Polyamine-dependent regulation of Vibrio cholerae biofilm formation

霍乱弧菌生物膜形成的多胺依赖性调节

• 批准号: 8179979
• 负责人: Ece Karatan
• 金额: $ 39.56万
• 依托单位: APPALACHIAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-17 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8179979
• 关键词: Affect; Antibodies; Attention; Bacteria; Binding; Binding Proteins; Biological Assay; Calorimetry; Cells; Cessation of life; Chemicals; Cholera; Desiccation; Development; Disease; Disease Progression; Engineering; Environment; Enzymes; Epitopes; Event; Genes; Goals; High Pressure Liquid Chromatography; Host Defense Mechanism; Human; Immune; In Vitro; Infection; Integral Membrane Protein; Intestines; Lead; Life Style; Ligands; Mediating; Membrane Proteins; Microbial Biofilms; Modeling; Organism; Oryctolagus cuniculus; Periplasmic Proteins; Polyamines; Process; Protein Family; Protein Sequence Analysis; Proteins; Reaction; Reading Frames; Regulation; Reporting; Role; Signal Transduction; Spermidine; Stomach; Stress; Surface; Survivors; System; Testing; Titrations; Ultraviolet Rays; Vibrio cholerae; Vibrio cholerae O139; Work; World Health Organization; antimicrobial; base; bis(3' ,5' )-cyclic diguanylic acid; carbene; combat; design; extracellular; insight; member; microbial community; mutant; norspermidine; novel strategies; periplasm; phosphoric diester hydrolase; research study; sensor; solute; sugar

DESCRIPTION (provided by applicant): Vibrio cholerae is the causative agent of the devastating diarrheal disease cholera. This organism is also a natural inhabitant of aquatic environments worldwide. V. cholerae is thought to exist in these environments primarily in microbial communities called biofilms. Biofilm bacteria are more tolerant to environmental stresses such as UV radiation, desiccation, low pH, antimicrobials, and the immune defenses of the human host. This tolerance protects V. cholerae not only from the harsh conditions in the environment, but also from the acidic conditions of the host stomach. There is also evidence of V. cholerae biofilms in the host intestinal environment, which suggests that V. cholerae might use biofilm formation as a long-term protective strategy in the human host. Development of biofilms is controlled by signaling networks that respond to chemical signals in the environment. Our long-term goal is to identify and understand the signaling systems that regulate biofilm formation in V. cholerae. In this proposal, we focus our attention on a specific signaling system that responds to the polyamines norspermidine and spermidine. These polyamines are thought to act as environmental signals that are detected and processed by a signaling system composed of the proteins NspS and MbaA. NspS is predicted to be the sensor protein or the system that detects these polyamines. MbaA is predicted to be a phosphodiesterase that breaks down cyclic-diguanylate-monophosphate (c-di-GMP), one of the central modulators of biofilm formation in V. cholerae. We hypothesize that norspermidine and spermidine bind directly to NspS; these binding events affect the interaction of NspS and MbaA, which then results in changes in its c-di-GMP phosphodiesterase activity. This change is manifested as increases or decreases in c-di-GMP levels in the cell that lead to corresponding increases or decreases in biofilm formation. We base this hypothesis on the following observations: 1) NspS is homologous to the spermidine binding protein PotD of Esherichia coli; 2) In the absence of NspS and MbaA, spermidine and norspermidine cannot affect biofilm formation when supplied extracellularly; 3) MbaA contains an EAL domain, found in c-di- GMP phophodiesterases, in which all the residues that contribute to enzymatic activity are conserved. The goal of the proposed project is to provide definitive evidence for this signaling system. The specific aims of this project are to i) Establish that norspermidine and spermidine are ligands for NspS by doing binding experiments using isothermal titration calorimetry; ii) Provide evidence for NspS-MbaA interaction using coimmunoprecipitation experiments; and iii) Confirm that MbaA is a phosphodiesterase by enzyme assays and correlate this activity to its role in biofilm formation using biofilm assays. PUBLIC HEALTH RELEVANCE: Vibrio cholerae is the causative agent of the devastating diarrhoeal disease cholera. According to the World Health Organization, cholera is estimated to result in approximately 100,000 deaths worldwide every year. V. cholerae can exist in the human host in microbial communities called biofilms, which can make this organism highly tolerant to host defense mechanisms and potentially aid in disease progression (Hang et al., 2003, Xu et al., 2003). The insight gained from the proposed project, which involves studying V. cholerae biofilm formation, is expected to provide a better understanding of cholera and yield novel strategies to combat this deadly disease.

描述（由申请人提供）：霍乱弧菌是毁灭性腹泻病霍乱的病原体。这种生物也是全世界水生环境的天然居民。人们认为霍乱弧菌主要存在于这些环境中称为生物膜的微生物群落中。生物膜细菌对环境压力（例如紫外线辐射、干燥、低pH值、抗菌剂和人类宿主的免疫防御）具有更强的耐受性。这种耐受性不仅可以保护霍乱弧菌免受环境中恶劣条件的影响，还可以使其免受宿主胃部酸性条件的影响。还有证据表明宿主肠道环境中存在霍乱弧菌生物膜，这表明霍乱弧菌可能利用生物膜形成作为人类宿主的长期保护策略。生物膜的形成是由响应环境中化学信号的信号网络控制的。我们的长期目标是识别和了解调节霍乱弧菌生物膜形成的信号系统。在本提案中，我们将注意力集中在对多胺去甲亚精胺和亚精胺作出反应的特定信号系统上。这些多胺被认为充当环境信号，由蛋白质 NspS 和 MbaA 组成的信号系统检测和处理。 NspS 预计是传感器蛋白或检测这些多胺的系统。 MbaA 预计是一种磷酸二酯酶，可分解环二鸟苷酸单磷酸 (c-di-GMP)，环二鸟苷酸是霍乱弧菌生物膜形成的主要调节剂之一。我们假设去甲亚精胺和亚精胺直接与 NspS 结合；这些结合事件影响 NspS 和 MbaA 的相互作用，从而导致其 c-di-GMP 磷酸二酯酶活性发生变化。这种变化表现为细胞中 c-di-GMP 水平的增加或减少，从而导致生物膜形成相应增加或减少。我们的这一假设基于以下观察：1）NspS 与大肠杆菌的亚精胺结合蛋白 PotD 同源； 2) 在缺乏NspS和MbaA的情况下，亚精胺和去甲亚精胺在细胞外提供时不能影响生物膜的形成； 3) MbaA 含有一个 EAL 结构域，存在于 c-di-GMP 磷酸二酯酶中，其中所有有助于酶活性的残基都是保守的。拟议项目的目标是为该信号系统提供明确的证据。该项目的具体目标是 i) 通过使用等温滴定量热法进行结合实验，确定去甲亚精胺和亚精胺是 NspS 的配体； ii) 使用免疫共沉淀实验提供 NspS-MbaA 相互作用的证据； iii) 通过酶测定确认 MbaA 是一种磷酸二酯酶，并使用生物膜测定将该活性与其在生物膜形成中的作用相关联。 公共卫生相关性：霍乱弧菌是毁灭性腹泻病霍乱的病原体。据世界卫生组织估计，霍乱每年导致全球约 10 万人死亡。霍乱弧菌可以存在于人类宿主中称为生物膜的微生物群落中，这可以使该生物体对宿主防御机制具有高度耐受性，并可能有助于疾病进展（Hang 等人，2003 年；Xu 等人，2003 年）。该项目涉及研究霍乱弧菌生物膜的形成，从中获得的见解预计将有助于更好地了解霍乱，并产生对抗这种致命疾病的新策略。