Rickettsia-induced transcriptional activation

描述（由申请人提供）：立克次体立克次体是一种专性细胞内细菌和落基山斑疹热的病原体，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途径作出反应。内皮细胞的立克次体感染导致核因子- kappab （NF-kappaB）的激活，这是一种控制一系列基因表达的转录因子，涉及细菌感染、免疫反应和细胞凋亡。NF-kappaB的抗凋亡功能在立克次体感染过程中保护宿主细胞免于凋亡死亡是至关重要的。本应用程序的目的是进一步了解立克次体诱导的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，并研究干扰这些信号是否会影响立克次体的复制。Aim 1将表征感染期间IkappaB激酶复合物（IKK）的激活和IkappaB蛋白的磷酸化/降解。我们将通过免疫沉淀（IP）：激酶测定来确定催化亚基IKKalpha和IKKbeta的激活动力学。调节亚基IKKgamma的作用将使用一种特异性的细胞渗透性肽进行评估，该肽阻断其与IKK复合物的关联。还将研究选定的IKK和NF-kappaB特异性抑制剂对立克次体生物复制的影响。目的2将研究有丝分裂原活化蛋白（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激酶级联反应ERK1/2和p38的调节将通过磷酸化状态特异性抗体的免疫印迹和免疫染色进行检测，并通过IP:western分析进行活性测定。目的3将定义趋化因子诱导对感染反应的调控，并探讨其对MAP激酶和NF-kappaB途径的依赖性。利用分子生物学和显微镜技术和不同致病性立克次体的物种/菌株，我们将研究感染、IKK/NF-kappaB和MAP激酶的激活以及趋化因子反应的诱导之间的关系。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.