Peptide Nucleic Acid Antisense Oligomer as a Therapeutic Strategy against Bacterial Infection: Proof of Principle Using Mouse Intraperitoneal Infection

Bacterial strains and growth conditions. E. coli strain SM105 [thr-1 araC14 tsx-78 Δ(galK-attλ)99 hisG4 rfbD1 rpsL136 sylA5 mtl-1 thi-1] and its isogenic derivative SM101 (9) with a defective outer membrane were purchased from the E. coli Genetic Stock Center (New Haven, CT). SM101 is deficient in the UDP-N-acetylglucosamine acyltransferase, and its lipid A content is only one-third of that of wild-type SM105. The low lipid A content causes the outer membrane of SM101 to be permeable to high-molecular-weight substances like PNA. SM101 and SM105 cells were grown at 30°C in Luria-Bertani (LB; Becton Dickinson and Company, Sparks, MD) medium containing 50 μg/ml streptomycin. E. coli strain K-12 was obtained from the American Type Culture Collection (ATCC 29425) and grown at 37°C in nutrient broth.

Chemical synthesis of PNA and peptide-PNA conjugate.PNA and peptide-PNA conjugate were synthesized by Bio-Synthesis (Dallas, TX). Briefly, PNA with the sequence CTCATACTCT and peptide-PNA conjugate with the sequence KFFKFFKFFK-CTCATACTCT were synthesized manually on a solid phase by following the procedures described by Good et al. (13) using PNA monomers from Applied Biosystems (Foster City, CA) and amino acid residues from Novabiochem (San Diego, CA). The crude PNA or conjugate was then purified using high-performance liquid chromatography and identified and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Inhibition of bacterial growth in vitro. E. coli SM101, SM105, or K-12 isolates were grown as described above (9, 22). Cell cultures were diluted to ∼10⁵ CFU/ml, and PNA or the peptide-PNA conjugate was added to a final concentration of 0, 4, 40, or 400 μM as described by Good and Nielsen (12). After incubation with shaking at 37°C for various times, aliquots of cell cultures were collected for cell growth assay by measuring viable cell counts. Viable cell counting was done by diluting the cultures and plating them in triplicate on LB plates. The plates were then incubated overnight at 37°C, and the colonies were enumerated by visual inspection.

Mouse intraperitoneal model of E. coli infection.Mice were infected intra-abdominally to induce peritonitis according to the established protocols (20, 29, 30) under University of Texas—Houston Health Science Center animal welfare guidelines (HSC-AWC-02-059). BALB/c mice were used for infection experiments due to genetic control of the endotoxic response linked to lipopolysaccharide and CD14 (33, 34). For each infection experiment, 6- or 8-week-old female BALB/c mice in groups of six to eight were used. Mice were housed four to a cage and kept in a laminar-flow cabinet under specific-pathogen-free conditions for 2 weeks before use and monitored throughout experimentation under Institutional Animal Care and Use Committee-approved guidelines. Bacterial cells of log-phase cultures (optical density at 600 nm = ∼1.0) were collected by centrifugation and then resuspended in sterile phosphate-buffered saline (PBS) at 4°C. This log-phase preparation of bacteria was serially diluted in PBS, and a 70% lethal dose (LD₇₀) of strain SM101 (approximately 1.2 × 10⁹ CFU) or an LD₉₀ of strain K-12 (approximately 8 × 10⁸ CFU) was injected intraperitoneally (i.p.) into mice in 500-μl aliquots. Infected mice were observed for up to 9 days. Blood samples (up to 300 μl) were collected at 24 h after bacterial challenge by puncturing the orbital plexus with a heparinized capillary tube. The collected blood samples were used for bacterial titer analysis and proinflammatory cytokine (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], IL-6, and IL-12) determination as described below.

PNA or peptide-PNA conjugate treatment.The therapeutic efficacy of PNA or peptide-PNA conjugate was evaluated using the mouse model above. Groups of mice were challenged by i.p. injection of E. coli SM101 or K-12. SM101-infected mice were treated with a single injection of PNA administered i.p. 30 min before the bacterial challenge. K-12-infected mice were treated with a single injection of peptide-PNA conjugate administered intravenously 30 min after the bacterial challenge. The control groups included mice treated with PBS alone.

Bacterial titer analysis.Blood samples collected at 24 h postinfection were serially diluted, plated onto LB agar plates, and incubated overnight at 37°C. The colonies on the plates were enumerated by visual inspection.

ELISA evaluation for proinflammatory cytokines.Blood was collected using heparinized tubes at 24 h postinfection. Cells were removed by centrifugation, and the remaining serum was assessed for production of proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-12. Evaluation was accomplished using commercially available enzyme-linked immunosorbent assay (ELISA) DuoSet kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Briefly, Costar 96-well vinyl assay plates were coated with capture antibody overnight (e.g., TNF-α at 0.5 μg/ml, IL-6 at 1 μg/ml). Plates were washed three times with wash buffer (0.05% Tween 20 in PBS). Blocking buffer (1% bovine serum albumin, 5% sucrose, 0.05% NaN₃ in PBS) was added for a 3-h incubation. After three washings, 10 μl serum (final dilution of 1:20 in PBS) was added for a 2-h incubation. Biotin-conjugated secondary antibodies were added, incubated for 2 h, washed, and then developed using streptavidin-horseradish peroxidase (Sigma, St. Louis, MO) and TMB Microwell peroxidase substrate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Absorbance was read at 570 nm and 450 nm on an ELISA plate reader (Molecular Devices, Sunnyvale, CA). Means of duplicate or triplicate wells were calculated based on a standard curve constructed for each assay, using recombinant murine cytokines (R&D Systems). The limit of sensitivity for these assays is 5 pg/ml.

Inhibition of SM101 growth in vitro by acpP-targeting PNA.Inhibition of E. coli SM101 growth was evaluated by examining the effects of various PNA concentrations. As shown in Fig. 1, compared with the cell culture without PNA, the viable cells in cultures with PNA were significantly reduced. The inhibition of bacterial growth was both time and concentration dependent. However, this inhibition was not observed in wild-type E. coli SM105 cells (data not shown).

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FIG. 1.

Inhibition of E. coli mutant SM101 growth in vitro by acpP-targeting PNA. PNA was added to cell cultures containing 5 × 10⁵ CFU/ml SM101 to a final concentration of 4, 40, or 400 μM as indicated on the graph, with addition of an equal volume of water as a control (0 μM). Cultures were incubated as described in Materials and Methods. Aliquots of each culture were collected at 2 and 4 h, diluted, and plated for viable cell determination. Error bars indicate standard deviations of the results from three experiments (*, P < 0.01 relative to 2 h control; **, P < 0.001 relative to 4-h control [as determined by Student's t test]).

Rescue of SM101-infected mice by acpP-targeting PNA.SM101 (LD₇₀) was i.p. introduced into mice, and animals were observed for 1 week. All mice showed evidence of infection soon after bacterial administration (6 h), with characteristics such as lethargy, warmth to the touch, and scruffiness. Of the control (untreated) mice, 62.5% succumbed to infection within 48 h after receipt of SM101 (Fig. 2). Untreated mice (four of six) demonstrated high levels of bacteria in serum at 24 h postinfection, while treatment of mice with 5 nmol or 50 nmol PNA led to complete absence of bacteria at that time (Table 1). Of significance, all of the six mice treated with PNA at either dose level survived (Fig. 2).

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FIG. 2.

Survival of SM101-infected mice due to PNA treatment. Six-week-old BALB/c were infected and treated as described in the text. The infected mice (LD₇₀) were monitored every 4 to 6 h for survival through 7 days. Six to eight mice were infected per group, and the experiments were repeated with nearly identical results.

As shown in Table 2, untreated mice exhibited marked increases in IL-6 and IL-12 and minor elevations in TNF-α and IL-1β. Treatment with PNA at a 5-nmol level led to significant reductions (P < 0.05) in serum IL-6 and IL-12. At a dose of 50 nmol, significant and substantial reductions were seen in TNF-α, IL-6, and IL-12.

Inhibition of K-12 growth in vitro by peptide-PNA conjugate.The cationic peptide with the sequence KFFKFFKFFK was previously shown to be effective in carrying PNA (as peptide-PNA conjugate) across the membrane barrier (13). The acpP-targeting peptide-PNA conjugate was produced to examine antibacterial efficacy against wild-type E. coli (strain K-12). As shown in Fig. 3, compared with the cell cultures in the absence of the conjugate, the viable cells in culture were significantly reduced in the presence of the conjugate; no viable K-12 cell was detected in the cultures with 40 μM or 400 μM conjugate. This inhibition was not observed in cell cultures with addition of peptide alone; after incubation for 4 h, 2.1 × 10⁷ CFU/ml and 1.4 × 10⁷ CFU/ml K-12 cells were detected in the cultures with 40 μM and 400 μM peptide, respectively (data not shown in Fig. 3).

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FIG. 3.

Inhibition of E. coli wild-type K-12 growth in vitro by acpP-targeting peptide-PNA conjugate. Peptide-PNA conjugate was added to cell cultures containing 5 × 10⁵ CFU/ml E. coli strain K-12 to a final concentration of 0, 4, 40, or 400 μM as indicated on the graph. Cell cultures were grown, and viable cells were determined as described in the legend to Fig. 1. Error bars indicate standard deviations of the results from two experiments (*, P < 0.001 relative to 2-h control; **, P < 0.005 relative to 4-h control [as determined by Student's t test]).

Rescue of K-12-infected mice by the peptide-PNA conjugate.As shown in Fig. 4, untreated mice (90%) succumbed to infection within 48 h. Mice treated with the low PNA conjugate dose (100 nmol) succumbed to infection in a manner similar to controls. However, all mice treated with 500 nmol peptide-PNA conjugate exhibited a survival benefit. Treatment with peptide-PNA conjugate also significantly reduced the bacterial load; serum collected at 24 h postinfection showed significantly reduced levels of bacteria for the groups treated with 100 or 500 nmol (P < 0.05) (Table 3).

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FIG. 4.

Survival of K-12-infected mice due to treatment with acpP-targeting peptide-PNA conjugate. Six-week-old BALB/c mice were i.p. infected with 8 × 10⁸ CFU E. coli strain K-12 in 500 μl PBS (LD₉₀). Infected mice were treated with the peptide-PNA conjugate (100 or 500 nmol) 30 min postinfection; comparisons are made to untreated mice. Survival and general health of these animals was monitored every 6 to 12 h through 9 days.

Although the level of bacteremia correlated well with survival, there was only a modest reduction in the concentrations of the proinflammatory cytokines; TNF-α, IL-1β, and IL-12 were all marginally reduced, but not significantly (Table 4).