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: 9090056
• 负责人: Jon Scott Blevins
• 金额: $ 18.27万
• 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 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9090056
• 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 Regulation; Gene Proteins; Genes; Genetic Transcription; Global Change; Growth; Health; High-Throughput Nucleotide Sequencing; In Vitro; Individual; Infection; Intention; Knowledge; Life; Life Cycle Stages; Liquid Chromatography; Lyme Disease; Mammals; Molecular; Mus; Mutagenesis; 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; conditional mutant; enhancer binding protein; enzootic; insight; interest; mutant; novel; novel therapeutics; overexpression; prevent; research study; response; tandem mass spectrometry; targeted treatment; transcriptome; 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命周期在自然界中存在。该细菌占据节肢动物蜱载体和脊椎动物库，并且为了在这两种截然不同的环境中持续存在，该细菌在整体基因表达方面经历了显着的适应性变化。这种适应性反应由增强子结合蛋白 Rrp2 调节，该蛋白激活 RpoN/RpoS 替代 sigma 因子级联。虽然 Rrp2 在激活 RpoN/RpoS 途径中的作用已非常明确，但最近的一项研究还表明，Rrp2 的功能也独立于 RpoN/RpoS，并且 Rrp2 调节的这一分支是博氏疏螺旋体的生存能力所必需的。迄今为止，该 Rrp2 依赖性、RpoN/RpoS 独立调节分支内的基因身份尚未确定。 具体目标 1 中的实验将鉴定伯氏疏螺旋体中受 Rrp2 调节的基因。 cDNA 文库高通量测序 (RNA-Seq) 和凝胶内消化以及液相色谱凝胶串联质谱 (GeLC/MS-MS) 的互补技术将分别用于定义伯氏疏螺旋体转录组和蛋白质组的整体变化。样品将从 rrp2 条件突变体以及可过度表达 rrp2 的克隆的体外培养物中产生。在具体目标 2 中，先前鉴定的基因和/或蛋白质 目标将针对诱变和表型分析。定向等位基因交换将用于在野生型伯氏疏螺旋体中产生突变体。由于细菌生长需要依赖 Rrp2、不依赖 RpoN/RpoS 的基因调控这一分支，因此可能需要条件突变方法来生成这些突变体中的一个或多个。首先将评估伯氏疏螺旋体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会表现出显着的体外缺陷。为了测试后面这些突变体在哺乳动物体内的生存能力，我们将表征伯氏疏螺旋体突变体感染小鼠引发疾病的能力。 从本提案提出的目标中获得的知识将有助于进一步了解伯氏疏螺旋体的生物学。定义这些基因对细菌活力和/或哺乳动物感染性的生理贡献将是未来至少一项 R01 提案的重点，最终目的是识别潜在的细菌 可以开发新疗法来预防或治疗莱姆病的目标。