Analyzing a novel mechanism of action of bacterial cAMP producing toxins

分析细菌 cAMP 产生毒素的新作用机制

基本信息

批准号：
8998800
负责人：
ETHAN BIER
金额：
$ 53.79万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
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：
2014-02-01 至 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8998800
关键词：
Adenylate Cyclase Anthrax disease Antibiotics Antibodies Asthma Autophagocytosis Bacillus anthracis Binding Biological Biological Models Bioterrorism Blood Circulation Blood Vessels Cadherins Cause of Death Cell Adhesion Molecules Cell membrane Cell physiology Cells Cessation of life Cholera Cholera Toxin Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Dermatitis Diarrhea Disease Docking Drosophila genus Drosophila melanogaster Edema Embryo Endosomes Epithelial Epithelial Cell Junction Epithelial Cells Extravasation Fishes Fluids and Secretions Genetic Genetic Models Grant Health Human Immune Infection Inflammatory Injection of therapeutic agent Intercellular Junctions Intervention Intestines Ions Ischemia Lead Ligands Link Liquid substance Malignant Neoplasms Mediating Medical Monomeric GTP-Binding Proteins Mus National Security Neurodegenerative Disorders Pathogenesis Pathology Pathway interactions Patients Phagocytosis Process Production Proteins Public Health Recycling Resources Sanitation Shock Signal Transduction Signaling Protein System Testing Toxin Translating Vascular Endothelial Cell Vascular Endothelium Vascular Permeabilities Vesicle Vibrio cholerae Virulence Factors Water Zebrafish anthrax edema factor base body system cytokine edema factor effusion fly human disease in vivo instrument intestinal epithelium intestinal homeostasis migration monolayer notch protein novel novel strategies overexpression pandemic disease pathogen prevent programs protein transport rab11 protein small molecule inhibitor trafficking translational approach

项目摘要

DESCRIPTION (provided by applicant): Our recent discovery that key virulence factors from Bacillus anthracis, edema factor (EF), and Vibrio cholerae, cholera toxin (Ctx), both inhibit protein trafficking to cell-cell junctions is paradigm shifting. EF is a highly active adenylate cyclase and Ctx ADP-ribosylates Gs¿ subunits to constitutively activate host adenylate cyclase. These two cAMP producing toxins reduce the levels and activity of a small GTPase (Rab11) required in the final step of endocytic recycling of cell adhesion molecules (e.g., cadherins) and signaling proteins (e.g., Notch components) to cell-cell junctions, resulting in disruption of the vascular endothelium (EF) or intestinal epithelium (Ctx). This novel effect of EF and Ctx was discovered and genetically dissected in the model system Drosophila melanogaster (fruit fly), and these cell biological mechanisms translate to toxin action in human vascular endothelial cells (EF) and intestinal epithelial cells (Ctx) as well as in vivo in mice (both EF and Ctx). Another key finding, with important practical implications, was that over-expression of Rab11 can reverse the junction disrupting effects of EF and Ctx in vivo in flies and in human cells. In the current revised grant, we propose three integrated aims to elucidate the pathways mediating the barrier disruptive actions of EF and Ctx and to analyze the consequences of this novel cell biological mechanism in disease pathogenesis. In Aim 1 we will examine the pathways by which high sustained levels of cAMP produced by EF/Ctx reduce Rab11 protein levels to derail junctional transport and explore new potential functions of EF and Ctx related to inhibition of exocyst function in immune cells. In Aim 2, we will investigate how inhibition of endocytic recycling promotes leakage across human cell monolayers and in the vasculature during anthrax infection. Since vascular collapse is a frequent cause of death in anthrax, we will also determine whether increasing Rab11 levels or treating with known traffic-promoting agents can reverse the vascular leakage caused by EF. In Aim 3, we will similarly examine the contribution of exocyst inhibition to the massive fluid secretion that is pathognomonic of cholera and whether elevating endocytic recycling via genetic or pharmacological means is protective in vivo. The proposed studies have important translational relevance to treating anthrax since toxins can reach critical lethal levels just as patients begin to seek medical intervention, when antibiotics are no longer effective. Thus, treatments based on restoring endocytic recycling could be used in conjunction with existing anti-toxin therapies (e.g., anti-toxin antibodies, small molecule inhibitors) to neutralize toxins already present in the circulation. An advantage of traffic-promoting agents is that they would intervene at the very last step when vascular integrity collapses and other organ systems fail. Such traffic-promoting compounds might also increase the efficacy of fluid replacements to treat cholera and to treat other barrier disruptive diseases including: ischemia, asthma, dermatitis, IBD, cancer, ciliary diseases, and neurodegenerative disorders.

描述（由申请人提供）：我们最近发现炭疽杆菌、水肿因子 (EF) 和霍乱弧菌、霍乱毒素 (Ctx) 的关键毒力因子均抑制蛋白质运输至细胞-细胞连接，这是一种范式转变。高活性腺苷酸环化酶和 Ctx ADP-核糖基化 Gs¿这两种产生 cAMP 的毒素可降低细胞粘附分子（例如钙粘蛋白）和信号蛋白（例如 Notch 成分）内吞回收最后步骤所需的小 GTP 酶 (Rab11) 的水平和活性。 ) 到细胞与细胞的连接，导致血管内皮 (EF) 或肠上皮的破坏EF 和 Ctx 的这种新作用是在模型系统果蝇（果蝇）中发现并进行基因剖析的，这些细胞生物学机制转化为人类血管内皮细胞 (EF) 和肠上皮细胞 (Ctx) 的毒素作用。）以及小鼠体内（EF 和 Ctx）的另一个重要发现，具有重要的实际意义，是 Rab11 的过度表达可以逆转连接破坏效应。在当前修订的资助中，我们提出了三种综合途径来阐明 EF 和 Ctx 介导的屏障破坏作用，并分析这种新的细胞生物学机制在疾病发病机制中的后果。在目标 1 中，我们将研究 EF/Ctx 产生的高持续水平的 cAMP 降低 Rab11 蛋白水平从而使连接运输脱轨的途径，并探索 EF 和 Ctx 与抑制相关的新潜在功能。在目标 2 中，我们将研究内吞再循环的抑制如何促进炭疽感染期间跨人体细胞单层和脉管系统的渗漏，因为血管塌陷是炭疽死亡的常见原因，我们还将确定是否会增加。 Rab11 水平或使用已知的交通促进剂治疗可以逆转 EF 引起的血管渗漏。在目标 3 中，我们将类似地检查外囊抑制对大量液体的贡献。霍乱特有的分泌物，以及通过遗传或药理学手段提高内吞循环是否具有体内保护作用，所提出的研究对于治疗炭疽具有重要的转化意义，因为当患者开始寻求医疗干预时，当抗生素使用时，毒素可能达到临界致命水平。因此，基于恢复内吞再循环的治疗可以与现有的抗毒素疗法（例如抗毒素抗体、小分子抑制剂）结合使用来中和。交通促进剂的一个优点是，当血管完整性崩溃和其他器官系统衰竭时，它们会在最后一步进行干预，这种促进交通的化合物也可能会提高液体替代品治疗霍乱的功效。并治疗其他屏障破坏性疾病，包括：缺血、哮喘、皮炎、炎症性肠病、癌症、纤毛疾病和神经退行性疾病。