Cell Death Induced by Yersinia YopH

鼠疫耶尔森纳是鼠疫或黑死病的人种学病原体，在历史上造成了其他任何传染病都无法比拟的毁灭性大流行。由于其快速复制和有效的免疫逃避能力，鼠疫耶尔森氏菌最近被认为是潜在的生物恐怖主义工具。特别是，细菌的雾化传播可能导致快速和暴发性的肺炎感染，随后可能在人与人之间传播。尽管存在几种疫苗，耶尔森氏菌通常对链霉素和四环素类抗生素敏感，但这种肺炎形式的疾病很难治疗，仍然经常导致死亡。额外的支持性治疗可能会显著降低这种致命性， 特别是在大规模接触武器化的耶尔西尼亚病毒的情况下。这个项目的重点是鼠疫耶尔森氏菌逃避免疫系统的分子机制的一个关键组成部分，一种被称为YopH的高活性蛋白酪氨酸磷酸酶(PTPase)。在受感染的宿主中，细菌在淋巴结内繁殖，在那里它们附着在T和B细胞上，并给它们注射YopH。在淋巴细胞内部，YopH有效地抑制了淋巴细胞的激活和免疫反应的发展。我们发现YopH干扰早期T细胞抗原受体信号转导和T细胞存活。我们的具体目标是：(1)YopH在T细胞抗原受体信号转导中的分子靶点。这些研究将试图通过底物捕捉技术、蛋白质组学、胰酶多肽图谱、共聚焦显微镜以及T细胞激活的功能分析和读出来确定YopH抑制TCR诱导的T细胞激活的分子机制。(2)YopH诱导细胞周期紊乱和细胞死亡的机制。我们将通过结合成像、生物化学、蛋白质组学和细胞死亡的功能分析来研究核YopH的作用和YopH诱导细胞死亡的机制。(3)YopH特异性抑制剂的开发。在P01的密切合作下，我们将通过高通量筛选、核磁共振、虚拟对接和约束多肽化学相结合的方式开发YopH特异性小分子抑制剂。将通过与其他PTPase的反筛选和表达YopH的T细胞的功能测试来优化和提取先导。

Yersina pestis, the ethiologic agent of plague or Black Death, has in historical times caused devastating pandemics unrivaled by any other infectious disease. Due to its very rapid replication and effective immune evading capacity, Yersinia pestis has recently been recognized as a potential tool for bioterrorism. In particular, an aerosolized delivery of the bacterium could cause a rapid and fulminant pneumonic infection, which subsequently may spread from person to person. Although several vaccines exist and Yersinia usually is sensitive to streptomycin and tetracycline type antibiotics, the pneumonic form of the disease is difficult to treat and still often results in death. Additional supportive treatments may significantly reduce this lethality, especially in the case of massive exposure of a population to weaponized Yersinia. This project focuses on a key component of the molecular machinery by which Yersinia pestis evades the immune system, a highly active protein tyrosine phosphatase (PTPase) termed YopH. In infected hosts, the bacteria multiply in lymph nodes, where they adhere to T and B cells and inject them with YopH. Inside the lymphocytes, YopH efficiently inhibits lymphocyte activation and the development of an immune response. We have found that YopH interferes with early T cell antigen receptor signaling and T cell survival. Our Specific Aims are: (1) Molecular targets of YopH in T cell antigen receptor signal transduction. These studies will attempt to determine the molecular mechanism by which YopH inhibits TCR-induced T cell activation using substrate-trapping technology, proteomics, tryptic peptide mapping, confocal microscopy, and functional assays and read-outs for T cell activation. (2) Mechanisms of cell cycle perturbation and cell death induced by YopH. We will investigate the role of nuclear YopH and the mechanism of YopH-induced cell death by a combination of imaging, biochemistry, proteomics, and functional assays for cell death. (3) Development of a YopH-specific inhibitor. In close collaborations within this P01, we will develop YopH-specific small molecule inhibitors by combinations of high through-put screening, NMR, virtual docking, and constrained peptide chemistry. Leads will be optimized and taken through counter-screening with other PTPases and functional tests in YopH-expressing T cells.