Role of MiT Transcription Factors in Host Defense Against Bacterial Infection

描述（由申请人提供）：肠上皮的一个关键功能是区分细菌和病原微生物，从而避免感染性疾病和慢性炎症;然而，关于肠上皮细胞（IEC）如何在顶端表面感知细菌并识别信号以驱动适当的转录反应，人们知之甚少。本研究的长期目标是阐明IEC和微生物调控网络之间的关系，这些网络决定了体内宿主-微生物相互作用的结果。本提案的总体目标是阐明由MITF-TFE（MiT）家族转录因子（TF）介导的途径，这些转录因子决定宿主IEC对感染的反应。由于MiT家族TF是高度保守的，我们选择使用秀丽隐杆线虫，一种遗传上易于处理的模式生物，作为解决这些问题的工具。C.线虫表现出病原体特异性应答，其独立于TLR、NLR和NF-kB，因此代表了以无偏倚的方式在体内研究进化上保守的新型宿主防御途径的有用工具。这一提议的中心假设是，在病原感染过程中激活的未知信号通路控制了C.线虫和人类IEC。这项研究的基本原理是，一旦了解了MiT TF在宿主反应中的功能以及它们在体内的调节方式，它们的活性可能会被人为操纵，从而产生预防和治疗各种感染性或炎症性疾病的新的创新方法。为了验证这一中心假设，我们提出了三个具体的目标：1）利用嵌合遗传分析、翻译后修饰的生化特征和候选上游通路的遗传分析来阐明HLH-30/MiT的上游调控因子; 2）使用全局基因表达谱和染色质免疫沉淀方法阐明HLH-30/MiT的下游靶途径，和3）使用与高通量基因表达谱结合的生物化学、分子和细胞生物学方法中的人IEC系，阐明MiT TF在人IEC宿主防御中的作用。预计拟议研究的贡献是阐明新的非TLR/NLR/NF-B，HLH-30/MiT介导的调节宿主对C中细菌反应的途径。线虫和人类IEC。这些贡献是重要的，因为它们是连续研究的第一步，最终将允许通过MiT信号传导对宿主防御进行药理学操纵，无论是积极的还是消极的，以治疗感染或炎症性疾病。这项研究在概念上是创新的，因为它首次表明，在微生物刺激和肠道炎症过程中，MiT TF在人类IEC中的差异表达。此外，这项研究是创新的，因为它代表了更直接的体外方法的偏离，并利用了C. elegans，因此是一种无偏见和有效的体内方法。 公共卫生相关性：拟议的研究与公共卫生有关，因为成功完成这些研究后确定的信号成分可能为预防和治疗肠道感染性或炎症性疾病提供新的靶点，并显着推进宿主-微生物相互作用领域。本文提出的研究有望进一步探索MITF/TFE（MiT）转录因子在脊椎动物肠道感染和炎症性疾病遗传模型中其他信号通路的作用。该项目与NIH的使命相关，因为通过拟议研究获得的知识将增加控制宿主防御和MRSA发病机制的途径库，从而更好地了解疾病，使研究能够确定新的治疗靶点，并有可能加速新疗法的开发，减少各种感染和炎症性疾病的负担。

DESCRIPTION (provided by applicant): A key function of the intestinal epithelium is to discriminate commensal from pathogenic microbes and thus avoid infectious disease and chronic inflammation; however, little is known about how intestinal epithelial cells (IECs) sense bacteria at the apical surface and transduce the signals to drive appropriate transcriptional responses. The long-term goal of this research is to elucidate relationships between IEC and microbe regulatory networks that determine the outcome of host-microbe interactions in vivo. The overall objective of this proposal is to elucidate pathways mediated by MITF-TFE (MiT) family transcription factors (TFs) that determine the host IEC response to infection. Because MiT family TFs are highly conserved, we chose to use Caenorhabditis elegans, a genetically tractable model organism, as a tool to address these questions. C. elegans exhibits pathogen-specific responses that are independent of TLR, NLR, and NF-kB, and thus represents a useful tool to study novel host defense pathways that are evolutionarily conserved, in an unbiased manner in vivo. The central hypothesis of this proposal is that unknown signaling pathways activated during pathogenic infection control MiT-mediated host defense in C. elegans and human IECs. The rationale for the proposed research is that, once it is understood how MiT TFs function in the host response and how they are regulated in vivo, their activity could likely be manipulated pharmacologically, resulting in new and innovative approaches for the prevention and treatment of a variety of infectious or inflammatory diseases. To test the central hypothesis, three specific aims are proposed: 1) Elucidate upstream regulators of HLH-30/MiT, using mosaic genetic analysis, biochemical characterization of post-translational modifications, and genetic analysis of candidate upstream pathways; 2) Elucidate downstream target pathways of HLH-30/MiT, using global gene expression profiling and chromatin immunoprecipitation approaches, and 3) Elucidate the role of MiT TFs in human IEC host defense, using human IEC lines in biochemical, molecular, and cell biological approaches cou- pled with high-throughput gene expression profiling. The contribution of the proposed research is expected to be the elucidation of novel, non-TLR/NLR/NF-¿B, HLH-30/MiT-mediated pathways regulating the host response to bacteria in C. elegans and human IECs. These contributions are significant because they are the first step in a continuum of research that will eventually allow pharmacologic manipulation of host defense via MiT signaling, either positively or negatively, to treat infection or inflammatory disease. The proposed research is conceptually innovative because it shows for the first time that MiT TFs are differentially expressed in human IECs during microbial stimulation and intestinal inflammation.Furthermore, the proposed research is innovative because it represents a departure from more directed in vitro approaches and takes advantage of the tractability of C. elegans, and thus is an unbiased and efficient in vivo approach. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the signaling components identified upon successful completion of these studies are likely to provide new targets for prevention and treatment of intestinal infectious or inflammatory disease and significantly advance the field of host-microbe interactions. The research proposed here is expected to enable further exploration of the role of MITF/TFE (MiT) transcription factors, in the context of other signaling pathways in vertebrate genetic models of intestinal infection and inflammatory disease. The project is relevant to the mission of the NIH because the knowledge acquired with the proposed studies will increase the repertoire of pathways that control host defense and MRSA pathogenesis, leading to greater understanding of disease, enabling research to identify new targets for treatment, and having the po- tential to accelerate the development of new therapies, reducing the burden of a variety of infections and in- flammatory diseases.