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 Borrelia hermsii是tick传播发烧的原因，这是细菌疾病 以严重的复发发作发作为特征。疾病特征的起伏热是 VMP抗原变异和病原体的顺序免疫进化的直接结果。我们实验室的最新工作 提供了证据，表明VMP基因座上游的区域可能参与VMP的调节 表达，并且VMP表达中的突变克隆默认值无法进行VMP重组。 此外，缺乏倒在下游同源性区域的倒DNA重复的突变克隆没有 展示VMP抗原变异。尽管取得了这些进步，但顺式作用通用的确切身份和作用 VMP调控表达和重组的元素仍然未知。我们应用的目的将 确定对VMP基因座的顺式作用DNA区域，这些区域对于VMP调节的表达和 重组。基于已发表的初步发现，我们的中心假设是串联倒置 重复序列参与VMP的调节表达，并且VMP转录是需要的 基因转换过程。此外，我们假设它是DHS-的次级发夹结构 居民反向重复序列，这对于VMP重组至关重要。解决这些问题的理由 知识差距是，由此产生的结果将为针对其他投资的基础奠定基础 破坏基于疾病发病机理的抗原变异系统。那，提议 研究与NIH任务的那部分有关，该任务与发展基本知识有关 有可能有助于减少人类疾病和残疾的伯恩斯。 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 倒的重复序列，用于调节VMP的表达，2）确定VMP表达的重要性 基因转化，3）建立了DHS居民倒置重复结构的要求 重组。在第一个目标下，将使用我们的端粒介导的单个倒置DNA重复序列删除 删除/互补技术。来自突变克隆或等生野生的VMP的转录和翻译 类型控制将分别通过QRT-PCR和Western印迹分析确定。另外，mRNA 从感染小鼠以不同的时间间隔和 与野生型控制的那些相比。在第二个目标下，将在VMP中产生突变 与野生型对照相比，启动子区域并用于感染小鼠，以寻找抗原切换的损失。 在第三个目标中，DHS内的反复重复将中断或DNA序列在保留时改变 总序列长度。与野生型控制相比，将在感染后监测抗原变异 免疫能力小鼠。总体而言，我们的实验方法将允许对推定的直接突变分析 顺式作用元素，以进一步了解B. hermsii的这种免疫进化系统。