Abstract
Reported here is the isolation and characterization of two antibacterial peptides synthesized in an ant Myrmecia gulosa in response to bacterial challenge. The peptides were purified by reversed-phase high performance liquid chromatography and characterized by peptide sequencing and mass spectrometry. Both peptides were formed from 16 amino acids, were rich in proline ( approximately 30%), and had N-acetylgalactosamine O-linked to a conserved threonine. The activity of a synthetic non-glycosylated isoform was markedly reduced demonstrating that glycosylation was necessary for maximum activity. The peptides were active only against growing Escherichia coli. They were inactive against stationary cells, Gram-positive bacteria, the yeast Candida albicans, two species of mammalian cells, and bovine pestivirus.
Highlights
Based on primary and secondary structure, four main classes of cationic peptide antibiotics are derived from metazoa [1, 2] as follows: (i) linear, mostly helical, peptides without cysteine; this group includes the cecropins from insects and pigs, the magainins from the frog Xenopus laevis, and pardaxin from Moses sole fish [1, 3]. (ii) Peptides with an antiparallel -sheet structure stabilized by two or three intramolecular disulfide bonds
The bond typically joins together the two ends of a secondary loop structure; bactenecin isolated from bovine neutrophils, for example, has a loop formed of 9 residues with 1 and 2 residues on each end [6]. (iv) Linear peptides containing high proportions of 1 or more amino acids comprise the fourth class
Because they are the first inducible peptide antibiotics recovered from Formicidae, the name formaecins is proposed for these peptides
Summary
Immunization—Adult workers of M. gulosa were collected from five field colonies in New South Wales. Antimicrobial Assays—The liquid inhibition growth assay used to detect antimicrobial activity was as follows: mid-exponential phase bacteria were suspended in 1/10 strength nutrient broth in 25 mM sodium phosphate buffer (pH 7) containing 15 mM NaCl at a concentration of approximately 200 cfus/100 l. Mid-exponential phase C. albicans were suspended in 1/20 strength Sabouraud broth in the same buffer at a concentration of approximately 40 cfus/100 l. The inhibitory kinetics of F1 were assayed as follows: 100-l aliquots of mid-exponential phase E. coli NCTC 8196 (ϳ400 cfus) suspended in 50 mM sodium phosphate buffer (pH 7) were pipetted into nine microcentrifuge tubes; six of the tubes contained 1.5 g of F1. Aliquots (100 l) of exponential phase E. coli NCTC 8196 (1.75 ϫ 106 cfus) in 1⁄2 strength nutrient broth were combined with peptide and incubated at 37 °C with agitation. Cells were stained by immunoperoxidase using pestivirus-specific monoclonal antibodies [27] and horseradish peroxidase-conjugated goat anti-mouse IgG and visualized with 3-amino-9ethyl carbazole chromogen
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