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

基本信息

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
项目类别：
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&apos ,5&apos )-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

项目摘要

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）：霍乱是一种因摄入细菌弧菌霍乱而感染的流行腹泻病。世卫组织估计，发展中国家每年有3-5万例霍乱病例，大约有100,000例死亡。尽管疫苗，补液疗法和抗生素有效地治疗霍乱，但由于当地资源不足，地区，国家，国家和国际反应的速度太慢，因此人口仍然受到破坏。在这里，我们将调查两个调节霍乱代谢，生物膜形成和毒力的两个碳水化合物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，以将V.霍乱链球菌转移到低感染率，低毒力状态。最终，我们想利用我们的发现来设计易于使用的，廉价的环境添加剂和饮食修饰，从而降低感染力和毒力。我们建议研究的两个信号转导级联反应称为磷酸烯醇丙酮酸磷酸转移酶（PTS）和碳储存调节（CSR）系统。碳水化合物转运和代谢的PTS中间和中央调节剂的葡萄糖特异性酶LLA（Ellaglc）通过与其他蛋白质的直接相互作用来施加其控制。在这里，我们提供了第一个证据，表明Ellaglc与CSR途径的组成部分MSHH直接相互作用，以加速小型监管CSR RNA的降解。这激活了毒力和生物膜形成。 Ellaglc还与腺苷酸环化酶（AC）相互作用，以增加环状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与这些相对途径的组成部分的相互作用是成反比的。在AIM 1中，我们将在各种生长条件下量化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的转录和翻译调节。通过与Ethan Garner合作，我们将使用单分子显微镜研究Ellaglc及其在细胞中的伴侣的重新分布，以响应环境信号。我们还将检查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共免疫沉淀并通过高吞吐量测序对CSRA靶标的全基因组鉴定。这些靶标将得到确认，并将研究它们在生物膜形成和毒力中的作用。我们假设哺乳动物疾病需要CSR和PTS系统。在AIM 3中，我们将使用霍乱的新生儿兔模型来评估PTS和CSR系统在肠道定植，毒力基因转录和腹泻的阐述中的作用。