Phosphotyrosine-mediated regulation of enterohemorrhagic E. coli virulence

 描述（由申请方提供）：肠出血性大肠杆菌（EHEC）血清型O 157：H7是食源性出血性腹泻和潜在致命性溶血性尿毒综合征暴发的重要原因。肠出血性大肠杆菌感染的特征是在肠上皮上形成附着和消退（A/E）病变。负责该组织病理学的III型分泌系统（T3 SS）由肠上皮细胞消失（LEE）致病性岛的位点编码。肠出血性大肠杆菌能够协调毒力因子的表达以应对不断变化的环境，这对它的生存和宿主的成功定殖至关重要，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。包括LEE在内的毒力基因的表达受到严格调控。虽然组氨酸上的反应调节剂的蛋白磷酸化是众所周知的调节毒力基因表达，蛋白酪氨酸磷酸化在细菌中的调节作用知之甚少，特别是考虑到磷酸酪氨酸介导的信号转导在真核生物中的突出作用。利用高灵敏度的基于质谱的磷酸化蛋白质组学方法，我们最近发现大肠杆菌中酪氨酸磷酸化蛋白质的数量与大肠杆菌中酪氨酸磷酸化蛋白质的数量相比有显著性差异。大肠杆菌的表达量比以前报道的高出约10倍。新鉴定的蛋白质与基本细胞功能和毒力有关，表明酪氨酸磷酸化的中心调节作用。该研究旨在进一步阐明磷酸酪氨酸介导的信号传导在EHEC毒力中的调节作用，重点是Cra，这是被鉴定为酪氨酸磷酸化的蛋白质之一。Cra是一种控制糖代谢的DNA结合全局糖酵解代谢物响应调节剂，在促凝条件下LEE表达需要Cra以确保成功定殖。鉴于我们的数据表明位于DNA结合结构域的功能重要的Cra Y 47残基的磷酸化对T3 SS蛋白的产生和分泌产生负面影响，我们假设Cra Y 47的磷酸化通过干扰Cra DNA结合能力来负面控制毒力基因的表达。我们还假设，Cra磷酸化发生在响应碳源的可用性。以下具体目的解决这些假设：目的1：i）确定Cra酪氨酸磷酸化是否由糖酵解条件诱导;和ii）确定磷酸酪氨酸介导的Cra调节的整体调节作用。目标二：i）确定酪氨酸磷酸化在Cra介导的T3 SS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;和ii）确定Cra的磷酸酪氨酸介导的调节是否影响DNA结合。为了成功实现这些目标，我们将使用创新方法，包括RNA-Seq技术和定量高分辨率质谱方法。对酪氨酸如何调节肠出血性大肠杆菌毒力的了解的增加有助于确定治疗肠出血性大肠杆菌的新治疗方法，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.