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The Journal of Immunology, 2002, 169: 7009-7018.
Copyright © 2002 by The American Association of Immunologists

Repression of rac2 mRNA Expression by Anaplasma phagocytophila Is Essential to the Inhibition of Superoxide Production and Bacterial Proliferation1

Jason A. Carlyon*, Wai-Tsing Chan{dagger}, Jorge Galán{dagger}, Dirk Roos{ddagger} and Erol Fikrig2,*

* Section of Rheumatology, Department of Internal Medicine, and {dagger} Section of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06520; and {ddagger} Sanquin Research at Central Laboratory of The Netherlands Blood Transfusion Service, and Lansteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Anaplasma phagocytophila, the etiologic agent of human granulocytic ehrlichiosis, is an emerging bacterial pathogen that invades neutrophils and can be cultivated in HL-60 cells. Infected neutrophils and HL-60 cells fail to produce superoxide anion (O2-), which is partially attributable to the fact that A. phagocytophila inhibits transcription of gp91phox, an integral component of NADPH oxidase. cDNA microarray and RT-PCR analyses demonstrated that transcription of the gene encoding Rac2, a key component in NADPH oxidase activation, was down-regulated in infected HL-60 cells. Quantitative RT-PCR demonstrated that rac2 mRNA expression was reduced 7-fold in retinoic acid-differentiated HL-60 cells and 50-fold in neutrophils following A. phagocytophila infection. Rac2 protein expression was absent in infected HL-60 cells. Rac1 and Rac2 are interchangeable in their abilities to activate NADPH oxidase. HL-60 cells transfected to express myc-tagged rac1 and gp91phox from the CMV immediate early promoter maintained the ability to generate O2- 120 h postinfection. A. phagocytophila proliferation was severely inhibited in these cells. These results directly attribute the inhibition of rac2 and gp91phox transcription to the loss of NADPH oxidase activity in A. phagocytophila-infected cells and demonstrate its importance to bacterial intracellular survival.




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