Structural Characterization of Bacterial Antimicrobial Peptides

  • Author / Creator
    Lohans, Christopher T
  • Paenibacillus polymyxa NRRL B-30509, Paenibacillus terrae NRRL B-30644 and P. polymyxa NRRL B-30507 were found to produce several bacteriocins and non-ribosomal peptides. All three strains produce tridecaptins, non-ribosomal lipopeptides antimicrobially active against food pathogen Campylobacter jejuni. Two of these strains also produce polymyxins, lipopeptides also active against Gram-negative bacteria. Highly cyclized lantibiotics paenicidins A and B were isolated from P. polymyxa NRRL B-30509 and P. terrae NRRL B-30644, respectively. The lanthionine (Lan) and methyllanthionine (MeLan) post-translational modifications of paenicidin A were characterized by a novel partial desulfurization strategy, revealing the connectivity of three interlocking thioether-containing rings. Biosynthetic gene clusters responsible for the production of the tridecaptins and paenicidins were identified in the genome sequences of these strains. Carnobacterium maltaromaticum C2 produces carnolysin, a novel two-component lantibiotic with homology to enterococcal cytolysin. The post-translational modifications of carnolysins A1′ and A2′ were characterized with NMR spectroscopy and tandem mass spectrometry, revealing the Lan and MeLan bridging patterns. Like cytolysin, carnolysin contains unusual LL-Lan and LL-MeLan stereoisomers in the corresponding positions. However, carnolysin also contains D-alanine and D-aminobutyrate residues not found in cytolysin. The carnolysin biosynthetic gene cluster was identified in the genome of C. maltaromaticum C2. Heterologous expression of carnolysin in Escherichia coli was achieved by expressing the carnolysin precursor peptides with lantibiotic synthetase CrnM and reductase CrnJ. Antimicrobially active products were obtained by proteolytically removing the N-terminal leader sequences of the carnolysins isolated from C. maltaromaticum C2. These digested peptides were active against an array of Gram-positive indicator organisms, while not demonstrating the hemolytic activity associated with cytolysin at the levels tested. Enterocin 7A and 7B are leaderless bacteriocins produced by Enterococcus faecalis 710C. The impact of solvent on the secondary structure of enterocin 7A was examined by circular dichroism spectroscopy, revealing a high degree of α-helicity in fully aqueous conditions. The solution structure of enterocin 7A was solved based on NMR spectroscopic data, demonstrating that this peptide consists primarily of three amphiphilic α-helices burying a hydrophobic core. The structures of enterocins 7A and 7B resembled a region of the circular bacteriocin carnocyclin A, potentially having implications regarding the mode of action of these leaderless bacteriocins. The cysteines involved in the N-terminal disulfide bridge of class IIa bacteriocin leucocin A were replaced with leucine residues to probe the impact of this substitution on structure. While this mutant peptide retained antimicrobial activity, consistent with similar leucocin A mutants. The solution structure of (C9L,C14L)-leucocin A in aqueous trifluoroethanol was studied by NMR spectroscopy. Instead of the N-terminal three-strand β-sheet found in wild-type leucocin A, the N-terminus of the mutant structure is α-helical. However, this may not represent the active conformation of this peptide, and the structure of the N-terminus may be influenced by the choice of solvent.

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  • Degree
    Doctor of Philosophy
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    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
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  • Institution
    University of Alberta
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  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • van der Donk, Wilfred (Chemistry)
    • McMullen, Lynn (Agricultural, Food and Nutritional Sciences)
    • Cairo, Christopher (Chemistry)
    • Harynuk, James (Chemistry)
    • Lowary, Todd (Chemistry)