Rickettsia-induced transcriptional activation

描述(申请人提供)：立克次体是一种专性细胞内细菌，也是落基山斑点热的病原体，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通路做出反应。立克次体感染内皮细胞导致核因子-kappaB(NF-kappaB)的激活，核因子-kappaB是一种转录因子，控制着一系列参与细菌感染、免疫反应和细胞凋亡的基因的表达。在立克次体感染过程中，核因子-kappaB的抗凋亡功能对于保护宿主细胞免于凋亡性死亡是至关重要的。这项应用的目的是进一步了解立克次体诱导转录激活的信号机制，评估它们在宿主细胞对感染的反应中的参与，并调查干扰这些信号是否会影响立克次体的复制。目的1研究IkappaB蛋白在感染过程中的活化和蛋白的磷酸化/降解。我们将通过免疫沉淀(IP)：激酶试验来确定催化亚基IKKalpha和IKKbeta的激活动力学。将使用一种特定的细胞通透性多肽来评估调节亚单位IKKGamma的作用，这种多肽可以阻断其与IKK复合体的联系。还将研究选定的IKK和NF-kappaB的特定抑制剂对立克次体生物复制的影响。目的2研究丝裂原活化蛋白(MAP)激酶的激活及其在立克次体侵袭内皮细胞和激活核因子-kappaB中的作用。蛋白印迹和免疫染色将通过磷酸化状态特异性抗体和IP：Western分析的活性分析来检测MAP激酶级联通路ERK1/2和p38的调节。目的3将确定趋化因子诱导对感染的反应的调节，并探讨其对MAP激酶和核因子-kappaB通路的依赖。我们将利用分子生物学和显微镜技术以及不同致病力的立克次体物种/菌株，研究感染、激活IKK/NF-kappaB和MAP激酶以及诱导趋化因子反应之间的关系。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，并可能导致确定补充化疗的新靶点。

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.