Studies described in this application are designed to understand mechanisms by which endothelial cell-rickettsia interactions elicit cellular responses critical to the pathogenesis of rickettsial disease. Intracellular infection of vascular endothelial cells with R. rickettsii results in changes in gene transcription leading to expression of procoagulant and proinflammatory molecules. These endothelial cell responses are likely important in the pathologic changes associated with human infection, which results in the disease known as Rocky Mountain Spotted Fever (RMSF). Preliminary studies indicate that R. rickettsii infection results in activation of the transcription factor, nuclear factor -kB (NF-kB), which controls expression of many inducible genes involved in early responses to inflammatory stimuli. The studies proposed will focus on mechanisms of R. rickettsii induced NF-kB activation. The proposal is divided into two specific aims. The first specific aim will concentrate on characterization of NF-kB activation which occurs during infection of cultured endothelial cells with R. rickettsii, and will include study of the kinetics of activation as well as molecular characterization of the activated complex. The second specific aim will utilize two experimental approaches to explore intracellular signaling pathways operative in R. rickettsii-induced NF-kB activation. To explore characteristics of the organism important in eliciting this cellular response, a system was developed to study activation of NF-kB in isolated host cell cytoplasm. By bypassing the entry process, this system will allow extensive manipulation of the rickettsia organisms yet still allow direct interaction of rickettsia with host cell signaling machinery. Inhibitors of key regulatory molecules will also be used in intact cells to identify intracellular """"""""targets"""""""" of infection resulting in activation. The investigators hypothesize that R. rickettsii induced NF-kB activation is mediated via interaction of the organism with host cell components, but that the response likely differs somewhat from known physiologic inducers with regard to kinetics and isoform specificity. Since a primary event in the course of RMSF is extensive microvascular thrombosis, insights gained from these studies may provide valuable insights not only in endothelial cell involvement in rickettsial disease, but in thrombotic disease in general.
