Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System

描述（由申请方提供）：沙门氏菌每年在美国引起140万例胃肠炎和肠热病，并导致所有其他食源性细菌病原体死亡。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岛1（SPI 1）上编码的三型分泌系统（T3 SS）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性腹泻和细菌入侵。SPI 1 T3 SS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SPI 1 T3 SS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制定一个新的模型的SPI 1的监管电路，其中三个AraC样调节HilD，HilC，和RtsA的行为在一个复杂的前馈调节回路控制hilA的表达，编码的SPI 1结构基因的直接调节。我们假设调控信号主要通过HilD的翻译后控制进入系统，这反过来又激活hilC，rtsA和hilA。但这些调节系统如何控制HilD尚不清楚。鞭毛蛋白FliZ和蛋白HilE在蛋白质水平上独立地控制HilD，最有可能是通过与HilD的N-末端结构域的蛋白质-蛋白质相互作用。由于这些代表近端调控输入，我们专注于了解FliZ和HilE如何控制HilD功能。这项建议的具体目标是：1.确定FliZ和HilE如何控制HilD活性。生物化学和遗传实验将剖析HilD激活hilA的每一步，以确定FliZ和HilE如何控制HilD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相互作用。否定调控的HilD点突变将用于鉴定HilE或FliZ依赖性调控特异性所需的HilD区域。这些突变体也将使我们能够测试FliZ和HilE依赖性调节SPI 1在肠道侵袭过程中的作用。3.确定输入HilD以控制SPI 1 T3 SS的信号转导途径。控制SPI 1 T3 SS表达的已知调节系统将筛选通过HilD起作用的那些。质谱和双杂交分析将用于鉴定与HilD蛋白相互作用的其他因子。这些因素的特性将导致我们的整体信号转导的理解。SP1 T3 SS的调节作为整合宿主环境信号以控制毒力基因表达的范例，并且分析该系统对于我们理解这种B类优先病原体至关重要。 公共卫生相关性：沙门氏菌是美国主要的食源性病原体。这种细菌侵入人体肠道细胞引起疾病。我们的目标是了解细菌入侵系统的调节，以改善预防和/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.