<p> Glycopeptide Antibiotics (GPAs) such as vancomycin are often used clinically as antibiotics of last resort against infections due to Gram-positive bacteria that are resistant to more commonly used antibiotics such as methicillin. The clinical emergence of vancomycin resistant enterococci (VRE) and vancomycin resistantS. aureus (VRSA) necessitates methods to evade this resistance. </p> <p> GP As consist ofa heptapeptide backbone that is cross-linked to create a pocket that binds the D-Alanyl-D-Alanine terminus of peptidoglycan intermediates, inhibiting strengthening ofthe cell wall and resulting in susceptibility to osmotic stress. Resistance to GPAs occurs when D-Ala-D-Lactate replaces D-Ala-D-Ala and the GPA pocket can no longer bind effectively. In order to create novel binding pockets, we must understand the specificity ofthe P450 monooxygenase enzymes that have been shown to catalyze the cross-links. The 4 P450-encoding genes ofthe GPA A47934 biosynthetic cluster of Streptomyces toyocaensis as well as genes encoding electron transport proteins necessary for P450 function from Streptomyces coelicolor were cloned in Escherichia coli for heterologous expression and characterization. One P450, StaJ was purified and shown to bind CO as expected using spectrophotometric tests. </p> <p> The genes responsible for GP A resistance are regulated by a two component regulatory system consisting ofa sensor kinase (VanS) and a response regulator (V an.R). In order to probe the events leading to VanS autophosphorylation and ultimately resistance activation we utilize a series of GP A derivatives harbouring the photo labile group benzophenone as well as the fluorescent and affinity moieties BODIPY and biotin. Benzophenone permits light controlled covalent binding of the GP A to proteins that bind them while BODIPY allows fluorescence detection and biotin allows enrichment and detection by Western analysis. We report that this system was insufficient to clearly identify vancomycin binding proteins due to background signals despite multiple rounds of troubleshooting. It must be our conclusion that under the conditions tested, there are no proteins that bind the GP A derivative used in this study. </p> / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21559 |
| Date | 04 1900 |
| Creators | Back, Jason |
| Contributors | Wright, G. D., Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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