Study of superoxide dismutase in Porphyromonas gingivalis 381 Numerous studies have shown the importance of superoxide dismutase (SOD) in cells for protection against oxidative stress. One microorganism, Porphvromonas gingivalis (P. gingivalis, an obligate anaerobe possesses considerable aerotolerance and it has been suggested that SOD may play a key role in this activity. One recent study demonstrated that insertional inactivation of the sod gene in P. gingivalis ATCC 3327, resulted in cell lysis and a rapid loss of viability. Recently, similar methods in our laboratory using P. gingivalis 381 yielded mutants that appear to have a diminished, but still detectable, SOD activity. Further these partially active mutants did not appear to lyse upon exposure to air. Hence, we are currently attempting to construct a mutant displaying no detectable SOD activity. This will allow for thorough examination an characterization of SOD activities and properties. For instance, in P. gingivalis and other organisms, this enzyme has been suggested to confer tolerance against phagocytic killing by neutrophils. These finding suggest that SOD is a potential virulence factor that functions to dismutate destructive oxygen radicals release by the host cell. By comparing SOD- mutants to wild type 381 in a neutrophil killing assay and an animal model, we plan to demonstrate that SOD indeed may be a virulence factor in P. gingivalis. Although SOD activity in P. gingivalis has been shown to be upregulated in response to environmental factors such a increased temperature and aerobic culture conditions, little else is known about the control of sod gene transcription and expression. Therefore, we will study the effects of numerous environmental conditions sod gene control. To facilitate these studies, the construction of a sod::lacZ fusion mutant will be useful confirmation of this mutant is pending. In addition, we plan to study transcriptional regulatory elements of the sod gene. For instance, sequence data suggests a potential Fur-box (iron uptake regulated) element in the promoter region of the P. gingivalis sod gene. In other organisms, this element has been shown to regulate transcription of various genes, including those encoding superoxide dismutases. Using the FURTA (Fur titration assay), preliminary results suggest that P. gingivalis sod transcription may not be regulated by a Fur-box element. Further examination of this putative Fur-box as well as other potential regulatory elements critical to transcriptional control of the sod gene is planned. By describing the potendial role of SOD in contributing to the virulence of P. gingivlis, we hope to further elucidate the complexities involved in this organism's ability to infect and colonize the host.
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