isobolograms of PPMOs and antibiotics that target similar cellular processes. (b) Fractional inhibitory concentration (FIC) indices of PPMOs and antibiotics that target similar cellular processes. (c) FIC indices of PPMOs and antibiotics that target dissimilar cellular processes. FIC indices were calculated as described in Materials and Methods. An FIC index is considered synergistic when ≤0.5. Data represent the average of three to four independent experiments, and FIC indices were calculated from the best interaction point of these averages. Pip/Tazo, piperacillin-tazobactam; Scr, Scrambled.

FIG 4

PPMOs decreased P. aeruginosa burden in the lungs of infected mice. BALB/c mice were infected intratracheal with P. aeruginosaPA103 and treated with PPMOs or PBS control at 6 h postinfection. (a) The CFU/g of whole lung were analyzed. Statistics were determined with a one-way ANOVA and a Holm-Ŝidák's multiple-comparison test where P < 0.001 (**) compared to PBS and Scr-1073 controls and P < 0.001 (*) compared to PBS control. (b) H&E stains of the left distal lungs of infected mice treated as indicated compared to a naive control. The magnification in the top row is at ×4, with black boxes to indicate the regions pictured at ×40 magnification below. These data are combined from three independent experiments, except for RpsJ-0066, which was one experiment.