Global regulators converge to orchestrate metabolism, biofilm, and pathogenesis

全球监管机构齐心协力协调代谢、生物膜和发病机制

• 批准号: 8748584
• 负责人: PAULA I WATNICK
• 金额: $ 47.07万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8748584
• 关键词: Adenylate Cyclase; Affect; Antibiotic Therapy; Area; Bacteria; Bacterial Physiology; Behavior; Binding; Binding Proteins; Carbohydrates; Carbon; Cell Density; Cells; Cessation of life; Cholera; Co-Immunoprecipitations; Collaborations; Complex; Contracts; Cues; Cyclic AMP; Cyclic AMP Receptor Protein; Data; Diarrhea; Diet; Diet Modification; Dietary Intervention; Disease; Enzymes; Epidemic; Escherichia coli; Gene Expression; Genetic Transcription; Glucose; Goals; Growth; Haiti; High-Throughput Nucleotide Sequencing; Image; Immunoprecipitation; Infant; Ingestion; International; Intervention; Intestines; Laboratories; Learning; Life; Mediating; Medical; Messenger RNA; Metabolism; Microbial Biofilms; Microscopy; Modeling; Modification; Mono-S; Neonatal; Nutrient; Oryctolagus cuniculus; Pathogenesis; Pathway interactions; Phenotype; Phosphoenolpyruvate; Phosphorylation; Phosphotransferases; Play; Population; Production; Proteins; Regulation; Regulatory Pathway; Rehydrations; Repression; Resources; Risk; Role; Sanitation; Second Messenger Systems; Signal Transduction; Spatial Distribution; Surface; System; Testing; Time; Transcriptional Regulation; Translational Regulation; Vaccines; Vibrio cholerae; Virulence; Water; base; bis(3' ,5' )-cyclic diguanylic acid; carbohydrate metabolism; carbohydrate transport; design; drinking water; genetic regulatory protein; genome wide association study; in vivo; mortality; mutant; pathogen; public health relevance; quorum sensing; research study; response; second messenger; single molecule; sugar; yeast two hybrid system

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.

描述（由申请人提供）：霍乱是一种因摄入霍乱弧菌而感染的流行性腹泻病。世界卫生组织估计，发展中国家每年有 3-500 万例霍乱病例，导致约 10 万人死亡。虽然疫苗、补液疗法和抗生素可以有效治疗霍乱，但由于当地资源不足以及区域、国家和国际反应过于缓慢，人们继续遭受破坏。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响应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变为低传染性、低毒力状态。最终，我们希望利用我们的发现来设计容易获得、廉价的环境添加剂和饮食调整，以降低传染性和毒力。 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 IIA (EllAGlc) 是一种 PTS 中间体，也是碳水化合物转运和代谢的中心调节剂，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 CSR 途径的一个组成部分 MshH 相互作用，以加速小调节 csr RNA 的降解。这会激活毒力和生物膜的形成。 EllAGlc 还与腺苷酸环化酶 (AC) 相互作用以增加环 AMP 的产生。这导致毒力和生物膜形成的抑制。 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受到反向调节。在目标 1 中，我们将量化各种生长条件下 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。我们将与 Ethan Garner 合作，利用单分子显微镜实时研究 EllAGlc 及其伙伴在细胞中响应环境信号的重新分布。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 途径的末端成分。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。在目标 2 中，我们将通过与 CsrA 免疫共沉淀对 CsrA 靶标进行全基因组鉴定，并通过高通量测序鉴定沉淀的 RNA。这些目标将得到确认，并研究它们在生物膜形成和毒力中的作用。 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，我们将使用新生兔霍乱模型来评估 PTS 和 CSR 系统在肠道定植、毒力基因转录和腹泻发生过程中的作用。