title: Cathelicidin antimicrobial peptide from Alligator mississippiensis has antibacterial activity against multi-drug resistant Acinetobacter baumanii and Klebsiella pneumoniae.
abstract: Alligator mississippiensis (American alligator), a member of order Crocodilia, lives in bacteria-laden environments but is not often known to succumb to bacterial infections. Their serum has been shown to have antibacterial activity beyond that of human serum, and it is believed that this activity is partially due to cationic antimicrobial peptides (CAMPs). CAMPs are produced by many organisms as part of the innate immune system. CAMPs are attractive possible therapies against multi-drug resistant bacteria, such as those found in biofilm-infected war wounds, because they seldom cause genetic resistance in bacteria and are effective against antibiotic resistant bacteria. In this work, we identified, synthesized, and characterized a cathelicidin and two shorter fragments from the American alligator. We discovered the cathelicidin using Basic Local Alignment Search Tool (BLAST) alignment and by comparing A. mississippiensis expressed sequence tags (ESTs) with propeptide cathelicidins of other reptiles. We analyzed the structure using bioinformatics tools and circular dichroism and predicted that the full-length cathelicidin peptide has a mixed structure, with an N-terminal α-helix and a center Pro hinge. In minimal inhibitory concentration (MIC) assays, it was determined that the cathelicidin and the two shorter fragments have strong activity against multiple Gram-negative bacteria, including clinical isolates of multi-drug resistant (MDR) Acinetobacter baumannii and carbapenem-resistant Klebsiella pneumoniae. Using the ethidium bromide uptake assay, it was found that these peptides permeabilize the bacterial membrane and are less sensitive to salt inhibition than many other known CAMPs. The alligator cathelicidin peptides were not hemolytic against sheep red blood cells at 300 μg/ml and were not significantly cytotoxic against A549 human lung epithelial cells after 24 h exposure in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. These alligator cathelicidin peptides have activity similar to other CAMPs from reptiles such as NA-CATH. It is possible that the alligator cathelicidins play an important role in the innate immune response of A. mississippiensis, similar to LL-37 in humans. In addition, due to their activities against MDR bacteria and lack of cytotoxicity, the AM-CATH peptides could be an attractive platform for further development as a potential therapeutic.
authors.affiliation: School of Systems Biology, George Mason University, Manassas, VA, USA.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Barksdale
authors.affiliation: College of Science, George Mason University, Manassas, VA, USA.
authors.forename: Evelyn J
authors.initials: EJ
authors.lastname: Hrifko
authors.affiliation: School of Systems Biology, George Mason University, Manassas, VA, USA; College of Science, George Mason University, Manassas, VA, USA; National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA, USA. Electronic address: mvanhoek@gmu.edu.
authors.forename: Monique L
authors.initials: ML
authors.lastname: van Hoek
mesh_terms: D000151:Acinetobacter Infections / Q000150:complications / Q000276:immunology*; D040981:Acinetobacter baumannii / Q000276:immunology*; D000489:Alligators and Crocodiles / Q000276:immunology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000276:immunology*; D023181:Antimicrobial Cationic Peptides / Q000276:immunology*; D060829:Arthropod Proteins / Q000276:immunology*; D018441:Biofilms / Q000254:growth & development*; D002470:Cell Survival; D019295:Computational Biology; D004352:Drug Resistance, Microbial; D053878:Hemolytic Agents / Q000378:metabolism*; D007113:Immunity, Innate; D007710:Klebsiella Infections / Q000150:complications / Q000276:immunology*; D007711:Klebsiella pneumoniae / Q000276:immunology*; D010802:Phylogeny; D016415:Sequence Alignment; D054804:Cathelicidins
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D060829:Arthropod Proteins; D053878:Hemolytic Agents; C034705:antilipopolysaccharide factor (Limulus); D054804:Cathelicidins