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Comparison of structurally related peptides in their antimicrobial and anti-endotoxic activity

University of British Columbia

Sepsis, a serious problem in the developed world, is often associated with Gram negative bacteria. Their pathogenesis is in part related to the release of an outer membrane component, endotoxin. There is great interest in the development of an anti-endotoxin due to the lack of an effective therapy. In this study a series of α-helical cationic peptides, based on hybrids of the moth peptide cecropin and the bee peptide melittin was created using as the parent peptides, MBI-27 and MBI-28. MBI-27 and MBI-28 had already been shown to have antimicrobial and anti-endotoxin activity. From these two peptides along with CP26 and CP29 (more amphipathic in nature), a series of 31 variants with small amino acid changes were designed. The antimicrobial activity of the peptides was studied by MIC determination, killing assays and inner membrane permeabilization studies. The peptides were also tested for their ability to bind LPS and block production of IL-6 and TNF by LPS-stimulated macrophages. Changes made to the variant peptides did not improve upon the antimicrobial activity of MBI-28. Many of the peptides lost Gram-positive activity and some of the peptides lost all antimicrobial activity. The addition of Mg²+ had a partial inhibitory effect on the killing of E. coli and P. aeruginosa by CP26 and MBI-28 as well as inhibiting inner membrane permeabilization of E.coli by CP26. The peptides were found to be non-toxic to the macrophage cells under the conditions tested. The peptides were found to inhibit TNF production measured by the L cell assay (bio-active TNF) to a larger extent than that tested by ELISA, although the trends were generally the same. The peptide effect on IL-6 production was very similar to that seen with the TNF ELISA data. The peptides that were good antimicrobials and had high binding affinity for E.coli LPS also were able to inhibit production of IL-6 and TNF by LPS-stimulated macrophages. Some of the peptides that had no antimicrobial activity and higher LPS binding affinities were not as effective in preventing production of cytokines although this was not always the case. There are a large number of factors involved in what makes a good peptide; length and charge but not hydrophobicity of the peptides were found to play a role. [Certain scientific formulae used in this abstract could not be reproduced.]

Thesis/Dissertation

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2009-05-26

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http://hdl.handle.net/2429/8217

Microbiology and Immunology

University of British Columbia

UBCV

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