Rickettsia-Induced Transcriptional Activation

描述(申请人提供)：落基山斑点热是美国最流行和最重要的立克次体病，是由立克次体引起的一种急性硬壳性发热性疾病。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做出反应，可能是通过不同的信号转导途径。我们已经证明，立克次体感染内皮细胞会导致核因子-kB(NF-kB)的激活，核因子-kB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。我们还表明，在立克次体感染过程中，核因子-kB的抗凋亡功能可以保护宿主细胞免于凋亡性死亡。这项应用的目的是加深我们对立克次体诱导转录激活的信号机制的理解，并研究它们在宿主细胞对感染的反应中的参与。由于核因子-kB活化的基础事件是IKB(核因子-kB抑制因子)蛋白被IKB激酶(IKK)复合体降解，因此AIM I将描述感染过程中IKK的激活和IKB蛋白的磷酸化/降解。我们将通过免疫沉淀(IP)激酶分析来确定催化亚基IKK-A和IKK-B的激活动力学。调节亚单位ikk-g的作用将使用一种特定的细胞通透性多肽来评估，该肽可阻断其与ikk复合体的联系。目的2研究丝裂原活化蛋白(MAP)激酶的激活特征，并探讨其在立克次体侵袭内皮细胞中的作用以及核转录因子-kB(NF-kB)对MAP激酶级联通路ERK1/2和p38的调节作用。目的3将定义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-kB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菌株，研究感染、激活IKK/NF-kB和MAP激酶以及诱导趋化因子反应之间的相关性。这些研究将为理解立克次体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.