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（由申请人提供）：莱姆病螺旋体，伯氏疏螺旋体，利用复杂的地方性生活周期存在于自然界中。该细菌占据节肢动物蜱虫载体和脊椎动物水库，并坚持在这两个非常不同的环境中，细菌经历了显着的适应性变化的全球基因表达。这种适应性反应由增强子结合蛋白Rrp 2调节，该增强子结合蛋白Rrp 2激活RpoN/RpoS交替σ因子级联。虽然Rrp 2在激活RpoN/RpoS通路中的作用是非常确定的，但最近的研究还表明Rrp 2也独立于RpoN/RpoS发挥作用，并且Rrp 2的这个分支调节是B中的活力所必需的。burgdorferi。到目前为止，在这个Rrp 2依赖的，RpoN/Rpos独立的调节分支的基因的身份尚未确定。 具体目标1中的实验将鉴定B中的基因。由Rrp 2调节的burgdorferi。将使用cDNA文库高通量测序（RNA-Seq）和凝胶内消化后液相色谱凝胶串联质谱（GeLC/MS-MS）的互补技术来确定B的总体变化。burgdorferi转录组和蛋白质组。将从rrp 2条件突变体的体外培养物以及可过表达rrp 2的克隆中产生样品。在具体目标2中，在现有技术中鉴定的基因和/或蛋白质 目标将用于诱变和表型分析。定向等位基因交换将用于在野生型B中产生突变体。burgdorferi。因为细菌生长需要Rrp 2依赖性、RpoN/RpoS非依赖性基因调控的这个分支，所以可能需要条件突变方法来产生这些突变体中的一个或多个。B。首先评估伯氏病突变体的体外生长缺陷，但这些突变体中的一种或多种可能不表现出显著的体外缺陷。为了测试哺乳动物中这些后一种突变体的生存能力，我们将表征B。Burgdorferi突变体在小鼠中感染诱发疾病的能力。 从本提案中提出的目的中获得的知识将有助于我们进一步了解B的生物学。burgdorferi。确定这些基因对细菌生存力和/或哺乳动物感染性的生理贡献将是至少一个未来R 01提案的重点，其最终目的是鉴定潜在的细菌感染性。 针对这些目标可以开发新的治疗方法来预防或治疗莱姆病。