Rickettsia-Induced Transcriptional Activation

描述（由申请人提供）：落基山斑疹热是美国最流行和最重要的立克次体病，是一种由立克次体引起的急性蜱传发热性疾病。该生物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防御机制（可能通过不同的信号转导途径）进行应答。我们证明了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，NF-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。我们还发现，NF-κ B的抗凋亡功能可以保护宿主细胞在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，并研究它们参与宿主细胞对感染的反应。由于NF-kB激活的关键事件是IkB激酶（IKK）复合物降解IkB（NF-kB抑制剂）蛋白，目的I将表征感染期间IKK的激活和IkB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-a和IKK-b的活化动力学。调节亚基IKK-g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。目的2：研究丝裂原活化蛋白（MAP）激酶的活化及其与立克次氏体侵袭内皮细胞和NF-κ B活化的关系。用磷酸化状态特异性抗体免疫印迹法检测MAP激酶级联、ERK 1/2和p38的调节，用IP蛋白质免疫印迹法检测活性。目的3阐明感染后趋化因子的诱导调控机制，并探讨其对MAP激酶和NF-κ B通路的依赖性。利用分子生物学和显微镜技术和不同致病性的立克次体菌株，我们将研究感染、IKK/NF-kB和MAP激酶的激活以及趋化因子应答的诱导之间的相关性。这些研究将为理解立克次体的发病机制提供重要的视角，并为作为宿主-寄生虫关系的一部分而发生的信号事件的复杂相互作用提供有价值的见解。

DESCRIPTION (provided by applicant): Rocky Mountain spotted fever, the most prevalent and important of the rickettsioses in the United States, is an acute tick-borne febrile illness caused by Rickettsia rickettsii. The organism infects and proliferates predominantly within vascular endothelial cells, which respond by activating a series of defense mechanisms, possibly via distinct signal transduction pathways. We have demonstrated that R. rickettsii infection of endothelial cells results in the activation of nuclear factor-kappaB (NF-kB a transcription factor which controls the expression of an array of genes involved in bacterial infections, immune response, and apoptosis. We have also shown that anti-apoptotic functions of NF-kB protect the host cell from apoptotic death during R. rickettsii infection. The objective of this application is to further our understanding of signaling mechanisms underlying Rickettsia-induced transcriptional activation and investigate their participation in the host cell response to infection. Since the seminal event in the activation of NF-kB is the degradation of IkB (inhibitors of NF-kB) proteins by IkB kinase (IKK) complex, Aim I will characterize the activation of IKK and phosphorylation/degradation of IkB proteins during infection. We will determine the kinetics of activation of catalytic subunits, IKK-a and IKK-b, by an immunoprecipitation (IP) kinase assay. The role of the regulatory subunit, IKK-g will be evaluated using a specific, cell permeable peptide that blocks its association with the IKK complex. Aim 2 will characterize the activation of mitogen activated protein (MAP) kinases and investigate their involvement in rickettsial invasion of endothelial cells and activation of NF-kB Modulation of MAP kinase cascades, ERK1/2 and p38, will be examined by immunoblotting 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-kB pathways. Using molecular biology and microscopy techniques and strains of Rickettsia with varying pathogenicity, we will investigate the correlation between infection, activation of IKK/NF-kB and MAP kinases, and induction of chemokine response. These studies will offer important perspectives in understanding rickettsial pathogenesis and provide valuable insight into the complex interplay of signaling events, which occur as part of the host-parasite relationship.