Global regulators converge to orchestrate metabolism, biofilm, and pathogenesis

描述（由申请人提供）：霍乱是一种由摄入霍乱弧菌感染的流行性腹泻疾病。世界卫生组织估计，发展中国家每年有300万至500万霍乱病例，导致大约10万人死亡。虽然疫苗、补液疗法和抗生素在治疗霍乱方面是有效的，但由于地方资源不足，区域、国家和国际反应过于缓慢，人口继续遭受重创。在这里，我们将研究调节霍乱弧菌代谢、生物膜形成和毒力的两个碳水化合物反应性信号转导级联的协调。我们的目标是学习如何操纵碳水化合物线索，将霍乱弧菌转移到低传染性、低毒性的状态。最终，我们希望利用我们的发现设计出易于获得、价格低廉的环境添加剂和饮食调整，以降低传染性和毒性。我们建议研究的两个信号转导级联被称为磷酸烯醇丙酮酸磷酸转移酶（PTS）和碳储存调节（CSR）系统。葡萄糖特异性酶llA （EllAGlc）是一种PTS中间和碳水化合物运输和代谢的中央调节剂，通过与其他蛋白质的直接相互作用来施加其控制。在这里，我们提出了第一个证据，证明EllAGlc直接与MshH （CSR途径的一个组成部分）相互作用，以加速CSR小调控RNA的降解。这激活了毒力和生物膜的形成。EllAGlc还与腺苷酸环化酶（AC）相互作用，增加环AMP的产生，从而抑制毒力和生物膜的形成。根据我们的初步数据，我们假设EllAGlc与这些相反途径的成分的相互作用是反向调节的。在Aim 1中，我们将量化在各种生长条件下EllAGlc与AC和MshH的相互作用。我们将评估EllAGlc及其合作伙伴AC和MshH的转录和翻译调控。与Ethan Garner合作，我们将使用单分子显微镜实时研究EllAGlc及其伙伴在细胞中对环境信号的再分配。我们还将研究EllAGlc与MshH相互作用的结构基础。CsrA是一种mrna结合蛋白，是CSR途径的末端组分。我们假设CsrA与参与霍乱弧菌代谢、生物膜形成和毒力的mRNA靶标结合。在Aim 2中，我们将通过与CsrA共免疫沉淀对CsrA靶点进行全基因组鉴定，并通过高通量测序对沉淀rna进行鉴定。这些靶点将被确认，它们在生物膜形成和毒力中的作用将被研究。我们假设CSR和PTS系统是哺乳动物疾病所必需的。在目标3中，我们将使用新生儿霍乱兔模型来评估PTS和CSR系统在肠道定植、毒力基因转录和腹泻细化中的作用。

DESCRIPTION (provided by applicant): Cholera is an epidemic diarrheal disease contracted by ingestion of the bacterium Vibrio cholerae. The WHO estimates that there are 3-5 million cases of cholera each year in the developing world resulting in approximately 100,000 deaths. While vaccines, rehydration therapy, and antibiotics are effective in the treatment of cholera, populations continue to be devastated because local resources are inadequate and the regional, national, and international responses too slow. Here we will investigate coordination of two carbohydrate-responsive signal transduction cascades that regulate V. cholerae metabolism, biofilm formation, and virulence. Our goal is to learn how to manipulate carbohydrate cues to shift V. cholerae to a low infectivity, low virulence state. Ultimately, we would like to use our findings to design readily available, inexpensive environmental additives and dietary modifications that decrease infectivity and virulence. The two signal transduction cascades we propose to study are known as the phosphoenolpyruvate phosphotransferase (PTS) and carbon storage regulatory (CSR) systems. Glucose-specific Enzyme llA (EllAGlc), a PTS intermediate and central regulator of carbohydrate transport and metabolism, imposes its control through direct interactions with other proteins. Here, we present the first evidence that EllAGlc interacts directly with MshH, a component of the CSR pathway, to accelerate degradation of the small regulatory csr RNA's. This activates virulence and biofilm formation. EllAGlc also interacts with adenylate cyclase (AC) to increase production of cyclic AMP. This results in repression of virulence and biofilm formation. Based on our preliminary data, we hypothesize that interaction of EllAGlc with the components of these opposing pathways is inversely regulated. In Aim 1, we will quantify the interaction of EllAGlc with AC and MshH under a variety of growth conditions. We will assess transcriptional and translational regulation of EllAGlc and its partners AC and MshH. In collaboration with Ethan Garner, we will use single molecule microscopy to study the redistribution of EllAGlc and its partners in the cell in real tim in response to environmental signals. We will also examine the structural basis of the interaction of EllAGlc with MshH. CsrA, an mRNA-binding protein, is the terminal component of the CSR pathway. We hypothesize that CsrA binds to mRNA targets that participate in V. cholerae metabolism, biofilm formation and virulence. In Aim 2, we will undertake genome-wide identification of CsrA targets by co-immunoprecipitation with CsrA and identification of precipitated RNAs by high throughput sequencing. These targets will be confirmed, and their role in biofilm formation and virulence will be investigated. We hypothesize that the CSR and PTS systems are required for mammalian disease. In Aim 3, we will use the neonatal rabbit model of cholera to assess the role of the PTS and CSR systems in intestinal colonization, virulence gene transcription, and elaboration of diarrhea.