Mechanism of anti-phagocytosis by Yersinia pestis

鼠疫耶尔森菌抗吞噬机制

• 批准号: 6719772
• 负责人: KRISTIINA VUORI
• 金额: $ 38.2万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-15 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6719772
• 关键词: SDS polyacrylamide gel electrophoresis; Yersinia pestis; biological signal transduction; electrospray ionization mass spectrometry; enzyme activity; guanosinetriphosphatases; integrins; matrix assisted laser desorption ionization; phagocytosis; protein structure; protein tyrosine phosphatase

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.

描述（由申请人提供）：鼠疫的致病药物耶尔西尼亚·佩斯蒂斯（Yersinia Pestis）是人类已知的最致病的细菌之一。由于其高致病性，耶尔森尼亚被认为是生物恐怖主义的潜在武器。尤其是细菌的雾化形式可能会引起肺炎感染，这可能会非常迅速地感染大量患者。虽然早期的抗生素治疗通常在斑块的泡沫形式中有效，但肺斑块的反应较差，常常导致死亡。因此，在人口接触武器耶尔森氏菌后，需要其他治疗方式才能打击瘟疫。 Yersinia的高致病性是由于其能力逃避免疫系统。因此，尽管大多数其他细菌被整联蛋白介导的吞噬有效摄入并被吞噬细胞破坏，但Yersinia阻止了吞噬作用。 “抗吞噬作用”的机制涉及高度），有效的酪氨酸磷酸酶的毒力因子YOPH。 Yoph的一个关键目标被认为是对接蛋白PL30CAS（CAS），但其作用的确切机制仍然未知。在AIM 1中，我们将利用对整联蛋白信号传导和CAS蛋白的熟悉来检查分子机制抗头细胞增多症。我们的研究表明，CA是响应整联蛋白连接而激活RAC GTPase所必需的，并且这种激活对于吞噬作用至关重要。我们的数据支持YOPH的N末端结构域与CAS结合的观念，该结构与Yoph介导的抗毒细胞增多症相关。我们将通过抑制CAS及其结合伴侣CRK和随后的RAC激活之间的复杂形成来检验YOPH的功能。我们还将测试一种替代性，尽管不是互斥的假设，即YOPH-CAS相互作用导致YOPH靶向吞噬作用部位，并且随后失活了尚未识别的靶标分子。底物捕获技术和蛋白质组学方法与功能测定法相结合，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YOPH结构生物学方面的专业知识。我们的目标是开发YOPH特异性的小分子抑制剂，以阻断Yoph的N末端结构域的功能。将使用化学库筛选和基于NMR的设计的组合。将评估铅化合物在上面AIM 1中建立的吞噬作用和信号传导测定中的功效。 该应用将结合细胞生物学和结构生物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理中的根本重要问题。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Yersinia阻止吞噬细胞的抗菌功能的机制。我们进一步期望确定抑制剂将是进一步开发新型药物的宝贵起点，可用于打击瘟疫死亡。