Polyamine-dependent regulation of Vibrio cholerae biofilm formation

描述（由申请人提供）：霍乱弧菌是毁灭性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民。霍乱弧菌被认为主要存在于这些环境中称为生物膜的微生物群落中。生物膜细菌更能耐受环境压力，如紫外线辐射、干燥、低pH值、抗菌剂和人类宿主的免疫防御。这种耐受性不仅可以保护霍乱弧菌免受恶劣环境的影响，还可以保护其免受宿主胃的酸性条件的影响。也有证据表明霍乱弧菌在宿主肠道环境中存在生物膜，这表明霍乱弧菌可能将生物膜的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理解调节霍乱弧菌生物膜形成的信号系统。在这个建议中，我们将注意力集中在一个特定的信号系统，响应多胺去亚精胺和亚精胺。这些多胺被认为是由蛋白质NspS和MbaA组成的信号系统检测和处理的环境信号。NspS被预测为传感器蛋白或检测这些多胺的系统。据预测，MbaA是一种磷酸二酯酶，它可以分解环二胍-单磷酸（c-di-GMP），而环二胍-单磷酸是霍乱弧菌生物膜形成的主要调节剂之一。我们假设去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-二gmp磷酸二酯酶活性的变化。这种变化表现为细胞中c-二gmp水平的增加或减少，从而导致相应的生物膜形成的增加或减少。该假设基于以下观察结果：1)NspS与大肠杆菌亚精胺结合蛋白pod同源；2)在没有NspS和MbaA的情况下，亚精胺和去精胺在细胞外供给时不能影响生物膜的形成；3) MbaA含有一个EAL结构域，该结构域存在于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这个信号系统提供明确的证据。该项目的具体目的是：i)通过等温滴定量热法进行结合实验，确定去亚精胺和亚精胺是NspS的配体；ii)通过共免疫沉淀实验提供NspS-MbaA相互作用的证据；iii)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种活性与其在生物膜形成中的作用联系起来。

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.