Rickettsia-induced transcriptional activation

描述（由申请方提供）：立克次氏体是一种专性细胞内细菌和落基山斑疹热病原体，主要在血管内皮细胞内感染和增殖，血管内皮细胞通过激活一系列不同的信号转导途径进行应答。R.内皮细胞的立克次氏体感染导致核因子-κ B（NF-κ B）的活化，核因子-κ B是一种转录因子，其控制参与细菌感染、免疫应答和细胞凋亡的一系列基因的表达。NF-κ B的抗凋亡功能对于保护宿主细胞在R.立克次体感染本申请的目的是进一步了解立克次体诱导的转录激活的信号转导机制，以评估其参与宿主细胞对感染的反应，并研究干扰这些信号是否会影响立克次体的复制。目的1研究感染过程中IkappaB激酶复合物（IKK）的活化和IkappaB蛋白的磷酸化/降解。我们将通过免疫沉淀（IP）：激酶试验确定催化亚基IKK α和IKK β的活化动力学。调节亚基IKK γ的作用将使用特异性细胞渗透性肽进行评价，该肽阻断其与IKK复合物的结合。还将研究所选的IKK和NF-κ B特异性抑制剂对立克次体微生物复制的影响。目的2研究丝裂原活化蛋白（MAP）激酶的活化及其在立克次体侵袭内皮细胞和NF-κ B活化中的作用。MAP激酶级联、ERK 1/2和p38的调节将通过蛋白质印迹和使用磷酸化状态特异性抗体的免疫染色以及通过IP：蛋白质分析的活性测定来检查。目的3将明确感染后趋化因子诱导的调控机制，并探讨其对MAP激酶和NF-κ B通路的依赖性。使用分子生物学和显微镜的专业技术和不同致病性的立克次体的种/株，我们将调查感染，IKK/NF-κ B和MAP激酶的激活，诱导趋化因子反应之间的相关性。这些研究将为我们理解立克次体的发病机制提供重要的视角，并可能导致补充化疗的新靶点的确定。

DESCRIPTION (provided by applicant): Rickettsia rickettsii, an obligate intracellular bacterium and etiologic agent of Rocky Mountain spotted fever, infects and proliferates predominantly within vascular endothelial cells, which respond by activating a series of distinct signal transduction pathways. R. rickettsii infection of endothelial cells results in the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor which controls the expression of an array of genes involved in bacterial infections, immune response, and apoptosis. The anti-apoptotic functions of NF-kappaB are critical for the protection of host cells from apoptotic death during R. rickettsii infection. The goal of this application is to further our understanding of signaling mechanisms underlying Rickettsia-induced transcriptional activation, to evaluate their participation in the host cell response to infection, and to investigate if interfering with these signals affects rickettsial replication. Aim 1 will characterize the activation of IkappaB kinase complex (IKK) and phosphorylation/degradation of IkappaB proteins during infection. We will determine the kinetics of activation of catalytic subunits, IKKalpha and IKKbeta by an immunoprecipitation (IP): kinase assay. The role of the regulatory subunit, IKKgamma, will be evaluated using a specific, cell permeable peptide, which blocks its association with the IKK complex. The effects of selected, specific inhibitors of IKK and NF-kappaB on replication of Rickettsia organisms will also be studied. Aim 2 will investigate the activation of mitogen activated protein (MAP) kinases and their involvement in rickettsial invasion of endothelial cells and activation of NF-kappaB. Modulation of MAP kinase cascades, ERK1/2 and p38, will be examined by western blotting and immunostaining using phosphorylation state specific antibodies and activity assays by IP:western analysis. Aim 3 will define the regulation of chemokine induction in response to infection and explore its dependence on the MAP kinase and NF-kappaB pathways. Using specialized techniques of molecular biology and microscopy and species/strains of Rickettsia with varying pathogenicity, we will investigate the correlation between infection, activation of IKK/NF-kappaB and MAP kinases, and induction of chemokine response. These studies will offer important perspectives in our understanding of rickettsial pathogenesis and may lead to the identification of novel targets for supplemental chemotherapy.