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Characterization of a Coxiella burne...

Brennan, Robert Emmet, Jr.

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Characterization of a Coxiella burnetii SodC and evaluation of the roles of iNOS and NADPH oxidase in controlling C. burnetii infections.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Characterization of a Coxiella burnetii SodC and evaluation of the roles of iNOS and NADPH oxidase in controlling C. burnetii infections./
Author:	Brennan, Robert Emmet, Jr.
Description:	94 p.
Notes:	Source: Dissertation Abstracts International, Volume: 65-06, Section: B, page: 2762.
Contained By:	Dissertation Abstracts International65-06B.
Subject:	Biology, Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3137256
ISBN:	049684444X

Characterization of a Coxiella burnetii SodC and evaluation of the roles of iNOS and NADPH oxidase in controlling C. burnetii infections.
Brennan, Robert Emmet, Jr.

Characterization of a Coxiella burnetii SodC and evaluation of the roles of iNOS and NADPH oxidase in controlling C. burnetii infections. - 94 p.

Source: Dissertation Abstracts International, Volume: 65-06, Section: B, page: 2762.

Thesis (Ph.D.)--The Texas A&M University System Health Science Center, 2004.

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 a 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 calculating 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-gamma treatment of infected cell lines and primary macrophages resulted in increased production of nitric oxide (NO) and hydrogen 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 NG-monomethyl-L-arginine (NGMMLA) or the H2O2 scavenger, catalase. IFN-gamma treated primary macrophages from iNOS-/- and p47 phox-/- mice significantly inhibited replication but were less efficient at controlling infection compared to IFN-gamma treated wild-type macrophages. C57BL/6 wild type and knockout (KO) mice lacking iNOS or p47 phox were less effective 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 expression 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.

ISBN: 049684444XSubjects--Topical Terms:

1017734
Biology, Microbiology.

Characterization of a Coxiella burnetii SodC and evaluation of the roles of iNOS and NADPH oxidase in controlling C. burnetii infections.
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100 1 $a Brennan, Robert Emmet, Jr. $3 1939260
245 1 0 $a Characterization of a Coxiella burnetii SodC and evaluation of the roles of iNOS and NADPH oxidase in controlling C. burnetii infections.
300 $a 94 p.
500 $a Source: Dissertation Abstracts International, Volume: 65-06, Section: B, page: 2762.
500 $a Chair: James E. Samuel.
502 $a Thesis (Ph.D.)--The Texas A&M University System Health Science Center, 2004.
520 $a 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 a 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 calculating 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-gamma treatment of infected cell lines and primary macrophages resulted in increased production of nitric oxide (NO) and hydrogen 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 NG-monomethyl-L-arginine (NGMMLA) or the H2O2 scavenger, catalase. IFN-gamma treated primary macrophages from iNOS-/- and p47 phox-/- mice significantly inhibited replication but were less efficient at controlling infection compared to IFN-gamma treated wild-type macrophages. C57BL/6 wild type and knockout (KO) mice lacking iNOS or p47 phox were less effective 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 expression 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.
590 $a School code: 1388.
650 4 $a Biology, Microbiology. $3 1017734
650 4 $a Chemistry, Biochemistry. $3 1017722
650 4 $a Biology, Cell. $3 1017686
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710 2 0 $a The Texas A&M University System Health Science Center. $3 1250793
773 0 $t Dissertation Abstracts International $g 65-06B.
790 1 0 $a Samuel, James E., $e advisor
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791 $a Ph.D.
792 $a 2004
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3137256