Characterization of a Coxiella burnetii SodC and evaluation of the roles of iNOS and NADPH oxidase in controlling C. burnetii infections : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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[College Station, Tex.] :
[Texas A&M University System Health Science Center],
[2004]
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| Subjects: |
| Abstract: | ABSTRACTS: Coxiella burnetii, the etiologic agent of Q fever, is an obligate intracellular bacterium that replicates within the acidified phagolysosome of monocytes/macrophages. However, host control of C. burnetii infections is believed to be mediated primarily by activated monocytes/macrophages. The goal of this study was to characterize the potential role of C. burnetii Cu/Zn SOD (SodC) may play in intracellular survival and examine the contributions of reactive oxygen and nitrogen intermediates to the inhibition of C. burnetii replication. The inability to cultivate this organism on axenic media makes calculation infectious units challenging. Therefore, a rapid and reliable real-time PCR assay was developed to quantify C. burnetii cells from infected tissue culture cells. IFN-γ treatment of infected cell lines and primary macrophages resulted in increased production of nitric oxide (NO) and hyrdrogen peroxide (H₂O₂) and significant inhibition of C. burnetii replication. Inhibition of replication was reversed in the murine cell line J774.16 upon addition of either the inducible nitric oxide synthase (iNOS) inhibitor N^G-monomethly-L-arginine (N^GMMLA) or the H₂O₂ scavenger, catalase. IFN-γ treated primary macrophages from iNOS⁻ʹ⁻ and p47^phox⁻ʹ⁻ mice significantly inhibited replication but were less efficient at controlling infection compared to IFN-γ treated wild-type macrophages. C57BL/6 wild type and knockout (KO) mice lacking iNOS or p47^phox were less effectives at controlling C. burnetii infection compared to wild type mice. These results strongly support a role for both RNI and ROI in the host control of infection. A C. burnetii Cu/Zn SOD (SodC) was cloned, expressed and characterized. Assays for SOD activity demonstrated that the cloned insert codes for a SOD that was active over a wide range of pH and inhibitable with 2 mM KCN, a characteristic of CuZnSODs that distinguishes them from Fe or Mn SODs. Over experssion of the C. burnetii SodC in an E. coli sodC mutant restored resistance to H₂O₂ killing to wild type levels. Expression of sodC by C. burnetii was demonstrated by Western blot analysis. The potential for regulation of this enzyme during oxidative stress and/or by rpoS was also investigated. No differential expression of SodC was detected in response to oxidative stress by Western blot. Additional studies are necessary to examine sodC regulation and determine the potential role of this enzyme in intracellular survival. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Graduate School of Biomedical Sciences of The Texas A&M University System Health Science Center in partial fulfillment for the requirements for the degree of Doctor of Philosophy May 2004." Approved as to style and content by: James E. Samuel, Vernon L. Tesh, Renée M. Tsolis, Jonathan T. Skare, Thomas A. Ficht, John M. Quarles. |
| Physical Description: | ix, 94 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 83-92). |