C. trachomatis-mediated signaling pathways required for Type I IFN responses

描述（由申请人提供）：先天免疫系统能够在细胞外空间和细胞内区室中检测独特的微生物成分，以诱导清除病原体的免疫反应。最近的研究表明，多种细胞质途径触发I型干扰素（IFN）响应细胞内DNA和RNA分子。I型干扰素在细菌感染结果中的确切作用尚不清楚，因为这些细胞因子既可以预防又可以增强病原体复制。沙眼衣原体在许多不同的细胞类型中诱导有效的I型IFN反应，这依赖于多种模式识别受体，包括toll样受体和Nod受体。然而，衣原体诱导I型IFN反应的调控机制和这种反应所需的细菌配体尚不清楚。本应用程序的目的是确定宿主细胞检测衣原体感染诱导I型干扰素反应的机制。我的假设是衣原体c-二核苷酸是通过细胞质感应途径检测到的，这对宿主I型干扰素应答至关重要。我们将使用稳定表达STING-myc或干扰素刺激反应元件(IRSE)-荧光素酶报告基因的小鼠胚胎成纤维细胞（mef）来确定衣原体感染反应中STING定位所需的信号通路，并通过细胞质内递送c-di-AMP。初步结果显示，在沙眼衣原体感染和c-di-AMP治疗中，I型IFN的诱导都需要钙依赖性磷脂酶cPLA2，这表明在衣原体感染期间检测c-di-AMP是I型IFN应答所必需的。我们计划通过纯化假定的衣原体二腺苷酸环化酶（DAC）蛋白CT012来进一步验证这一点，并使用薄层色谱测试放射性标记ATP向c-二腺苷酸环化酶的转化。为了补充这个实验，我将在MEFS中过度表达假定的磷酸二酯酶recJ，并筛选衣原体诱导的I型IFN反应的缺失。我们已经组装了一个化学衍生衣原体突变体的大型文库，并将使用该文库筛选改变I型IFN反应所需的细菌因子。我们将使用表达ISRE-luc的mef筛选诱导I型IFN的突变体，并确定这些反应是否与STING易位相关。显示异常I型IFN表型的突变体将被测序以确定致病snp。最近与标记菌株的特征遗传交换将使我们能够将突变体与野生型菌株回交，以确定单个致病SNP。我们将对诱导I型IFN反应改变的EBs和EBs的上清液使用固相萃取。分离后的上清液将通过地黄皂苷渗透到宿主细胞质中，以鉴定I型IFN诱导生物活性分子。这些实验将大大促进我们对衣原体如何诱导先天免疫反应的理解。此外，我们将定义感知细胞内分子（如c-di-AMP）所需的宿主细胞途径。

DESCRIPTION (provided by applicant): The innate immune system is capable of detecting unique microbial components in both the extracellular space and intracellular compartments for induction of immune responses for clearance of pathogens. Recent studies have indicated that multiple cytosolic pathways trigger Type I Interferon (IFN) in response to intracellular DNA and RNA molecules. The exact role Type I IFNs play in the outcome of bacterial infections remains unclear as these cytokines can both prevent and enhance pathogen replication. Chlamydia trachomatis induces a potent Type I IFN response in many different cell types that is dependent on multiple pattern recognition receptors including Toll-like and Nod receptors. However, the regulatory mechanisms underlying Chlamydia-induced Type I IFN responses and the bacterial ligands necessary for this response are poorly understood. The objective of this application is to define the mechanism by which host cells detect Chlamydia infection to induce Type I Interferon responses. My hypothesis is that Chlamydia c-di- nucleotides are detected through cytosolic sensing pathways, which are critical for the host Type I interferon response. We will use murine embryonic fibroblasts (MEFs) stably expressing STING-myc or an Interferon Stimulated Response Element (IRSE)-luciferase reporter to define the signaling pathways that are required for proper STING localization in response to Chlamydia infection and through cytosolic delivery of c-di-AMP. Preliminary results show that the calcium-dependent phospholipase cPLA2 is required for Type I IFN induction in response to both C. trachomatis infection and treatment with c-di-AMP, suggesting that detection of c-di-AMP during Chlamydia infection is required for Type I IFN responses. We plan to test this further by purifying the putative Chlamydia di-adenylate cyclase (DAC) protein CT012 and testing for conversion of radiolabeled ATP to c-di-AMP using thin layer chromatography. To complement this assay I will over express the putative phosphodiesterase recJ in MEFS and screen for the loss of Chlamydia-induced Type I IFN responses. We have assembled a large library of chemically derived Chlamydia mutants and will use this library to screen for bacterial factors that are required for altered Type I IFN responses. We will screen our mutants for induction of Type I IFN using MEFs expressing ISRE-luc and also identify whether these responses correlate with STING translocation. Mutants showing aberrant Type I IFN phenotypes will be sequenced to identify the causative SNPs. The recently characterized genetic exchange with marked strains will allow us to backcross our mutants with wildtype strains to identify a single causative SNP. We will use solid phase extraction on supernatants from EBs and EBs that induce altered Type I IFN responses. The fractionated supernatants will be delivered by digitonin permeabilization to the host cytosol to identify the Type I IFN inducing bioactive molecule. These experiments will significantly advance our understanding of how Chlamydia induces innate immune responses. In addition, we will define the host cellular pathways required for sensing intracellular molecules such as c-di-AMP. PUBLIC HEALTH RELEVANCE: The objective of this application is to define the mechanism by which host cells detect Chlamydia infection to induce Type I Interferon responses. My hypothesis is that Chlamydia c-di-nucleotides are detected through cytosolic sensing pathways, which are critical for the host Type I interferon response.