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

PlzA、环二 GMP 和莱姆病的地方性循环

• 批准号: 10608622
• 负责人: Christopher Davies
• 金额: $ 74.6万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-18 至 2027-10-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10608622
• 关键词: Alleles; Amino Acids; Bacteria; Binding; Binding Proteins; Binding Sites; Biological; Biological Assay; Biological Process; Biopsy; Borrelia burgdorferi; CISH gene; Cell physiology; Chemotaxis; Co-Immunoprecipitations; DNA; Data; Electrophoretic Mobility Shift Assay; Environment; Gene Expression; Genes; Genome; Growth; Hand; In Vitro; Incidence; Infection; Ixodes; Ligand Binding; Ligands; Lyme Disease; Mammals; Mediating; Midgut; Molecular; Molecular Chaperones; Morphology; Mus; Mutate; Mutation; Mutation Analysis; Nature; Northern Blotting; Organ; Outcome; Outcome Study; Periodicity; Plasmids; Process; Property; Proteins; Public Health; RNA; RNA Binding; RNA annealing; RNA immunoprecipitation sequencing; Regulation; Regulator Genes; Sequence Analysis; Series; Site; Site-Directed Mutagenesis; Structure; Structure-Activity Relationship; Surface; Surface Plasmon Resonance; Tertiary Protein Structure; Testing; Tick-Borne Diseases; Ticks; Tissues; Transgenic Organisms; Translations; Validation; Work; cell motility; comparative; diguanylate cyclase; environmental change; enzootic; gene replacement; lyme pathogenesis; mutant; overexpression; posttranscriptional; protein protein interaction; seroconversion; transmission process

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.

项目概述：莱姆病是北方最常见的蜱传疾病 其发病率正在稳步上升。莱姆病螺旋体的能力 适应不断变化的环境条件部分取决于调节介导的 第二信使分子，环二GMP（c-di-GMP）。该提案将确定关键的 PlzA的功能决定簇，唯一确定的c-di-GMP结合蛋白产生的所有莱姆 疾病螺旋体分离物。基于最近确定的PlzA的原子结构， 表明PlzA属于c-di-GMP结合蛋白的独特xPilZ结构域类别， 数据表明PlzA具有RNA分子伴侣活性，我们将进行全面的 分析以剖析蛋白质的apo和holo形式两者的结构-功能关系。 基于比较序列和结构分析， 将作为定点诱变的靶点。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-蛋白质相互作用将被 体外评估。在此基础上，提出了一系列的B.其中 野生型plzA被plzA基因取代，该基因编码活性改变的PlzA蛋白 生成的.每种菌株在蜱和哺乳动物之间感染和传播的能力将被评估。 测定这些分析将定义功能域和生物学机制， apo和holo PlzA调节完成地方性流行病所需的细胞过程， 周期