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.
Highlights
Our group has developed a new process for isolating and identifying novel cationic antimicrobial peptides from small amounts of biological samples
Cationic antimicrobial peptides (CAMPs) are short proteins produced by the innate immune system of virtually all eukaryotic organisms [1]
Hemolysis and MTT assays To measure the hemolytic activity of peptides, 2 % sheep red blood cells were added to various dilutions of peptide reconstituted in phosphate buffered saline (PBS) in a sterile U-bottom 96 well plate. 2 % RBCs with PBS 1X alone served as the negative control, and 2 % RBC in water as the positive control
Summary
Bioinformatics The native, plasma-derived peptide sequences from the de novo sequencing [8] were used to predict the secondary structure and placement within the parent proteins. Other groups have studied synthetic mimetics of apolipoprotein E, which have been found to have anti-infective effects against HIV-1 and HSV-1 [48] and antimicrobial activity against MDR bacteria [14] This characterization of the broad antimicrobial activity of apolipoprotein C1-derived peptides in the present work adds to the understanding of this family of CAMPs. A1P is a fragment of a predicted alpha-1-proteinase. Apo and Apo had comparable activity against strains of P. aeruginosa, A. baumannii, and MRSA (ATCC 33592), but were less active against strains of E. coli tested, with EC50 values ranging from 2–20 μg/ml These peptides had similar membrane perturbation activity as LL-37, with membrane depolarization at low concentrations and pore formation and disruption at very high concentrations. Considering the strength of Apo and Apo on MDR A. baumannii in particular and the low cytotoxicity of these peptides, these are strong candidates for in vivo testing, or to use as scaffolds for stronger synthetic CAMPs
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