Bacterial effectors targeting the IKK/NF-kB pathway

描述（申请人提供）：尽管为减少水、肉和蔬菜的细菌污染进行了昂贵的尝试，但产滋贺毒素的E.大肠杆菌（STEC）和相关的肠道病原体（例如沙门氏菌、志贺氏菌、耶尔森氏菌）引起食源性腹泻病的日益频繁的爆发，从而构成巨大的健康负担。这些病原体使用III型分泌系统（T3 SS）将毒力蛋白（效应子）注入宿主细胞。虽然T3 SS效应子在细菌毒力中发挥重要作用，但它们破坏宿主细胞功能以促进病原体存活的机制尚不完全。我们正在研究STEC效应子靶向的哺乳动物信号转导途径，最终目标是提高我们预防和治疗STEC的能力。 细菌感染我们已经做出了重要且独特的发现，即与人类严重腹泻病爆发相关的STEC菌株表达一对同源效应子，其通过破坏通常由NF-κ B途径协调的对感染的转录应答来差异调节宿主先天免疫。在关键的先天免疫应答基因的NF-κ B活性由核糖体蛋白S3（RPS 3）调节，其具有作为NF-κ B亚基的辅助核功能。我们发现，NleH 1效应蛋白抑制RPS 3核转位，降低特定启动子的NF-κ B活性。具体而言，NleH 1抑制IKK复合物（IKK）磷酸化RPS 3，这是其核转位的关键要求。STEC菌株还编码名为NleH 2的同源效应子。尽管与NleHl共享84%的同一性，但NleH 2刺激而不是抑制RPS 3/NF-kB依赖性转录。我们假设NleH 1和NleH 2效应子通过破坏通常由RPS 3/NF-kB调节的对感染的促炎反应来促进细菌毒力，并提出了以下具体目标：1）使用肠炎疾病的动物模型量化NleH效应子对细菌毒力的重要性。2)表征NleH 1抑制RPS 3的IKK？磷酸化以阻止其核输入的分子机制。3)阐明NleH 1和NleH 2之间的机制差异及其在调节RPS 3/NF-kB依赖性信号传导中的病理生理学意义。成功完成拟议的研究将1）揭示这些细菌效应子如何选择性地调节先天免疫; 2）阐明病原体如何进化以吸收核糖体蛋白的辅助核功能; 3）表征细菌如何将其毒力蛋白整合到特定时空背景下的宿主信号转导途径中。

DESCRIPTION (provided by applicant): Despite costly attempts to reduce bacterial contamination of water, meat, and vegetables, 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. These pathogens use a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While T3SS effectors clearly play essential roles in bacterial virulence, the mechanisms by which they subvert the functions of host cells to promote pathogen survival are incompletely characterized. We are studying the mammalian signal transduction pathways targeted by STEC effectors, with the ultimate goal of improving our ability to prevent and to treat bacterial infections. We have made the important and unique discovery that the STEC strains associated with severe diarrheal disease outbreaks in humans express a pair of homologous effectors that differentially regulate host innate immunity by disrupting the transcriptional responses to infection that are normally coordinated by the NF-kB pathway. NF-kB activity at key innate immune response genes is regulated by ribosomal protein S3 (RPS3), which possesses an accessory nuclear function as an NF-kB subunit. We discovered that the NleH1 effector protein inhibits RPS3 nuclear translocation, reducing NF-kB activity at specific promoters. Specifically, NleH1 inhibits the Ik¿ kinase complex (IKK¿) from phosphorylating RPS3, a critical requirement for its nuclear translocation. STEC strains also encode a homologous effector named NleH2. Despite sharing 84 % identity with NleH1, NleH2 stimulates rather than inhibits RPS3/NF-kB-dependent transcription. We hypothesize that the NleH1 and NleH2 effectors promote bacterial virulence by subverting the pro- inflammatory responses to infection normally regulated by RPS3/NF-kB and propose the following specific aims: 1) Quantify the importance of NleH effectors to bacterial virulence using animal models of diarrheal disease. 2) Characterize the molecular mechanism by which NleH1 inhibits IKK¿ phosphorylation of RPS3 to prevent its nuclear import. 3) Elucidate mechanistic differences between NleH1 and NleH2 and their pathophysiological significance in regulating RPS3/NF-kB-dependent signaling. Successful completion of the proposed research will 1) reveal how these bacterial effectors selectively modulate 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 host signal transduction pathways in specific spatiotemporal contexts.