Mutational Analysis of Putative Genetic Elements Required for Vmp Regulated Expression and Antigenic Variation by the Relapsing Fever Agent, Borrelia hermsii

回归热病原赫氏疏螺旋体 Vmp 调节表达和抗原变异所需的推定遗传元件的突变分析

• 批准号: 10473671
• 负责人: Troy Michael Bankhead
• 金额: $ 22.95万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-24 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10473671
• 关键词: Address; Alleles; Animals; Antigenic Switching; Antigenic Variation; Area; Attenuated; Blood specimen; Borrelia; Characteristics; Clinical; Complement; DNA; DNA Sequence; Disease; Elements; Event; Fever; Future; Gene Conversion; Gene Expression; Genetic; Genetic Recombination; Genetic Transcription; Goals; Headache; Health; Human; Immune Evasion; Immune response; Immunocompetent; Individual; Infection; Interruption; Inverted Repeat Sequences; Investigation; Knowledge; Length; Mediating; Membrane Proteins; Messenger RNA; Mission; Monitor; Mus; Musculoskeletal Pain; Mutation; Mutation Analysis; Nausea; Outcome; Pathogenesis; Process; Promoter Regions; Proteins; Public Health; Publishing; Quantitative Reverse Transcriptase PCR; Recurrence; Regulation; Relapse; Relapsing Fever; Research; Role; Sequence Homology; Series; Structure; Symptoms; System; Techniques; Testing; Tick-Borne Relapsing Fever; Time; Transcript; Translations; United States; United States National Institutes of Health; Variant; Western Blotting; Work; base; cis acting element; disability; genetic element; genetic manipulation; human pathogen; mutant; pathogen; promoter; protein expression; relapsing fever borrelia; telomere; time interval; tool

Summary Borrelia hermsii is a causative agent of tick-borne relapsing fever, which is a bacterial illness characterized by severe recurrent febrile episodes. The undulating fever that is characteristic of the disease is a direct result of Vmp antigenic variation and sequential immune evasion by the pathogen. Recent work in our lab has provided evidence that a region upstream from the vmp locus may be involved in the regulation of Vmp expression, and that a mutant clone deficient in vmp expression was unable to undergo vmp recombination. Additionally, a mutant clone lacking an inverted DNA repeat residing in a downstream homology region did not exhibit Vmp antigenic variation. Despite these advancements, the exact identity and role of cis-acting genetic elements in vmp regulated expression and recombination remain unknown. The objective of our application will be to identify cis-acting DNA regions of the vmp locus that are important for vmp regulated expression and recombination. Based on published and preliminary findings, our central hypothesis is that the tandem inverted repeat sequences are involved in the regulated expression of vmp, and that vmp transcription is required for the gene conversion process. Additionally, we hypothesize that it is the secondary hairpin structure of the DHS- resident inverted repeat sequence that is essential for vmp recombination. The rationale for addressing these knowledge gaps is that the resulting outcomes will lay the groundwork for additional investigations aimed at disrupting the antigenic variation system that underpins the pathogenesis of disease. Thus, the proposed research is relevant to that part of NIH’s mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness and disability. Our central hypothesis will be tested by pursuing three specific aims: 1) Determine the role of the tandem inverted repeat sequences for regulated expression of vmp, 2) Determine the importance of vmp expression for gene conversion, and 3) Establish the requirement of the DHS-resident inverted repeat structure for vmp recombination. Under the first aim, individual inverted DNA repeats will be deleted using our telomere-mediated deletion/complementation technique. Transcription and translation of vmp from mutant clones or an isogenic wild type control will be determined via qRT-PCR and Western blot analysis, respectively. Additionally, mRNA transcript levels will be quantified from blood samples obtained from infected mice at varying time intervals and compared to those from the wild type control. Under the second aim, mutations will be generated within the vmp promoter region and used to infect mice to look for a loss of antigenic switching compared to a wild type control. In the third aim, the inverted repeat within the DHS will be interrupted or DNA sequence altered while retaining the overall sequence length. Antigenic variation compared to the wild type control will be monitored after infecting immunocompetent mice. Overall, our experimental approach will allow for direct mutational analysis of putative cis-acting elements to gain further understanding of this immune evasion system of B. hermsii.

概括 赫氏疏螺旋体是蜱传回归热的病原体，这是一种细菌性疾病 该病的特征是严重的反复发热。 Vmp 抗原变异和病原体连续免疫逃避的直接结果。 已提供证据表明 vmp 位点上游的区域可能参与 Vmp 的调节 表达，并且缺乏 vmp 表达的突变克隆无法进行 vmp 重组。 此外，下游同源区缺乏反向 DNA 重复序列的突变克隆不会 尽管有这些进步，顺式作用遗传的确切身份和作用仍然存在。 vmp 调节表达和重组中的元件仍然未知，我们的应用目标将是未知的。 鉴定对 vmp 调节表达很重要的 vmp 基因座的顺式作用 DNA 区域 根据已发表的初步结果，我们的中心假设是串联反转。 重复序列参与 vmp 的调节表达，并且 vmp 转录是 此外，我们勇敢地说它是 DHS- 的二级发夹结构。 常驻反向重复序列对于 vmp 重组至关重要。 知识差距在于，由此产生的结果将为旨在开展更多调查奠定基础 破坏支撑疾病发病机制的抗原变异系统。 研究与 NIH 使命的一部分相关，该使命涉及发展基础知识，这些知识将 可能有助于减轻人类疾病和残疾的负担。 我们的中心假设将通过追求三个具体目标来检验：1）确定串联的作用 vmp 调节表达的反向重复序列，2) 确定 vmp 表达的重要性 基因转换，以及 3) 建立 DHS 驻留反向重复结构对 vmp 的要求 在第一个目标下，将使用我们的端粒介导删除单个反向 DNA 重复序列。 来自突变克隆或同基因野生型的 vmp 的转录和翻译。 类型对照将分别通过 qRT-PCR 和蛋白质印迹分析确定。 转录水平将从不同时间间隔从受感染小鼠获得的血液样本中进行量化 与野生型对照相比，在第二个目标下，vmp 内将产生突变。 启动子区域并用于感染小鼠以寻找与野生型对照相比抗原转换的损失。 在第三个目标中，DHS 内的反向重复序列将被打断或改变 DNA 序列，同时保留 感染后将监测与野生型对照相比的整体序列长度。 总体而言，我们的实验方法将允许对假定的小鼠进行直接突变分析。 顺式作用元件，以进一步了解赫氏芽孢杆菌的这种免疫逃避系统。