Functional characterization of a peptide fragment, Os(3-12), derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi

Abstract

The rapid increase of multi-drug resistant bacteria and associated deaths has stimulated research into the development of novel therapeutic options. Antimicrobial peptides (AMPs) display a high therapeutic potential in solving this problem. Research focuses on new ways to enhance the antibacterial activity of AMPs and this includes the amidation of the C-terminus. Once the structure of an AMP is altered it is altered it is necessary to revaluate the properties of this AMP compared to the unaltered peptide. In this study, a peptide fragment Os(3-12), based on a defensin from the tick Ornithodoros savignyi, was amidated at the C-terminus. The effect of C-terminal amidation on the structural, antibacterial, cytotoxic and antioxidant activities of Os(3-12)NH2 was investigated and compared to Os(3-12) as well as the parent peptide Os. Mode of action related to membrane permeabilization was evaluated. The effect of serum and on the antibacterial activity of Os(3-12)NH2 was also determined.

Circular dichroism experiments indicated Os(3-12) and Os(3-12)NH2 to be unstructured in sodium dodecyl sulphate micelles and 50% trifluoroethanol, unlike Os which was predominantly α-helical. Although still less potent than Os, the determined minimum bactericidal concentration (MBC) for each peptide indicated that amidation increases the bactericidal activity of Os(3-12) by 16-fold against Escherichia coli and by 8-fold against both Pseudomonas aeruginosa and Bacillus subtilis. In comparison amidation enhanced the activity of the peptide towards Staphylococus aureus by only 2-fold. The kinetics of bactericidal activity revealed that Os(3-12)NH2 killed E. coli within 10 minutes and B subtilis within 60 minutes. SYTOX green was applied to evaluate the effects of the peptides on the membrane integrity of the bacterial cells. LL-37, a peptide known to disrupt microbial membranes, induced membrane permeabilization of both E. coli and S. aureus membranes. Both Os and Os(3-12)NH2 were found to also cause membrane permeabilization of these bacteria, albeit not to the same extent as LL-37, thus suggesting possible internal targets subsequent to membrane permeabilization. In the presence of 30% human serum and a physiological salt mixture comprising of 145 mM NaCl, 2.5 mM CaCl2 and 1 mM MgCl2 the bactericidal activity of Os(3-12)NH2 was lost. The amidated peptide was found to be non-toxic towards human erythrocytes and Caco-2 cells. Os(3-12)NH2 showed strong antioxidant activity and was found to be 15-fold more active than glutathione (GSH), a known antioxidant.

In conclusion Os(3-12)NH2 has been identified as a multifunctional AMP that is nontoxic to mammalian cells. However the therapeutic potential of Os(3-12)NH2 may be restricted to topical applications due to the peptide’s inactivity under physiological conditions. Although Os(3-12)NH2 causes membrane permeabilization, indications are that there are additional intracellular targets that need to be identified.