Cecropin-like antimicrobial peptide protects mice from lethal E.coli infection

Abstract

Resistance of pathogenic bacteria to standard antibiotics is an issue of great concern, and new treatments for bacterial infections are needed. Antimicrobial peptides (AMPs) are small, cationic, and amphipathic molecules expressed by metazoans that kill pathogens. They are a key part of the innate immune system in both vertebrates and invertebrates. Due to their low toxicity and broad antimicrobial activities, there has been increasing attention to their therapeutic usage. Our previous research demonstrated that four peptides-DAN1, DAN2, HOLO1 and LOUDEF1-derived from recently sequenced arthropod genomes exhibited potent antimicrobial effects in-vitro. In this study, we show that DAN2 protected 100% of mice when it was administered at a concentration of 20 mg/kg thirty minutes after the inoculation of a lethal dose of E. coli intraperitoneally. Lower concentrations of DAN2-10mg/kg and 5mg/kg protected more than 2/3s of the mice. All three dose levels reduced bacterial loads in blood and peritoneal fluid by 10-fold or more when counted six hours after bacterial challenge. We determined that DAN2 acts by compromising the integrity of the E. coli membrane. This study supports the potential of DAN2 peptide as a therapeutic agent for treating antibiotic resistant Gram-negative bacterial infections.

Fig 1. The infected mice exhibited dose-dependent response to survival.

All the mice of four groups were injected intraperitoneally (i. p.) with a lethal dose, 2.2 × 10⁷ CFU of E. coli and treated with different concentration of DAN2 after 30 minutes of bacterial challenge. The control group only received bacterial suspension and PBS. All the treated mice were monitored for five days. There were 6 mice/group.

Fig 2. DAN2 treated mice have reduced bacterial loads both in peritoneal fluid and blood.

Four mice were infected and treated with 0 mg/kg, 20 mg/kg, 10 mg/kg and 5 mg/kg of DAN2 individually and bacterial count was determined after 6 hours of infections. The error bars represent the standard deviation of the mean. The number of asterisks was used to denote the extent of statistical significance amongst groups (* denotes p < 0.05, ** denotes p < 0.005, *** denotes p < 0.0005).

Fig 3. DAN2 disrupts the integrity of E.coli cell membrane.

The DNA binding dye propidium iodide (PI) was used to evaluate cell membrane permeability of E. coli ATCC 25922 via flow cytometry. 2.0 × 10⁶ CFU/ml was incubated with varying concentrations of peptide for an hour and PI added subsequently. Flow cytometry was performed using a FACScan instrument. (A) Bacteria; (B) Heat treated (positive control); (C) DAN2 (48 μg/ml); (D) DAN2 (36 μg/ml); (E) DAN2 (24 μg/ml). Bacterial cells treated with either peptide or heat shocked have increased cellular fluorescence intensity of PI.