Polyamine-dependent regulation of Vibrio cholerae biofilm formation

描述(由申请人提供)：霍乱弧菌是毁灭性的腹泻疾病霍乱的病原体。这种生物也是世界范围内水生环境的自然居住者。霍乱弧菌被认为主要存在于这些环境中，主要存在于被称为生物膜的微生物群落中。生物膜细菌对环境压力，如紫外线辐射、干燥、低pH、抗菌剂和人类宿主的免疫防御具有更强的耐受性。这种耐受性不仅保护霍乱弧菌免受环境恶劣条件的影响，还保护宿主胃的酸性条件。在宿主肠道环境中也有霍乱弧菌生物膜存在的证据，这表明霍乱弧菌可能在人类宿主中使用生物膜形成作为一种长期保护策略。生物膜的发展是由信号网络控制的，信号网络对环境中的化学信号做出反应。我们的长期目标是识别和了解调控霍乱弧菌生物膜形成的信号系统。在这个提案中，我们将注意力集中在一个特定的信号系统上，该系统对多胺、去甲精胺和亚精胺做出反应。这些多胺被认为是环境信号，由蛋白质NSPS和MbaA组成的信号系统检测和处理。NSPS被预测为传感器蛋白或检测这些多胺的系统。MbaA是一种磷酸二酯酶，能分解霍乱弧菌生物膜形成的中枢调节剂--环二鸟苷一磷酸(c-di-GMP)。我们假设去甲精胺和亚精胺直接与NSPS结合，这些结合事件影响NSPS与MbaA的相互作用，从而导致其c-di-GMP磷酸二酯酶活性的变化。这种变化表现为细胞内c-di-GMP水平的增加或减少，从而导致生物被膜形成的相应增加或减少。我们基于以下观察结果提出了这一假说：1)NSPS与大肠杆菌的亚精胺结合蛋白PotD同源；2)在没有NSPS和MbaA的情况下，亚精胺和去甲精胺在细胞外供应时不会影响生物膜的形成；3)MbaA含有一个EAL结构域，在c-di-GMP磷酸二酯酶中发现，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确凿的证据。本项目的具体目标是：1)通过等温滴定量热法进行结合实验，确定去甲精胺和亚精胺是NSPS的配体；2)通过共免疫沉淀实验为NSPS-MbaA相互作用提供证据；3)通过酶分析确认MbaA是一种磷酸二酯酶，并通过生物膜分析将这一活性与其在生物膜形成中的作用联系起来。 公共卫生相关性：霍乱弧菌是毁灭性的腹泻疾病霍乱的病原体。根据世界卫生组织的数据，据估计，全球每年约有10万人死于霍乱。霍乱弧菌可以存在于人类宿主中被称为生物膜的微生物群落中，这可以使这种微生物对宿主防御机制高度耐受，并有可能有助于疾病的发展(Hang等人，2003，Xu等人，2003)。从拟议的项目中获得的洞察力，包括研究霍乱弧菌生物膜的形成，预计将提供对霍乱的更好了解，并产生抗击这种致命疾病的新策略。

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.