Peptides from American alligator plasma are antimicrobial against multi-drug resistant bacterial pathogens including Acinetobacter baumannii

Abstract

BackgroundOur group has developed a new process for isolating and identifying novel cationic antimicrobial peptides from small amounts of biological samples. Previously, we identified several active antimicrobial peptides from 100 μl of plasma from Alligator mississippiensis. These peptides were found to have in vitro antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In this work, we further characterize three of the novel peptides discovered using this process: Apo5, Apo6, and A1P.ResultsWe examined the activity of these peptides against multi-drug resistant strains and clinical isolates of common human pathogens. We investigated their structural characteristics using circular dichroism and tested for membrane disruption and DNA binding. These peptides were found to have strong in vitro activity against multi-drug resistant and clinically isolated strains of S. aureus, Escherichia coli, P. aeruginosa, and Acinetobacter baumannii. Apo5 and Apo6, peptides derived from alligator apolipoprotein C-1, depolarized the bacterial membrane. A1P, a peptide from the serpin proteinase inhibitor, did not permeabilize membranes. Performing circular dichroism analysis, Apo5 and Apo6 were found to be predominantly helical in SDS and TFE buffer, while A1P has significantly different structures in phosphate buffer, SDS, and TFE. None of these peptides were found to be hemolytic to sheep red blood cells or significantly cytotoxic up to 100 μg/ml after 24 h exposure.ConclusionsOverall, we suggest that Apo5 and Apo6 have a different mode of action than A1P, and that all three peptides make promising candidates for the treatment of drug-resistant bacteria, such as A. baumannii.

Intraperitoneal treatment with antimicrobial peptide rescues mice from a pulmonaryFrancisellainfection

“…Apo6 has been shown to have activity against a broad range of pathogens ( Bacillus cereus , Escherichia coli , Pseudomonas aeruginosa , and Staphylococcus aureus , as well as drug-resistant strains of E. coli, P. aeruginosa, S. aureus , and Acinetobacter baumannii ) in low salt buffer (EC 50 ) [16, 19]. Apo6 shares a salt-sensitive phenotype with LL-37 [14, 35] and was found to be inactive in Muller-Hinton broth against these bacteria in MIC assays [16, 19]. We first tested Apo6 and the GATR peptides in MIC assays against F. tularensis LVS.…”

“…Next, we tested the antimicrobial activity of Apo6 and its derivatives against F. tularensis LVS and F. tularensis NIH B38 strain in a low salt buffer, which is an alternate measure of antimicrobial activity reported as EC 50 values, shown in Table 2 [16, 19, 26, 27, 34-36]. We performed these experiments using LL-37 as a positive control, which was found to be highly effective against F. tularensis LVS (EC 50 = 0.209 µg/ml), similar to the EC 50 reported for F. novicida [27].…”

“…Membrane potential was measured using a fluorescent assay utilizing DiSC 3 (5) dye as previously described with some modification [19, 28]. F. tularensis LVS was grown on chocolate II agar (48 h, 37°C, 5% CO 2 ), and the colonies were suspended in 10 mM phosphate buffer to 0.5 McFarland standard.…”

“…Pore formation in F. tularensis LVS cytoplasmic membrane was assessed using ethidium bromide as described previously with some modification [19, 28]. tularensis LVS was grown on chocolate II agar (48 h, 37°C, 5% CO 2 ) and colonies scraped into solution.…”

“…We have discovered a number of antimicrobial peptides with strong antimicrobial activity against a range of bacteria. Previously, we found that peptides representing the C-terminal fragments of an apolipoprotein in A. mississippiensis had antimicrobial activity against Bacillus cereus , Escherichia coli , Pseudomonas aeruginosa , and Staphylococcus aureus , as well as drug-resistant strains of E. coli, P. aeruginosa, S. aureus , and Acinetobacter baumannii [16, 19].…”