Salmonella SopA is a functional mimicry of eukaryotic E3 protein ubiquitin ligase

描述(申请人提供)：鼠伤寒沙门氏菌在人和家畜中引起轻度胃肠炎和更严重的系统性感染。它编码两个专门的III型蛋白分泌系统，这两个系统是细菌入侵上皮细胞、在宿主细胞内生存和诱导胃肠炎所必需的。通过III型分泌系统，沙门氏菌注入一组细菌效应蛋白，利用宿主细胞功能诱导肠道炎症。以往的工作表明，SOPA是III型效应分子之一，在趋化因子的产生和肠炎的诱导中发挥关键作用，并且是有效的多形核白细胞(PMN)跨上皮间迁移所必需的。我们的初步数据表明SOPA是一种E3泛素连接酶。我们还发现SOPA E3连接酶活性参与了沙门氏菌诱导的PMN跨上皮迁移。此外，我们还发现SOPA能够从未感染的哺乳动物细胞提取物中泛素化蛋白(S)。此外，我们还证明了SOPA被宿主泛素E3连接酶HsRMA1泛素化。沙门氏菌E3泛素连接酶SOPA的发现为我们提供了一个研究SOPA功能的独特机会，有助于揭示沙门氏菌和SOPA引起肠道炎症和肠炎的分子和生化机制。我的工作假设是，SOPA泛素化细菌和/或宿主底物蛋白(S)，这与沙门氏菌诱导的炎症反应有关。我们试图确定由沙门氏菌SOPA泛素化的细菌和/或宿主蛋白。在这项提议中，我们计划：1)确定SOPA和HsRMA1介导的泛素化中的泛素赖氨酸连接；2)鉴定被沙门氏菌泛素化的细菌或宿主蛋白(S)。从这项研究中确定的SOPA泛素化蛋白将为更长期的研究奠定基础，这些研究旨在了解SOPA是如何导致胃肠炎的，以及沙门氏菌是如何导致人类的肠道炎症的。 公共卫生相关性：我们对沙门氏菌、沙门氏菌、沙门氏菌沙门氏菌是如何引起胃肠炎的，人们知之甚少。沙门氏菌毒力蛋白宿主细胞靶点的确定将有助于临床治疗药物的设计和多重耐药细菌的治疗。这项研究的结果将有助于我们了解沙门氏菌是如何诱导人类肠道炎症的。

DESCRIPTION (provided by applicant): Salmonella enterica serovar Typhimurium causes mild gastroenteritis and more severe systematic infections in both humans and domestic animals. It encodes two specialized type III protein secretion systems that are required for bacterial invasion into epithelial cells, for survival inside the host cells, and for the induction of gastroenteritis. Through the type III secretion system, Salmonella injects a panel of bacterial effector proteins to exploit the host cell function to induce intestinal inflammation. Previous work has shown that SopA, one of the type III effectors, plays a key role in chemokine production, induction of enteritis, and is required for efficient polymorphonuclear leukocytes (PMN) trans-epithelial migration. Our preliminary data demonstrated that SopA is an E3 ubiquitin ligase. We also found that the SopA E3 ligase activity is involved in Salmonella- induced PMN transepithelial migration. Furthermore, we showed that SopA is capable of ubiquitinating protein(s) from uninfected mammalian cell extracts. In addition, we demonstrated that SopA is ubiquitinated by HsRMA1, a host ubiquitin E3 ligase. The discovery of the Salmonella E3 ubiquitin ligase, SopA, has provided us with a unique opportunity to study SopA function and to help unravel the molecular and biochemical mechanisms by which Salmonella and SopA induce intestinal inflammation and enteritis. My working hypothesis is that SopA ubiquitinates bacterial and/or host substrate protein(s), which are involved in Salmonella-induced inflammatory responses. We seek to identify bacterial and/or host proteins that are ubiquitinated by Salmonella SopA. In this proposal, we plan to: 1) determine the ubiquitin lysine linkages in SopA and HsRMA1-mediated ubiquitination; 2) identify bacterial or host protein(s) that are ubiquitinated by Salmonella SopA. SopA-ubiquitinated proteins identified from this study will lay the foundation for more long- term studies aimed at understanding how SopA induces gastroenteritis and how Salmonella induces intestinal inflammation in humans. PUBLIC HEALTH RELEVANCE: the understanding gained by our long-standing interest in studying Salmonella, salmonellosis continues to pose worldwide medical concerns and remains the number one cause of food-borne diseases even in developed countries. How Salmonella cause gastroenteritis is poorly understood. The identification of host cellular targets of Salmonella virulence proteins will aid clinical therapeutic drug designs and treatment of multidrug-resistant bacteria. Results from this study will help us understand how Salmonella induces intestinal inflammation in humans.