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. / Dissertation (MSc)--University of Pretoria, 2016. / Biochemistry / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/49614 |
| Date | January 2016 |
| Creators | Omar Ismail, Naadhira |
| Contributors | Gaspar, A.R.M. (Anabella Regina Marques), Neitz, A.W.H. (Albert Walter Herman), Bester, Megan J. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2016 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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