Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System

描述（由申请人提供）：沙门氏菌在美国每年导致140万例肠胃炎和肠热病例，并且是导致死亡的所有其他食源性细菌病原体的主要原因。沙门氏菌引起肠道和全身性疾病的先决条件是通过沙门氏菌致病性岛1 （SPI1）编码的3型分泌系统（T3SS）将效应蛋白直接注射到宿主肠上皮细胞中。这些效应蛋白诱导炎症性腹泻和细菌侵袭。SPI1 T3SS的表达受到来自各种全球调控系统的环境信号的严格调控。我们的长期目标是全面了解控制SPI1 T3SS的信号转导途径在感染过程中是如何整合的。广泛的遗传分析使我们能够为SPI1调控回路制定一个新的模型，其中三个类似arac的调控因子HilD， HilC和RtsA在一个复杂的前馈调控回路中起作用，控制hilA的表达，编码SPI1结构基因的直接调控因子。我们假设调节信号主要通过HilD的翻译后控制进入系统，进而激活hilC、rtsA和hilA。但是这些监管系统如何控制HilD是未知的。鞭毛蛋白FliZ和蛋白HilE在蛋白水平上独立控制HilD，很可能是通过与HilD的n端结构域的蛋白-蛋白相互作用。由于这些代表了近端调节输入，我们将重点了解FliZ和HilE如何控制HilD功能。本建议的具体目的是：1。确定FliZ和HilE如何控制HilD活动。生化和遗传实验将解剖hilA的HilD激活的每个步骤，以确定FliZ和HilE如何控制HilD的功能或稳定性。2. 描述HilD与HilE或FliZ之间交互的性质。共同免疫沉淀和双杂交分析将用于表征HilE-HilD和FliZ-HilD相互作用。否定调控的HilD点突变将用于识别HilD中特定的HilD区域，这些区域是HilD或fliz依赖的调控所必需的。这些突变体也将使我们能够测试FliZ和hile依赖性的SPI1在肠道入侵过程中的作用。3. 确定进入HilD以控制SPI1 T3SS的信号转导途径。已知的控制SPI1 T3SS表达的调控系统将通过HilD进行筛选。质谱和双杂交分析将用于确定与HilD蛋白相互作用的其他因素。这些因素的表征将导致我们对全局信号转导的全面理解。SP1 T3SS的调控是整合宿主环境信号以控制毒力基因表达的范例，分析该系统对我们了解这种B类优先病原体至关重要。

DESCRIPTION (provided by applicant): Salmonella cause 1.4 million cases of gastroenteritis and enteric fever per year in the US and lead all other foodborne bacterial pathogens as a cause of death. A prerequisite for Salmonella to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a Type Three Secretion System (T3SS) encoded on Salmonella Pathogenicity Island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals from a variety of global regulatory systems. Our long term goal is to obtain a comprehensive understanding of how the signal transduction pathways that control the SPI1 T3SS are integrated during the infection process. Extensive genetic analysis has allowed us to formulate a new model for the SPI1 regulatory circuit in which the three AraC-like regulators HilD, HilC, and RtsA act in a complex feed-forward regulatory loop to control expression of hilA, encoding the direct regulator of the SPI1 structural genes. We hypothesize that regulatory signals feed into the system primarily via post-translational control of HilD, which in turn activates hilC, rtsA, and hilA. But how these regulatory systems control HilD is unknown. The flagellar protein FliZ and the protein HilE independently control HilD at the protein level, most likely via protein-protein interaction with the N-terminal domain of HilD. As these represent proximal regulatory inputs, we focus on understanding how FliZ and HilE control HilD function. The specific aims of this proposal are to: 1. Determine how FliZ and HilE act to control HilD activity. Biochemical and genetic experiments will dissect each step in HilD activation of hilA to determine how FliZ and HilE act to control HilD function or stability. 2. Characterize the nature of the interaction between HilD and HilE or FliZ. Co-immunoprecipation and two-hybrid analysis will be used to characterize HilE-HilD and FliZ-HilD interactions. HilD point mutations that negate regulation will be used to identify regions of HilD that are specifically required for HilE- or FliZ-dependent regulation. These mutants will also allow us to test the role of FliZ- and HilE-dependent regulation of SPI1 during intestinal invasion. 3. Determine the signal transduction pathways that feed into HilD to control the SPI1 T3SS. Known regulatory systems that control SPI1 T3SS expression will be screened for those that function through HilD. Mass spectrometry and two-hybrid analysis will be used to identify additional factors that interact with HilD protein. Characterization of these factors will lead to our overall understanding of global signal transduction. The regulation of the SP1 T3SS serves as a paradigm for the integration of host environmental signals to control virulence gene expression and analysis of this system is critical to our understanding of this Class B priority pathogen. PUBLIC HEALTH RELEVANCE: Salmonella are major food-borne pathogens in the US. The bacteria invade the human intestinal cells to cause disease. Our goal is to understand the regulation of the bacterial invasion system to improve prevention and/or treatment.