The proline-rich peptide Bac7(1-35) reduces mortality from Salmonella typhimurium in a mouse model of infection

Abstract

Background: Bac7 is a proline-rich peptide with a potent in vitro antimicrobial activity against Gram-negative bacteria. Here we investigated its activity in biological fluids and in vivo using a mouse model of S. typhimurium infection.

Results: The efficacy of the active 1-35 fragment of Bac7 was assayed in serum and plasma, and its stability in biological fluids analyzed by Western blot and mass spectrometry. The ability of the peptide to protect mice against Salmonella was assayed in a typhoid fever model of infection by determination of survival rates and bacterial load in liver and spleen of infected animals. In addition, the peptide's biodistribution was evaluated by using time-domain optical imaging. Bac7(1-35) retained a substantial in vivo activity showing a very low toxicity. The peptide increased significantly the number of survivors and the mean survival times of treated mice reducing the bacterial load in their organs despite its rapid clearance.

Conclusions: Our results provide a first indication for a potential development of Bac7-based drugs in the treatment of salmonellosis and, eventually, other Gram-negative infections. The in vivo activity for this peptide might be substantially enhanced by decreasing its excretion rate or modifying the treatment schedule.

Figure 1

Antimicrobial activity of Bac7(1-35) in the presence of biological fluids. Kinetics of the bactericidal activity of 10 μM Bac7(1-35) against S. enterica ATCC 14028 in the absence (filled squares) or in the presence of 66% murine serum (filled circles), or 66% murine plasma (filled triangles). Bacterial growth without peptide is indicated by empty symbols. Results represent the mean ± SD of three independent determinations performed in triplicate.

Figure 2

Bac7(1-35) stability in blood fractions. (A) Western blot analysis of Bac7(1-35) incubated for different times at 37°C in 25% murine serum or plasma. Lane 1: 0.5 μg Bac7(1-35); lanes 2-6: Bac 7(1-35) after incubation with murine serum or plasma for respectively 0, 1, 4, 8, 24 hrs; lane 7: serum or plasma alone. (B) MC-LC chromatograms of Bac7(1-35) incubated at 37°C in 25% murine serum or plasma. (C) The percentages of Bac7(1-35) with respect to the t₀ control were calculated following LC-MS analysis (see section Methods for further details) after incubation of the peptide with murine serum (filled squares) or plasma (filled triangles) for different times.

Figure 3

In vivo activity of Bac7(1-35). Survival curves (A) and viable bacterial counts in liver and spleen homogenates (B) of mice infected with S. enterica after treatment via i.p. with Bac7(1-35) are shown. CBA/Ca mice were infected via i.p. with S. enterica ATCC 14028 (10² CFU/mouse) and Bac7(1-35) at 30 mg/kg was immediately injected via i.p. after bacterial challenge (dotted line). Control mice were given 0.2 ml of PBS (continuous line). Mice were monitored for survival over a 60-day period after infection. *p < 0.05 treated vs untreated mice. Three days after bacterial infection, untreated (squares) and peptide-treated (triangles) mice were killed, and liver (full symbols) and spleen (empty symbols) homogenates were prepared as described in section Methods. Results are expressed as number of CFU/g organ; bars represent the mean value for each group.

Figure 4

Biodistribution of Bac7(1-35)-Alexa680 in healthy mice after i.p. injection. (A) The animal was placed in prone position, fluorescence emission in regions of interest encompassing the kidneys were acquired at indicated times post-injection and normalized. (B) The animal was placed in supine position, fluorescence emission in regions of interest encompassing the thorax and abdomen was acquired at indicated times post-injection and normalized. (C) Ex vivo images of organs at 5 hours after i.p. injection. Imaging of the organs was performed immediately after sacrifice: laser power and integration time were optimized while keeping constant scan step to compare fluorescence intensities after normalization. The images are representative of two independent experiments with comparable results.