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

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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.