Role of MiT Transcription Factors in Host Defense Against Bacterial Infection

描述(申请人提供)：肠上皮的一个关键功能是区分共生微生物和致病微生物，从而避免传染病和慢性炎症；然而，关于肠上皮细胞(IECS)如何感知根尖表面的细菌并传递信号以驱动适当的转录反应，人们知之甚少。这项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。这项建议的总体目标是阐明由MITF-TFE(MIT)家族转录因子(TF)介导的决定宿主IEC对感染的反应的途径。由于麻省理工学院家族的转录因子高度保守，我们选择使用秀丽隐杆线虫，一种遗传上易驯化的模式生物，作为解决这些问题的工具。线虫表现出独立于TLR、NLR和NF-kB的病原体特异性反应，因此是在体内以无偏见的方式研究进化保守的新的寄主防御途径的有用工具。这一建议的中心假设是，在致病感染过程中激活的未知信号通路控制着线虫和人类IECS中MIT介导的宿主防御。这项拟议研究的基本原理是，一旦了解了麻省理工学院转录因子如何在宿主反应中发挥作用，以及它们在体内是如何调节的，它们的活性很可能被药理学操纵，从而产生预防和治疗各种传染病或炎症性疾病的新的创新方法。为了验证这一中心假说，我们提出了三个具体目标：1)利用镶嵌遗传分析、翻译后修饰的生化特征以及候选上游途径的遗传分析，阐明HLH-30/MIT的上游调控因子；2)利用全球基因表达谱和染色质免疫沉淀方法，阐明HLH-30/MIT的下游靶通路；以及3)利用人IEC系在生化、分子和细胞生物学方面的研究，结合高通量基因表达谱，阐明MIT因子在人类IEC宿主防御中的作用。这项拟议研究的贡献有望阐明线虫和人类IECS中新的、非TLR/NLR/NF-B、HLH-30/MIT介导的调节宿主对细菌的反应的途径。这些贡献意义重大，因为它们是一系列研究的第一步，最终将允许通过麻省理工学院的积极或消极信号对宿主防御进行药理学操作，以治疗感染或炎症性疾病。这项拟议的研究在概念上是创新的，因为它首次表明MIT TF在微生物刺激和肠道炎症过程中在人的IECs中差异表达。此外，拟议的研究具有创新性，因为它偏离了更直接的体外方法，并利用了线虫的易驯化特性，从而是一种公正和有效的体内方法。 公共卫生相关性：拟议的研究与公共卫生相关，因为成功完成这些研究后确定的信号成分可能为肠道传染病或炎症性疾病的预防和治疗提供新的靶点，并显著推进宿主-微生物相互作用领域。这项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的其他信号通路中的作用。该项目与NIH的使命相关，因为通过拟议的研究获得的知识将增加控制宿主防御和MRSA致病机制的途径库，导致对疾病的更好理解，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，减轻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.