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(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在自然界中存在。这种细菌既占据了节肢动物扁虱的媒介，也占据了脊椎动物的储藏库，为了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方面经历了显著的适应性变化。这种适应性反应由增强子结合蛋白RRP2调节，RRP2激活RpoN/rpos替代sigma因子级联反应。虽然RRP2在激活RpoN/rpos途径中的作用已被证实，但最近的一项研究也表明，RRP2也独立于RpoN/rpos发挥作用，并且RRP2调控的这一分支是伯氏杆菌生存所必需的。到目前为止，这个依赖RRP2、RpoN/rpos独立的调控分支中的基因的身份还没有确定。特定目标1的实验将确定伯氏杆菌中受RRP2调控的基因。高通量文库测序(RNA-Seq)和凝胶内消化-高效液相色谱-串联质谱仪(GeLC/MS-MS)的互补技术将分别用于确定伯氏杆菌转录组和蛋白质组的全球变化。样本将来自RRP2条件突变的体外培养物，以及能够过表达RRP2的克隆。在特定目标2中，先前 AIM将针对突变和表型分析。定向等位基因交换将被用于在野生型Burgdorferi中创造突变体。由于细菌生长需要依赖于RRP2、RpoN/rpos的这一分支基因调控，因此可能需要一种条件突变方法来产生一个或多个这些突变体。伯氏杆菌突变体将首先接受体外生长缺陷的评估，但这些突变体中的一个或多个可能不会表现出显著的体外缺陷。为了测试这些后一种突变在哺乳动物中的生存能力，我们将表征伯氏杆菌突变体在小鼠中感染一种诱发疾病的能力。从这项提案中提出的目标所获得的知识将有助于我们进一步了解伯氏杆菌的生物学。确定这些基因对细菌活性和/或哺乳动物感染性的生理贡献将是至少一个未来R01提案的重点，最终目的是识别潜在的细菌。 可针对其开发新疗法以预防或治疗莱姆病的靶点。