The rifamycins are a class of antibiotics which were once used almost exclusively to treat tuberculosis, but are currently receiving renewed interest. Resistance to rifamycins is most commonly attributed to mutations in the drug target, RNA polymerase. Yet environmental isolates are also able to enzymatically inactivate rifamycins in a number of ways. Recently, rifamycin resistance determinants from the environment were found to be closely associated with a so called rifamycin associated element (RAE). The region containing the RAE from an environmental strain was shown to induce gene expression in the presence of rifamycins, hinting at an inducible system for rifamycin resistance. In this work, we examine the RAE from a model organism for Streptomyces genetics, Streptomyces venezuelae. We confirm that the promoter region containing the RAE upstream of a rifamycin monooxygenase rox is inducible by rifamycins. The strains of S. venezuelae generated in this work can be used in future genetic studies on the RAE.
As well, the rifamycin monooxygenase Rox was purified for the first time and characterized biochemically. The structure of Rox was obtained with and without the substrate rifampin. Steady state kinetics for the enzyme were determined with a number of substrates, and its ability to confer resistance to rifamycins was examined. Monooxygenated rifamycin SV compound was purified and structurally characterized by NMR analysis. We proposed an aromatic hydroxylase type mechanism for Rox, in which the enzyme hydroxylates the aromatic core of the rifamycin scaffold and causes a non-enzymatic C-N bond cleavage of the macrolactam ring. This is a new mechanism of rifamycin resistance, and sheds some light on the decomposition of rifamycins mediated by monooxygenation, which is still poorly understood. / Thesis / Master of Science (MSc) / Antibiotic resistance represents a major threat to global health. Infections that were once readily treatable are no longer so due to the rise in multidrug resistant bacteria. As our arsenal of effective antibiotics is depleted, new drugs are being discovered less and less frequently. This has caused the scientific community to get creative in coming up with treatments: trying combinations of antibiotics, using antibiotics which were once considered too toxic, and repurposing antibiotics for different bacteria.
Rifamycins are a class of antibiotics most commonly used in the treatment of tuberculosis. However, they are becoming more widely used as a result of antibiotic resistance. There are a number of different ways bacteria can become resistant to the harmful effects of rifamycins: by modifying the target so the drug can no longer bind to it, actively pumping the drug out of the cell, or by changing the drug in some way so it is no longer effective. Bacteria in the environment use antibiotics as a form of chemical warfare to gain an advantage over their neighbours; therefore, they have had millions of years to evolve very effective methods of antibiotic resistance. By surveying what kinds of antibiotic resistance are in the environment, we can predict what we might see one day in a medical setting.
In this thesis, I have studied a protein that bacteria make to inactivate rifamycins. The rifamycin monooxygenase Rox adds an oxygen to the rifamycin scaffold; this causes spontaneous cleavage of the rifamycin backbone and changes the conformation of the drug so it can no longer bind to its target. I have also investigated the regulation of this and other genes in the bacterial strain Streptomyces venezuelae. By understanding how this process works, we can potentially design inhibitors to stop this from happening, should this method of resistance ever become clinically prevalent.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/20423 |
| Date | 11 1900 |
| Creators | Kelso, Jayne |
| Contributors | Wright, Gerard D, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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