UBC Theses and Dissertations
The bacterial production of antimicrobial, cationic peptides and their effects on the outer membranes of Gram-negative bacteria Piers, Kevin Lee
Natural polycationic antibiotic peptides have been found in many different species of animals and insects and shown to have broad antimicrobial activity. To permit further studies on these peptides, bacterial expression systems were developed. Attempts to express these peptides with an N-terminal signal sequence were unsuccessful due to the lability of the basic peptides. Therefore, different fusion protein systems were tested, including fusions to glutathione-S-transferase (GST) and Staphylococcus aureus protein A. For GST, fusions to the defensin, human neutrophil peptide 1 (HNP- 1), or a synthetic cecropin/melittin hybrid (CEME) were generally unstable if found in the soluble fraction of lysed cells, but were stable if found as insoluble inclusion bodies. In the course of these studies, we developed a novel method of purilring inclusion bodies, using the detergent octyl-polyoxyethylene, as well as establishing methods for preventing fusion protein proteolytic breakdown. Cationic peptides could be successfully released from the GST carrier protein with high efficiency by chemical means (cyanogen bromide digestion) and with low efficiency by enzymatic cleavage (using factor Xa). Fusions of protein A to cationic peptides were expressed in the culture supernatant of S. aureus clones and after affinity purification, CNBr digestion and column chromatography, pure cationic peptide was obtained. CEME produced by this procedure had the same amino acid content, amino acid sequence, gel electrophoretic mobility and antibacterial activity as CEME produced by protein chemical procedures. Three catiorilc peptides, CEME, CEMA and melittin, were all found to have a broad range of antibacterial activity at concentrations that were comparable to conventional antibiotics. All three were found to permeabiize the outer membrane of Pseudomorias aeruginosa and Enterobacter cloacae to lysozynie and the hydrophobic probe l-N-phenylnaphthylamine. CEMA permeabiized membranes at concentrations 2- to 5-fold lower than CEME and 20-fold lower than melittin. In some cases, it disrupted membranes better than polymyxin B, a known potent permeabilizer. CEMA also had the highest binding affinity for purified P. aeruginosa LPS and whole cells, although CEME and melittin also bound strongly. These data are discussed with special reference to the mechanism by which these peptides cross the outer membrane of Gram-negative bacteria. It is proposed that they utilize the self-promoted uptake pathway which has been suggested previously for other cationic antibiotics. As well, the potential for the use of cationic peptides as therapeutic antibiotics is discussed.
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