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Characterization of bactenecin : a small antimicrobial cationic peptide

University of British Columbia

Bactenecin is a 12-amino acid-long cyclic peptide from bovine neutrophils. It has the sequence: RLCRIVVIRVCR, and was cyclized by formation of a single disulphide bond. Computer modeling suggested it was amphipathic, with a central hydrophobic ring and positive charges located at both the C- and N-termini. Circular dichroism (CD) spectral studies were consistent with the computer model, in showing that bactenecin is rigid, β-turn structure regardless of its environment. CD spectral studies showed that two linear variants of bactenecin were more flexible, then adopted random structure in phosphate buffer, α-helical structure in trifluoroethanol and β-sheet structure in the presence of liposomes. Bactenecin was shown to have moderate antimicrobial activity against three wildtype Gram-negative bacteria, but was inactive against several Gram-positive bacteria. Disruption of the disulphide bond abolished the antimicrobial activity against the wild type Gram-negative bacteria, but had in improved antimicrobial activity against Staphylococcus epidermis and Entercoccus faecalis. Both the native cyclic and linear bactenecins interacted with the outer and cytoplasmic membranes of Escherichia coli differently. Nature bactenecin bound and permeabilized the outer membrane better than the linear variants, which explains its better activity against the wild-type Gram-negative bacteria. However, bactenecin had poor activity in depolarizing the cytoplasmic membrane. Conversely the linear variants demonstrated poor interaction with the outer membrane, yet were effective in depolarizing the cytoplasmic membrane. These results suggest that cyclization was important for bactenecin to interact with the outer membrane, and furthermore, that cyclic bactenecin could act by a fundamentally different mechanisms from its linear variants. Bactenecin was proposed to have a mechanism of action which involves other cellular targets than the bacterial membrane. Structure:function studies implied that increasing the number of positive charges, by introduction of additional arginine residues at both the N - and C-termini, resulted in improved activity. Linear variants were more active against Gram-positive bacteria. Introduction of the hydrophobic residue, tryptophan, into the loop of cyclic bactenecin improved the antimicrobial activity and dramatically broadened the antimicrobial spectrum. Two interesting candidates as potential therapeutic agents were identified, Bac2A-CN and Bac2R,W. These two peptides had good antimicrobial activity against a wide range of bacteria, including some clinically significant pathogens, and low toxicity.

Thesis/Dissertation

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2009-07-02

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http://hdl.handle.net/2429/9911

Microbiology and Immunology

University of British Columbia

UBCV

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