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Infection and Immunity, January 2005, p. 155-165, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.155-165.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

NF-{kappa}B Activation during Rickettsia rickettsii Infection of Endothelial Cells Involves the Activation of Catalytic I{kappa}B Kinases IKK{alpha} and IKKß and Phosphorylation-Proteolysis of the Inhibitor Protein I{kappa}B{alpha}

Dawn R. Clifton,1,{dagger} Elena Rydkina,2 Robert S. Freeman,3 and Sanjeev K. Sahni2*

Department of Environmental Medicine,1 Hematology-Oncology Unit, Department of Medicine,2 Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York3

Received 22 June 2004/ Returned for modification 27 July 2004/ Accepted 22 September 2004

Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-{kappa}B (NF-{kappa}B) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-{kappa}B subunits, p65 and p50, and I{kappa}B proteins, I{kappa}B{alpha} and I{kappa}Bß. The transcript and protein levels of p50, p65, and I{kappa} remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of I{kappa}B{alpha} at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of I{kappa}B{alpha} mRNA. The level of I{kappa}B{alpha} mRNA gradually returned toward baseline, whereas that of total I{kappa}B{alpha} protein remained lower than the corresponding controls. The activities of IKK{alpha} and IKKß, the catalytic subunits of I{kappa}B kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-{kappa}B response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKK{alpha} and IKKß, leading to attenuation of nuclear translocation of NF-{kappa}B. Also, increased activity of IKK{alpha} was evident later during the infection, coinciding with the late phase of NF-{kappa}B activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-{kappa}B activation. Since NF-{kappa}B is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.


* Corresponding author. Mailing address: Hemostasis and Thrombosis Program, Hematology-Oncology Unit, Department of Medicine, P.O. Box 610, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Phone: (585) 275-0439. Fax: (585) 473-4314. E-mail: Sanjeev_sahni{at}urmc.rochester.edu.

Editor: J. B. Bliska

{dagger} Present address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.


Infection and Immunity, January 2005, p. 155-165, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.155-165.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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