Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System

描述(申请人提供)：沙门氏菌在美国每年导致140万例胃肠炎和肠热病，并导致所有其他食源性细菌致死。沙门氏菌引起肠道和全身疾病的先决条件是通过沙门氏菌致病岛1(SPI1)编码的三型分泌系统(T3SS)将效应蛋白直接注射到宿主肠道上皮细胞。这些效应蛋白可诱导炎症性腹泻和细菌入侵。SPI1 T3SS的表达受到严格调控，以响应来自各种全球调控系统的环境信号。我们的长期目标是全面了解控制SPI1 T3SS的信号转导通路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调控回路建立一个新的模型，其中三个AraC样调节因子Hild、HilC和RTSA在一个复杂的前馈调控环中作用，控制Hila的表达，编码SPI1结构基因的直接调节因子。我们假设，调控信号主要通过对Hild的翻译后控制进入系统，这反过来又激活了HilC、RTSA和Hila。但这些监管系统是如何控制希尔德的，目前尚不清楚。鞭毛蛋白Fliz和HILE在蛋白质水平上独立控制HILDs，很可能是通过与HILDN-末端结构域的蛋白质相互作用来实现的。由于这些代表了近端的调控输入，我们重点了解Fliz和HILE如何控制HILD的功能。这项建议的具体目的是：1.确定Fliz和Hile如何采取行动控制HIRD活动。生化和遗传学实验将剖析HILA激活HILD的每一步，以确定Fliz和HILE如何控制HILD的功能或稳定性。2.描述HILD和HILE或FLIZ之间相互作用的性质。免疫共沉淀和双杂交分析将用于表征hILE-Hild和Fliz-Hild的相互作用。取消调控的Hild点突变将被用来确定hILL或FliZ依赖的调控所特别需要的Hild区域。这些突变还将使我们能够测试FliZ和HILE依赖的SPI1调控在肠道侵袭中的作用。3.确定调控SPI1 T3SS的信号转导通路。已知的控制SPI1 T3SS表达的调控系统将被筛选出那些通过HILD发挥作用的系统。质谱学和双杂交分析将被用来确定与希尔德蛋白相互作用的其他因素。对这些因素的表征将有助于我们对全球信号转导的整体理解。SP1T3SS的调控是整合寄主环境信号以控制毒力基因表达的范例，对该系统的分析对于我们理解这种B类优先病原体是至关重要的。 公共卫生相关性：在美国，沙门氏菌是主要的食源性病原体。这种细菌侵入人体肠道细胞导致疾病。我们的目标是了解细菌入侵系统的调节，以改进预防和/或治疗。

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.