Phosphotyrosine-mediated regulation of enterohemorrhagic E. coli virulence

 描述(申请人提供)：肠出血性大肠埃希氏菌(EHEC)O157：H7血清型是食源性血性腹泻暴发和潜在致命溶血性尿毒症综合征的重要原因。EHEC感染的特征是在肠上皮形成附着和消失(A/E)病变。负责这种组织病理学的III型分泌系统(T3SS)是由肠细胞消失(Lee)致病岛编码的。EHEC协调毒力因子的表达以应对环境变化的能力对于它的生存和寄主的成功定植至关重要，这就是为什么了解毒力的调控基础对于鉴定 新的治疗策略。毒力基因的表达，包括李氏杆菌的基因，都受到严格的调控。虽然组氨酸反应调节因子的蛋白磷酸化调节毒力基因的表达是众所周知的，但蛋白质酪氨酸磷酸化在细菌中的调节作用却知之甚少，特别是考虑到磷酸酪氨酸介导的信号在真核生物中的重要作用。利用基于高灵敏质谱学的磷酸蛋白质组学方法，我们最近发现在大肠杆菌中酪氨酸磷酸化蛋白的数量比以前报道的高出约10倍。新发现的蛋白质与细胞的基本功能和毒力有关，表明酪氨酸磷酸化具有中心调节作用。这项拟议的研究旨在进一步阐明磷酸酪氨酸介导的信号在EHEC毒力中的调节作用，重点是CRA，它是被鉴定为酪氨酸磷酸化的蛋白质之一。CRA是一种DNA结合的全球糖酵解代谢产物反应调节因子，控制糖代谢，是Lee在糖异生条件下表达所必需的，以确保成功的定植。鉴于我们的数据表明位于DNA结合区的具有重要功能的CRA Y47残基的磷酸化对T3SS蛋白的产生和分泌产生负面影响，我们假设CRA Y47的磷酸化通过干扰CRA DNA结合能力来负向控制毒力基因的表达。我们还假设，CRA磷酸化发生是对碳源可获得性的响应。以下特定目的针对这些假说：目的1)确定CRA酪氨酸磷酸化是否由糖酵解条件诱导；以及ii)确定磷酸酪氨酸介导的CRA调节的整体调控效果。目的：1)确定酪氨酸磷酸化在CRA介导的T3SS调节中的作用，包括评估CRA酪氨酸磷酸化对Lee表达和A/E损伤形成的影响；以及ii)确定磷酸酪氨酸介导的CRA调节是否影响DNA结合。为了成功地实现这些目标，我们将使用创新的方法，包括RNA-Seq技术和定量高分辨率质谱学方法。增加关于酪氨酸如何调节EHEC毒力的知识有助于确定治疗EHEC的新治疗方法，对于EHEC，抗生素治疗是禁忌的。

 DESCRIPTION (provided by applicant): Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 are an important cause of food-borne outbreaks of bloody diarrhea and the potentially fatal hemolytic uremic syndrome. EHEC infection is characterized by the formation of attaching and effacing (A/E) lesions on the intestinal epithelium. A type III secretion system (T3SS) responsible for this histopathology is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. The ability of EHEC to co-ordinate the expression of virulence factors in response to changing environments is crucial for its survival and successful colonization of the host, which is why understanding of the regulatory basis for virulence is important for identifying new therapeutic strategies. The expression of virulence genes including those of the LEE is tightly regulated. While protein phosphorylation of response regulators on histidine is well-known to regulate virulence gene expression, the regulatory role of protein tyrosine phosphorylation in bacteria is poorly understood, especially considering the prominent role of phosphotyrosine-mediated signaling in eukaryotes. Using a highly sensitive mass spectrometry-based phosphoproteomics approach, we recently found that the number of tyrosine-phosphorylated proteins in E. coli was about 10-fold higher than previously reported. The newly identified proteins relate to fundamental cell functions and virulence, indicating a central regulatory role of tyrosine phosphorylation. The proposed study aims to further elucidate the regulatory role of phosphotyrosine-mediated signaling in EHEC virulence with the focus being on Cra, which was among proteins identified as tyrosine phosphorylated. Cra, a DNA-binding global glycolytic metabolite-responsive regulator controlling sugar metabolism, is required for LEE expression under gluconeogenic conditions to ensure successful colonization. Given our data indicating that phosphorylation of the functionally important Cra Y47 residue, located in the DNA-binding domain, negatively affects the production and secretion of T3SS proteins, we hypothesize that phosphorylation of Cra Y47 negatively controls expression of virulence genes by interfering with Cra DNA-binding capacity. We also hypothesize that Cra phosphorylation occurs in response to carbon source availability. The following Specific Aims address these hypotheses: Aim 1: i) Determine whether Cra tyrosine phosphorylation is induced by glycolytic conditions; and ii) Determine the global regulatory effect of phosphotyrosine-mediated regulation of Cra. Aim 2: i) Determine the role of tyrosine phosphorylation in Cra-mediated regulation of the T3SS including assessing the effect of Cra tyrosine phosphorylation on LEE expression and A/E lesion formation; and ii) Determine if phosphotyrosine-mediated regulation of Cra affects DNA-binding. To successfully address these aims, we will use innovative approaches including the RNA-Seq technology and a quantitative high resolution mass spectrometry approach. Increased knowledge on how tyrosine regulates EHEC virulence can facilitate the identification of novel therapeutic approaches to treat EHEC, for which antibiotic therapy is contraindicated.