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Structural and Mechanistic Studies on Antimicrobial Lipopeptides Open Access


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Type of item
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University of Alberta
Author or creator
Cochrane, Stephen A.
Supervisor and department
Vederas, John C. (Chemistry)
Examining committee member and department
Mobashery, Shahriar (Chemistry and Biochemistry, University of Notre Dame)
Li, Liang (Chemistry)
Clive, Derrick L. J. (Chemistry)
McMullen, Lynn M. (Agricultural, Food and Nutritional Science)
Department of Chemistry

Date accepted
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Doctor of Philosophy
Degree level
The lipopeptides, tridecaptin A1, tridecaptin B1 and cerexin A1 were isolated from Paenibacillus terrae NRRL B-30644, Paenibacillus polymyxa NRRL B-30507 and Bacillus mycoides ATCC 21929, respectively. A combination of chemical synthesis, peptide derivatization, high performance liquid chromatography and nuclear magnetic resonance was used to elucidate the absolute stereochemistry of these peptides, which were not known prior to this work. Furthermore, new cerexin analogues bearing a succinyl group on the lysine or 4-hydroxylysine residues were isolated from B. mycoides ATCC 21929 and fully characterized. A new synthesis of orthogonally protected Fmoc-L-threo-4-hydroxylysine has been designed, which is shorter than literature procedures, giving comparable yields and easy access to both the threo- and erythro-isomers. Tridecaptin A1 was found to have strong activity against Gram-negative bacteria. Structural studies revealed the lipid tail could be replaced with octanoic acid, yielding Oct-TriA1, which is synthetically more accessible and retains all antimicrobial activity. Oct-TriA1 has strong activity against strains of multidrug resistant Gram-negative bacteria, including Klebsiella pneumoniae and Acinetobacter baumannii. This peptide is stable to proteases and has low cytotoxicity and hemolytic activity. Activity is retained in vivo, increasing the survival rate of mice infected with Klebsiella pneumoniae. An unacylated analogue of tridecaptin A1, H-TriA1, has low antimicrobial, hemolytic and cytotoxic activity, but was found to sensitize Gram-negative bacteria to antibiotics typically used for Gram-positive infections. In particular, the activity of rifampicin and vancomycin are increased 512- and 256-fold against Klebsiella pneumoniae. It was found that covalently linking H-TriA1 to erythromycin led to enhanced activity in vitro, greater than that of the synergistic mixture. In contrast to the tridecaptins, the cerexins were found to have poor antimicrobial activity against most bacteria. An alanine scan was used to identify critical residues in Oct-TriA1 for antimicrobial activity. We found that D-Dab8 is essential for activity and substitution of this residue destroys the ability of the peptide to adopt a stable defined secondary structure in a model membrane environment. We identified the mode of action of the tridecaptins as disruption of the proton-motive force on the inner-membrane of Gram-negative bacteria. This halts the synthesis of adenosine triphosphate, the essential energy source of the cell, and kills Gram-negative bacteria in approximately 30 min. The tridecaptins bind to lipopolysaccharide on the surface of the outer-membrane and use this anchor to traverse the outer-membrane and enter the periplasm. We also identified the selective binding of tridecaptin A1 to the Gram-negative analogue of peptidoglycan precursor lipid II, which contains diaminopimelic acid, rather than lysine found in the Gram-positive version. In vitro assays were used to show that Gram-negative lipid II significantly enhances the ability of the tridecaptins to disrupt a proton gradient compared to Gram-positive lipid II. This explains the selective activity of the tridecaptins against Gram-negative bacteria. Finally, a solution structure of Oct-TriA1 was determined by nuclear magnetic resonance, and Gram-negative lipid II modeled into this structure. This preliminary model shows a key interaction between D-Dab8 on Oct-TriA1 and diaminopimelic acid on lipid II, which is only present in the Gram-negative version.
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. 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.
Citation for previous publication
Cochrane, S.A.; Li, X.; He, S.; Yu, M.; Wu, M.; and Vederas, J.C. Synthesis of tridecaptin-antibiotic conjugates with in vivo activity against gram-negative bacteria. J. Med. Chem. DOI: 10.1021/acs.jmedchem.5b01578.Cochrane, S.A.; Surgenor, R.R.; Khey, K.M.W.; and Vederas, J.C. Total Synthesis and Stereochemical Assignment of the Antimicrobial Lipopeptide Cerexin A1. Org Lett. 17, 5428-5431 (2015).Cochrane, S.A.; Lohans, C.T.; van Belkum, M.J.; Bels, M.A.; and Vederas, J.C. Studies on Tridecaptin B1, a New Tridecaptin Analogue with Activity Against Multidrug Resistant Gram-Negative Bacteria. Org. Biomol. Chem. 13, 6073-6081 (2015).Cochrane, S.A.; and Vederas, J.C. Unacylated Tridecaptin A1 Acts as an Effective Sensitizer of Gram-Negative Bacteria to Other Antibiotics. Int. J. Antimicrobial Agents 44, 493-499 (2014).Cochrane, S.A.; Findlay, B.; Vederas, J.C.; and Ratemi, E.S. Key Residues in Octyl-tridecaptin A1 Analogs Linked to Stable Secondary Structure in the Membrane. ChemBioChem 15, 1295-1299 (2014).Cochrane, S.A.; Lohans, C.T.; Brandelli, J.R.; Mulvey, G.; Armstrong, G.D.; and Vederas, J.C. Synthesis and Structure-Activity Relationship Studies of N-Terminal Analogues of the Antimicrobial Peptide Tridecaptin A1. J. Med. Chem. 57, 1127-1131 (2014).Lohans, C.T.; van Belkum, M.J.; Cochrane, S.A.; Huang, Z.; Sit, C.S.; McMullen, L.M.; and Vederas, J.C. Biochemical, Structural, and Genetic Characterization of Tridecaptin A1, an Antagonist of Campylobacter jejuni. ChemBioChem 15, 243-249 (2014).Cochrane, S.A.; Huang, Z.; and Vederas, J.C. Investigation of the Ring-Closing Metathesis of Peptides in Water. Org. Biomol. Chem. 11, 630-639 (2013).

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