Mechanism of anti-phagocytosis by Yersinia pestis

性状（由申请人提供）：鼠疫耶尔森氏菌是鼠疫的病原体，是人类已知的致病性最强的细菌之一。由于其高致病性，耶尔森菌已被公认为生物恐怖主义的潜在武器。特别是细菌的气溶胶形式可能会导致肺炎感染，这可能会非常迅速地感染大量人群。虽然早期抗生素治疗通常对腺泡状斑块有效，但肺炎斑块的反应较低，通常导致死亡。因此，在人群暴露于武器化的耶尔森氏菌后，需要额外的治疗模式来对抗鼠疫。 耶尔森氏菌的高致病性是由于其逃避免疫系统的能力。因此，虽然大多数其他细菌被整合素介导的吞噬有效地摄入并被吞噬细胞破坏，但耶尔森氏菌阻断吞噬作用。“抗吞噬作用”的机制涉及毒力因子YopH，一种高效的酪氨酸磷酸酶。YopH的一个关键靶点被认为是对接蛋白pl 30 cas（Cas），但其作用的确切机制仍然未知。在目标1中，我们将利用我们对整合素信号传导和Cas蛋白的熟悉来研究抗吞噬作用的分子机制。我们的研究表明，Cas是响应于整合素连接而激活Rac GT3所必需的，并且这种激活是吞噬作用所必需的。我们的数据支持YopH的N-末端结构域与Cas结合的观点，这与YopH介导的抗吞噬作用相关。我们将测试这一假设，即YopH的功能，抑制Cas和它的结合伙伴Crk和随后的Rac激活之间的复合物的形成。我们还将测试另一种假设，尽管不是相互排斥的，即YopH-Cas相互作用导致YopH靶向吞噬作用的位点，并随后使尚未鉴定的靶分子失活。底物捕获技术和蛋白质组学方法与功能测定相结合，将用于识别和表征这些靶蛋白。Aim 2将利用我们的合作者Maurizio Pellecchia博士在基于NMR的药物发现和结构生物学方面的专业知识。我们的目标是开发YopH特异性小分子抑制剂，阻断YopH的N-末端结构域的功能。将使用化学文库筛选和基于NMR的设计的组合。将在上述目标1中建立的吞噬作用和信号传导测定中评价先导化合物的功效。 该应用将联合收割机细胞生物学和结构生物学实验室的互补专业知识，以独特的方式解决耶尔森氏菌发病机制中的根本性重要问题。我们预计，这些研究得到了大量初步数据的支持，将使我们能够更好地了解耶尔森氏菌阻断吞噬细胞抗菌功能的机制。我们进一步期望，待鉴定的抑制剂将是进一步开发可用于对抗鼠疫死亡率的新药的有价值的起点。

DESCRIPTION (provided by applicant): Yersinia pestis, the causative agent of plague, is one of the most pathogenic bacteria known to mankind. Due to its high pathogenicity, Yersinia has been recognized as a potential weapon for bioterrorism. An aerosolized form of the bacteria in particular could cause a pneumonic infection, which could infect a large number of people very rapidly. While early antibiotic treatment is usually effective in the bubonic form of plaque, the pneumonic plaque is less responsive and often results in death. Additional modes of treatment would therefore be required to combat plague following exposure of a population to weaponized Yersinia. Yersinia's high pathogenicity is due to its capability to evade the immune system. Thus, while most other bacteria are effectively ingested by integrin-mediated engulfment and destroyed by phagocytes, Yersinia blocks phagocytosis. The mechanism of "anti-phagocytosis" involves the virulence factor YopH, a highly), effective tyrosine phosphatase. One crucial target for YopH is thought to be the docking protein pl30cas(Cas), but the precise mechanism of its action remains unknown. In Aim 1, we will utilize our familiarity with integrin signaling and the Cas protein to examine the molecular mechanism antiphagocytosis. Our studies indicate that Cas is required for activation of the Rac GTPase in response to integrin ligation, and that this activation is essential for phagocytosis. Our data support the notion that the N-terminal domain of YopH binds to Cas, which correlates with YopH-mediated anti-phagocytosis. We will test the hypothesis that YopH functions by inhibiting a complex formation between Cas and its binding partner Crk and subsequent Rac activation. We will also test an alternative, although not mutually exclusive hypothesis that the YopH-Cas interaction results in targeting of YopH to the sites of phagocytosis, and in the subsequent inactivation of yet-to-be-identified target molecules. Substrate-trapping technology and proteomics approaches combined with functional assays will be used to identify and characterize these target proteins. Aim 2 will take advantage of the expertise of Dr. Maurizio Pellecchia, our collaborator, in NMR-based drug discovery and structural biology with YopH. Our goal is to develop YopH-specific small molecule inhibitors that block the function of the N-terminal domain of YopH. A combination of chemical library screening and NMR-based design will be used. Lead compounds will be evaluated for efficacy in the phagocytosis and signaling assays established in Aim 1 above. This application will combine complementary expertise of a cell biological and a structural biological laboratory to address a fundamentally important question in the Yersinia pathogenesis in a unique manner. We anticipate that these studies, which are supported by a significant amount of preliminary data, will allow us to better understand the mechanisms by which Yersinia block the antimicrobial function of phagocytes. We further expect that the inhibitors to be identified will be a valuable starting point for the further development of novel drugs that can be used to combat plague mortality.