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

摘要 赫氏疏螺旋体是扁虱传播的回归热的病原体，回归热是一种细菌性疾病。 以严重的反复发烧为特征的。这种疾病的特征是起伏不定的发热 这是病原体对VMP抗原变异和顺序免疫逃避的直接结果。我们实验室最近的工作 已经提供了证据表明，Vmp位点上游的一个区域可能参与了Vmp的调节 缺乏VMp表达的突变克隆不能进行VMp重组。 此外，缺乏位于下游同源区域的反向DNA重复的突变克隆没有 表现出Vmp抗原变异。尽管有这些进展，顺式作用基因的确切身份和作用 VMP调控表达和重组中的元件尚不清楚。我们申请的目标将是 目的：确定Vmp基因座上对Vmp调控的表达和 重组。根据已发表的和初步的发现，我们的中心假设是串联倒置 重复序列参与了VMp的调控表达，并且VMp转录是必需的 基因转化过程。此外，我们假设它是国土安全部的二级发夹结构- 驻留反向重复序列是VMP重组所必需的。解决这些问题的理由是 知识差距是，由此产生的结果将为旨在 破坏支撑疾病发病机制的抗原变异系统。因此，拟议的 研究与NIH使命中与发展基础知识有关的部分相关，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)确定串联的角色 VMp调节表达的反向重复序列，2)决定VMp表达对 基因转化；3)建立了VMP的DHS驻留反向重复结构要求 重组。在第一个目标下，单个倒置的dna重复序列将通过我们的端粒介导被删除。 缺失/互补技术。突变克隆或等基因野生型VMP的转录和翻译 类型控制将分别通过qRT-PCR和Western印迹分析来确定。此外，mRNA. 转录水平将从感染小鼠的血液样本中以不同的时间间隔进行量化 与野生型对照相比。在第二个目标下，突变将在VMP内产生 启动子区域与野生型对照相比，用于感染小鼠寻找抗原切换的丢失。 在第三个目标中，DHS内的反向重复序列将被中断或DNA序列改变，同时保留 总序列长度。感染后将监测与野生型对照相比的抗原变异 免疫能力强的小鼠。总体而言，我们的实验方法将允许对推定的 顺式作用元件，以进一步了解赫氏杆菌的这种免疫逃避系统。

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.