Periodontal diseases are bacterially induced, inflammatory diseases which are responsible for loss of alveolar bone and connective tissue supporting the teeth which results in loss of teeth. Gram negative anaerobic bacteria are highly associated with these diseases. One of them is Porphyromonas gingivalis belonging to the phylum Bacteroidetes. Infection by P. gingivalis is recurrent after physical removal of the bacteria from the oral cavity and even after antibiotic treatment as development of resistance is not rare. Hence complete understanding the biology of this bacterium is of significance. This gram negative obligate anaerobe, being aerotolerant, manages to survive inside the oral cavity, where oxidative stress is ubiquitous. Genome sequence of P. gingivalis shows the presence of a transcriptional regulator OxyR which is a homologue of OxyR present in E. coli. P. gingivalis OxyR induces the expression of antioxidant defense genes like sod, ahpC-F, dps to protect the bacteria from oxidative stress. Expression of P. gingivalis OxyR regulon is not very well understood. Microarray studies carried out in our lab using P. gingivalis W83 to study gene regulation by OxyR, indicated that several genes in P. gingivalis are co-regulated by iron-and OxyR. Literature also supports that in iron deplete conditions genes involved in oxidative stress are down-regulated. These studies formed the basis of our hypothesis that OxyR might regulate the genes in P. gingivalis in an iron dependent manner. To study the mechanism of regulation by P. gingivalis OxyR and to determine whether OxyR regulation is iron dependent, two approaches were applied - in vitro characterization of binding and in vivo characterization. First step of in vitro characterization was to perform CHIP-chip assay to determine OxyR-binding sites present on the genomic DNA of P. gingivalis. As this assay was performed under completely anaerobic conditions, the target fragments to which OxyR was found to bind during this assay were not same as reported in literature. These and the fragments reported in literature were used for EMSA. EMSAs carried out using crude cell lysates and in vitro OxyR protein preparations showed expected results but the results were not reproducible. In vivo expressed and purified P. gingivalis OxyR never bound to the target fragments used. Preparation of a stable protein preparation and improvement in the parameters of EMSA is very important to further investigate the binding in vitro. The second approach is based on in vivo characterization of binding. This requires tagging the P. gingivalis OxyR at its C-terminus with fluorescent protein to observe its binding to the target DNA sequences. Fluorescently tagged OxyR, is expected to emit fluorescence from a highly localized area to produce sharp fluorescent spots when it is bound to its target sequences. Unbound OxyR is expected to emit a fluorescent signal which is spread over the entire area of the cell. This technique will help to determine the conditions under which OxyR binds to its target DNA sequences. This provides a means to confirm the results obtained from in vitro characterization instead of just extrapolating them.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1412 |
| Date | 06 August 2012 |
| Creators | Paranjape, Anuya R. |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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