Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System

沙门氏菌致病性岛1型III型分泌系统的调控

• 批准号: 8490284
• 负责人: JAMES M. SLAUCH
• 金额: $ 34.11万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8490284
• 关键词: Affect; Amplifiers; Bacteria; Biochemical Genetics; Biosensor; Cause of Death; Cells; Co-Immunoprecipitations; Complex; Diarrhea; Disease; Epithelial Cells; Gastroenteritis; Gene Expression Profiling; Genetic; Genetic Transcription; Goals; Half-Life; Human; Individual; Infection; Inflammatory; Injection of therapeutic agent; Intestinal Diseases; Intestines; Invaded; Knowledge; Lead; Mass Spectrum Analysis; Measures; Mediating; Modeling; Mutation; N-terminal; Nature; Pathogenesis; Pathogenicity Island; Point Mutation; Prevention; Process; Proteins; Regulation; Research; Role; SP1 gene; Salmonella; Salmonella enterica; Signal Transduction; Signal Transduction Pathway; Site; Structural Genes; System; Systemic disease; Systemic infection; Testing; Type III Secretion System Pathway; Typhoid Fever; Virulence; Work; base; feeding; foodborne; foodborne pathogen; genetic analysis; improved; mutant; novel; pathogen; protein protein interaction; public health relevance; research study; response; yeast two hybrid system

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.

描述（由申请人提供）：沙门氏菌每年在美国导致140万例胃肠炎和肠发烧，并带领所有其他食源性细菌病原体作为死亡原因。沙门氏菌同时引起肠道和全身性疾病的先决条件是通过在沙门氏菌致病岛1（SPI1）上编码的三型分泌系统（T3S）直接注射效应蛋白在宿主肠上皮细胞中。这些效应蛋白诱导炎症性腹泻和细菌侵袭。 SPI1 T3SS的表达受到各种全球调节系统的环境信号的严格调节。我们的长期目标是对如何在感染过程中整合控制SPI1 T3SS的信号转导途径的全面了解。广泛的遗传分析使我们能够为SPI1调节电路制定新的模型，其中三个类似ARAC的调节剂HILD，HILC和RTSA ACT在复杂的前馈调节循环中控制HILA的表达，编码SPI1结构基因的直接调节剂。我们假设调节信号主要通过对HILD的翻译后控制为系统，后者又激活了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和蛋白质Hile在蛋白质水平上独立控制HILD，这很可能是通过蛋白质 - 蛋白质与HILD的N末端结构域的相互作用。由于这些代表近端调节输入，因此我们专注于了解Fliz和Hile Control如何控制HILD功能。该提案的具体目的是：1。确定Fliz和Hile如何控制Hild活动。生化和遗传实验将在Hild激活HILA的每个步骤中剖析HILA的每个步骤，以确定FLIZ和HILE如何用来控制HILD功能或稳定性。 2。表征Hild和Hile或Fliz之间相互作用的性质。共免疫沉淀和两种杂交分析将用于表征Hile-Hild和Fliz-Hild相互作用。否定调节的HILD点突变将用于识别依赖Hile或Fliz依赖性调节所需的HILD区域。这些突变体还将使我们能够测试肠侵袭过程中SPI1的Fliz和Hile依赖性调节的作用。 3。确定馈入HILD以控制SPI1 T3SS的信号转导途径。控制SPI1 T3SS表达的已知调节系统将被筛选为通过HILD发挥作用的系统。质谱和两杂交分析将用于识别与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球信号转导的整体理解。 SP1 T3SS的调节是整合宿主环境信号以控制毒力基因表达和对该系统的分析的范例至关重要的范式。