Salmonella Invasion Gene Regulation

描述(申请人提供)：致病性沙门氏菌菌株是全世界传染病的重要原因。这种细菌拥有一种入侵相关的III型分泌系统，编码在沙门氏菌致病岛L上，主要功能是将效应蛋白转运到宿主细胞的胞浆中。这些分泌的效应蛋白具有诱导细菌进入肠道细胞的细胞活性。此外，效应蛋白具有诱导小肠内液体分泌和炎症的活性，这些都是小肠结肠炎的特征。III型分泌系统和相关效应蛋白的表达依赖于Hila转录激活因子，Hila转录激活因子是OmpR-ToxR转录激活因子家族的成员。Hila基因的过度表达导致了高侵袭性表型，而鼠伤寒沙门氏菌突变体对组织培养细胞的侵袭能力比野生型菌株低500倍，它们侵袭小鼠Peyer‘s斑块M细胞的能力显著减弱，对小鼠的口服LDs0值大约是亲本菌株的60倍。Hila的表达(以及Hila激活的III型分泌系统)受到许多环境信号(渗透压、pH、氧气、生长状态)和遗传元件(HilC、Hild、Sira/Bara、Fliz、PhoBR、FADD、envZ、PhoPQ、Fis、Rose、PAg和胆汁)的调节。我们的研究主要集中在识别和表征Hila表达的调控因子。最近，我们发现FIMZ反应调节因子激活了一个hile-lacZY报告基因。后来的工作证明，PhoPQ双组分调节系统和PhoBR双组分系统都使用hile/fimZ调控途径来抑制Hila的表达。因此，人们提出了各种实验来详细描述这些相互作用。此外，我们建议识别由新的重要的FimZ反应调节因子调控的基因，以及使用FimZ来处理来自环境的信号的基因。最后，先前报道的PAG抑制基因已被证明具有调节组织培养细胞内侵袭转录激活因子Hila的能力。还提出了实验来详细地表征这种阻遏因子。 为了确定这些调节因子影响Hila表达及其相关毒力表型的机制，提出了以下具体目标： 1)鉴定和表征将环境信号转化为入侵基因表达变化的信号通路。 2)FimZ调节子的特征。 3)pAg基因的鉴定、鉴定及其在细胞内Hla表达中的作用。 该提案的总体目标是了解控制SPI-1 hiIA激活物表达的环境信号和调控机制，以及HILA控制的毒力机制，包括侵袭和胃肠炎。对这一调控系统的了解将有助于更好地了解这种病原体与宿主之间的复杂相互作用，并可能导致识别新的治疗靶点来控制沙门氏菌病。

DESCRIPTION (provided by applicant): Pathogenic Salmonella strains are an important cause of infectious disease throughout the world. The bacteria possess an invasion-associated type III secretion system, encoded on Salmonella pathogenicity island l, that functions primarily to translocate effector proteins into the cytosol of host cells. These secreted effector proteins have cellular activities that induce uptake of the bacteria into intestinal cells. In addition, the effector proteins have activities that induce fluid secretion and inflammation within the small intestine that are hallmarks of enterocolitis. The expression of the type III secretion system and the associated effector proteins is dependent upon the HilA transcriptional activator, which is a member of the OmpR-ToxR family of transcriptional activators. Overexpression of the hilA gene results in a hyperinvasive phenotype while S. typhimurium hilA mutants are up to 500-fold less invasive than wild type strains for tissue culture cells, are significantly impaired in their ability to invade M cells of murine Peyer's patches and have oral LDs0 values for mice approximately 60-fold higher than the parent strain. The expression of hilA (and the type III secretion system that hilA activates) is regulated by a number of environmental signals (osmolarity, pH, oxygen, growth state) and genetic elements (hilC, hilD, sirA/barA, fliZ, phoBR, fadD, envZ, phoPQ, fis, arosE, pag and bilE). Our studies have focused on identifying and characterizing regulators of hilA expression. Recently, we found the fimZ response regulator activates a hilE-lacZY reporter. Subsequent work demonstrated that both the phoPQ two component regulator system and the phoBR two component system use the hilE/fimZ regulatory pathway to repress hilA expression. As a result, a variety of experiments are proposed to characterize those interactions in detail. In addition, we propose to identify genes regulated by the newly important FimZ response regulator, as well as genes that use FimZ to process signals from the environment. Finally, a previously reported pag repressor gene has been shown to have the ability to regulate the invasion transcriptional activator hilA within tissue culture cells. Experiments are proposed to characterize this repressor in detail as well. To determine the mechanisms by which these regulators affect hilA expression and its associated virulence phenotypes the following specific aims are proposed: 1) Identification and characterization of the signaling pathways that translate environmental signals into alterations in invasion gene expression. 2) Characterization of the FimZ regulon. 3) Identification and characterization of the pag gene and its role in intracellular hilA expression. The overall goal of this proposal is to understand the environmental signals and regulatory mechanisms that control expression of the SPI-1 hiIA activator, and the virulence mechanisms that hilA controls including invasion and gastroenteritis. An understanding of this regulatory system will lead to a better understanding of the complex interactions between this pathogen and the host and may lead to the identification of new therapeutic targets to control salmonellosis.