Bacterial effectors targeting the IKK/NF-kB pathway

描述（由申请人提供）：尽管为减少水、肉和农产品的细菌污染进行了昂贵的尝试，但产志贺毒素的大肠杆菌（STEC）和相关的肠道病原体（如沙门氏菌、志贺氏菌、耶尔森氏菌）正在引起越来越频繁的食源性腹泻病爆发，从而构成巨大的健康负担。这些病原体都使用III型分泌系统（T3SS）将毒力蛋白（效应器）注入宿主细胞。虽然T3SS效应物在细菌毒力中明显发挥重要作用，但其生化机制尚不完全清楚。我们正在表征由产志贺毒素大肠杆菌效应物靶向的哺乳动物信号转导通路。我们最近描述了一种名为NleH1的产志贺毒素大肠杆菌效应蛋白，它通过一种独特的机制，靶向NF-KB转录因子，抑制宿主对感染的先天免疫反应。NF-KB转录因子家族调节许多重要的促炎宿主防御微生物病原体的激活。NF-KB在参与细菌感染先天反应的关键基因上的活性受核糖体蛋白S3 （RPS3）的调控，RPS3作为NF-KB亚基具有辅助核功能。我们发现NleH1抑制RPS3的核输入，导致NF-KB活性的选择性丧失。我们发现NleH1通过抑制Ik¿激酶复合物（IKK¿）磷酸化RPS3来阻止RPS3的核易位，这是RPS3核易位所必需的。与人类出血性结肠炎爆发最常见的产大肠杆菌血清型也编码一种名为NleH2的同源效应物。尽管NleH2与NleH1有84%的同源性，但NleH2刺激而不是抑制RPS3/ nf - kb依赖性转录。该研究的中心假设是，产志贺毒素大肠杆菌NleH1和NleH2效应物通过破坏RPS3/NF-KB控制的感染炎症反应调节来促进细菌存活。以下具体目标旨在定义这些新蛋白的分子机制：目标1。表征NleH1抑制RPS3的IKK¿磷酸化以阻止其核输入的分子机制。目标2。阐明NleH1和NleH2在调控RPS3/ nf - kb依赖性信号传导中的机制差异及其病理生理意义。目标3。量化NleH效应物对细菌毒力和传播的重要性，使用细菌腹泻病的动物模型。成功完成拟议的研究将1)揭示这组新的细菌效应物如何选择性地调节先天免疫；2)阐明病原体如何进化到选择核糖体蛋白的辅助核功能；3)描述细菌如何整合其毒力蛋白来颠覆宿主信号转导级联。

DESCRIPTION (provided by applicant): Despite costly attempts to reduce bacterial contamination of water, meat, and produce, Shiga toxin-producing E. coli (STEC) and related enteric pathogens (e.g. Salmonella, Shigella, Yersinia) are causing increasingly frequent outbreaks of food borne diarrheal disease, thus constituting enormous health burdens. Each of these pathogens uses a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While T3SS effectors clearly play essential roles in bacterial virulence, their biochemical mechanisms are incompletely characterized. We are characterizing mammalian signal transduction pathways targeted by STEC effectors. We have recently described an STEC effector protein named NleH1 that inhibits the host innate immune response to infection by targeting the NF-KB transcription factor using a unique mechanism. The NF-KB family of transcription factors regulates the activation of many crucial pro-inflammatory host defenses to microbial pathogens. The activity of NF-KB at key genes involved in the innate response to bacterial infection is regulated by ribosomal protein S3 (RPS3), which possesses an accessory nuclear function as an NF-KB subunit. We discovered that NleH1 inhibits the nuclear import of RPS3, leading to the selective loss of NF-KB activity. We found that NleH1 prevents the nuclear translocation of RPS3 by inhibiting the Ik¿ kinase complex (IKK¿) from phosphorylating RPS3, which is required for its nuclear translocation. The STEC serotypes that are most commonly implicated in causing deadly outbreaks of hemorrhagic colitis in humans also encode a homologous effector named NleH2. Despite sharing 84 % identity with NleH1, NleH2 stimulates rather than inhibits RPS3/NF-KB-dependent transcription. The central hypothesis for the proposed research is that the STEC NleH1 and NleH2 effectors promote bacterial survival by subverting the regulation of inflammatory responses to infection controlled by RPS3/NF-KB. The following specific aims are designed to define the molecular mechanism of these novel proteins: Aim 1. Characterize the molecular mechanism by which NleH1 inhibits IKK¿ phosphorylation of RPS3 to prevent its nuclear import. Aim 2. Elucidate mechanistic differences between NleH1 and NleH2 and their pathophysiological significance in regulating RPS3/ NF-KB-dependent signaling. Aim 3. Quantify the importance of NleH effectors to bacterial virulence and transmission using animal models of bacterial diarrheal disease. Successful completion of the proposed research will 1) reveal how this novel group of bacterial effectors selectively modulates innate immunity; 2) clarify how pathogens have evolved to co-opt the accessory nuclear functions of ribosomal proteins; and 3) characterize how bacteria have integrated their virulence proteins into subverting host signal transduction cascades.