PlzA, cyclic-di-GMP and the enzootic cycle for Lyme disease

项目摘要：莱姆病是北方地区最常见的壁虱传播疾病 而且它的发病率正在稳步上升。莱姆病螺旋体的致病能力 要适应不断变化的环境条件，在一定程度上取决于由 第二信使分子，环-二-GMP(c-di-GMP)。这份提案将确定关键的 PlzA的功能决定因素，PlzA是唯一被发现的由所有莱姆产生的c-di-GMP结合蛋白 疾病螺旋体分离株。根据最近测定的Plza的原子结构， PlzA属于c-di-GMP结合蛋白的xPilZ结构域， 数据显示Plza有RNA伴侣活动，我们将进行全面的 分析蛋白质的载脂蛋白和全息蛋白的结构-功能关系。 根据比较序列和结构分析，表面暴露的氨基酸残基 将被作为定点突变的目标。这些突变对Plza结构的影响， C-di-GMP结合、RNA缠绕、RNA解旋和蛋白质-蛋白质相互作用 在体外进行评估。在这些分析的基础上，获得了一系列伯氏杆菌转基因菌株 野生型Plza被编码Plza蛋白的Plza基因取代，活性将发生变化 已生成。每种毒株感染扁虱和哺乳动物的能力将是 下定决心。这些分析将通过以下方式定义功能域和生物学机制 哪种apo和holo plza调节完成地方病所需的细胞过程？ 周而复始。

Project summary: Lyme disease is the most common tick-borne disease in the northern hemisphere and its incidence is steadily increasing. The ability of the Lyme disease spirochetes to adapt to changing environmental conditions is dependent in part on regulation mediated by the secondary messenger molecule, cyclic-di-GMP (c-di-GMP). This proposal will identify the critical functional determinants of PlzA, the sole identified c-di-GMP binding protein produced by all Lyme disease spirochete isolates. Based on the recently determined atomic structure of PlzA which revealed that PlzA belongs to the unique xPilZ domain class of c-di-GMP binding proteins and data demonstrating that PlzA has RNA chaperone activities, we will perform a comprehensive analysis to dissect structure-function relationships of both apo and holo forms of the protein. Based on comparative sequence and structural analyses, surface-exposed amino acid residues will be targeted for site-directed mutagenesis. The impact of these mutations on PlzA structure, c-di-GMP binding, RNA winding, RNA unwinding, and protein-protein interactions will be assessed in vitro. Based on these analyses, a series of B. burgdorferi transgenic strains in which wild-type plzA is replaced with plzA genes that encode PlzA proteins with altered activity will be generated. The ability of each strain to infect and transmit between ticks and mammals will be determined. These analyses will define the functional domains and biological mechanisms by which apo and holo PlzA regulate cellular processes required for the completion of the enzootic cycle.