Rrp2-dependent gene regulation in Borrelia burgdorferi

伯氏疏螺旋体中 Rrp2 依赖性基因调控

• 批准号: 8951367
• 负责人: Jon Scott Blevins
• 金额: $ 21.99万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8951367
• 关键词: Arthropods; Automobile Driving; Bacteria; Bacteria sigma factor KatF protein; Biology; Borrelia burgdorferi; Complex; Data; Defect; Digestion; Disease; Environment; Europe; Exhibits; Future; Gel; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Proteins; Genes; Genetic Transcription; Global Change; Growth; High-Throughput Nucleotide Sequencing; In Vitro; Individual; Infection; Intention; Knowledge; Life; Life Cycle Stages; Liquid Chromatography; Lyme Disease; Mammals; Molecular; Mus; Mutagenesis; Mutation; Nature; Parents; Pathway interactions; Phenotype; Physiological; Proteins; Proteome; RNA; Regulation; Regulon; Role; Sampling; Sigma Factor; Techniques; Testing; Therapeutic Intervention; Ticks; Time; Transactivation; United States; Vector-transmitted infectious disease; Work; cDNA Library; enhancer binding protein; enzootic; insight; interest; mutant; novel; novel therapeutics; overexpression; prevent; public health relevance; research study; response; tandem mass spectrometry; transcriptome sequencing; vector

 DESCRIPTION (provided by applicant): The Lyme disease spirochete, Borrelia burgdorferi, utilizes a complex enzootic life cycle to exist in nature. The bacterium occupies both an arthropod tick vector and a vertebrate reservoir, and to persist in these two very distinct environments, the bacterium undergoes significant adaptive changes in global gene expression. This adaptive response is regulated by an enhancer-binding protein, Rrp2, which activates the RpoN/RpoS alternative sigma factor cascade. While the role of Rrp2 in activating the RpoN/RpoS pathway is very well-established, a recent study has also shown that Rrp2 also functions independent of RpoN/RpoS and that this branch of Rrp2 regulation is required for viability in B. burgdorferi. To date, the identities of the genes within this Rrp2-dependent, RpoN/RpoS-independent regulatory branch have not been determined. Experiments in Specific Aim 1 will identify genes in B. burgdorferi that are regulated by Rrp2. The complementary techniques of high-throughput sequencing of cDNA libraries (RNA-Seq) and in-gel digestion followed by liquid chromatography gel-tandem mass spectrometry (GeLC/MS-MS) will be used to define global changes in the B. burgdorferi transcriptome and proteome, respectively. Samples will be generated from in vitro grown cultures of an rrp2 conditional mutant, as well as a clone that can overexpress rrp2. In Specific Aim 2, genes and/or proteins identified in the prior Aim will be targeted for mutagenesis and phenotypic analyses. Directed allelic exchange will be used to create mutants in wild type B. burgdorferi. Because this branch of Rrp2-dependent, RpoN/RpoS-independent gene regulation is required for bacterial growth, a conditional mutation approach might be required to generate one or more of these mutants. B. burgdorferi mutants will first be assessed for in vitro growth defects, but it is possible that one or more of these mutants might not exhibit a significant in vitro defect. To test the viability of these latter mutats within the mammal, we will characterize the B. burgdorferi mutants for their abilities to infect an elicit disease in mice. The knowledge gained from the Aims presented in this proposal will help further our understanding of the biology of B. burgdorferi. Defining the physiological contributions of these genes to bacterial viability and/or mammalian infectivity will be the focus of at least one future R01 proposal, with the ultimate intention of identifying potential bacterial targets against which new therapeutics could be developed to prevent or treat Lyme disease.

 描述（由适用提供）：莱姆病螺旋体，Borrelia burgdorferi，利用自然存在的复杂的enzootic生命周期。该细菌既占据了节肢动物tick矢量，又占据脊椎动物的晶状体，并且在这两个非常不同的环境中持续存在，细菌在全球基因表达中经历了显着的适应性变化。这种自适应反应受增强剂结合蛋白RRP2的调节，该蛋白激活RPON/RPOS替代Sigma因子级联。虽然RRP2在激活RPON/RPOS途径中的作用非常完善，但最近的一项研究还表明，RRP2还与RPON/RPOS无关，并且RRP2调节的这个分支在B. burgdorferi中需要可行性。迄今为止，尚未确定此RRP2依赖性RPON/RPOS独立的调节分支中基因的身份。特定目标1中的实验将鉴定由RRP2调节的伯氏芽孢杆菌中的基因。 cDNA库（RNA-SEQ）和凝胶消化的高通量测序的完整技术，然后使用液相色谱凝胶倾斜度质谱法（GELC/MS-MS）分别定义B. burgdorferi转录组和蛋白质组的全局变化。样品将是由RRP2条件突变体的体外种植培养物以及可以过表达RRP2的克隆产生的。在特定目标2中，在先前确定的基因和/或蛋白质 目标将针对诱变和表型分析。定向的Allic Exchange将用于在B. Burgdorferi野生型中创建突变体。由于细菌生长需要RRP2依赖性，RPON/RPOS独立的基因调节的分支，因此可能需要采用有条件的突变方法来产生其中一个或多个这些突变体。 B. burgdorferi突变体将首先评估为体外生长缺陷，但是这些突变体中的一个或多个可能以测试这些后来的突变体在哺乳动物内的生存能力，我们将表征伯格多代芽孢杆菌突变体的能力来感染小鼠引起疾病的能力。从本提案中提出的目标中获得的知识将有助于我们进一步理解B. burgdorferi的生物学。定义这些基因对细菌生存能力和/或哺乳动物感染的物理贡献将是至少一个将来的R01提案的重点，最终的意图是确定潜在的细菌 可以开发新疗法以预防或治疗莱姆病的靶标。