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

Abstract

Background: Our 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.

Results: We 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.

Conclusions: Overall, 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.

Keywords: Acinetobacter baumannii; Alligator mississippiensis; Antimicrobial peptides; Escherichia coli; Multi-drug resistant bacteria; Staphylococcus aureus.

Fig. 1

Primary and tertiary structure of parental proteins and peptide placement within. a Amino acid sequence of A. mississippiensis apolipoprotein C-1 and the Apo5 and Apo6 fragments. b Amino acid sequence of A. mississippiensis alpha-1-proteinase 2-like protein and the A1P fragment. c Ribbon model of apolipoprotein C-1 showing cleavages points of Apo5 (long dashes) and Apo6 (short dashes). d Ribbon model of alpha-1-proteinase showing cleavage point of A1P

Fig. 2

CD spectra of peptides to determine secondary structure. a Apo5, b Apo6, c A1P. All spectra were taken with peptide concentrations of 100 μg/ml in a 1 mm pathway cuvette. Spectra were read in 10 mM phosphate buffer (long dashes), 50 % TFE in 10 mM phosphate buffer (dash dot dash), and 60 mM SDS in 10 mM phosphate buffer (solid line)

Fig. 3

Secondary structure prediction by I-TASSER [24], visualized by Chimera [25]. a Apo5, b Apo6, c A1P

Fig. 4

Helical wheels of a Apo5 and b Apo6, modified from Heliquest

Fig. 5

Pore-forming activity by Apo5, Apo6, and A1P. An increase in fluorescence demonstrates greater binding of DNA by ethidium bromide, which indicates the formation of pores in the bacterial membrane. a. Kinetics of permeabilization and binding of ethidium bromide to DNA after treatment with Apo5 (dashes), Apo6 (solid line), A1P (dots), and LL-37 (dash dot) b. Permeabilization of membrane after 20 min

Fig. 6

Membrane depolarization activity by Apo5, Apo6, and A1P. Depolarization determined using DiSC₃(5) for each peptide at 50 μg/ml (■), 5 μg/ml (), 0.5 μg/ml (), as well as no treatment ()

Fig. 7

DNA binding by a Apo5, b Apo6, c A1P, and d LL-37 as measured using a gel shift assay. Ratio of DNA:CAMP is shown

Fig. 8

Peptide Cytotoxicity a. Hemolysis of sheep red blood cells was measured using a photometric assay after exposure to 300 μg/ml for 1 h and b. Inhibition of Proliferation of A549 human lung epithelial cells measured using the MTT assay after 24 h exposure. Apo5 (■), Apo6 (), A1P (), LL-37 ()