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

总结 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 以严重的反复发热为特征。这种疾病的特征是起伏的发热， Vmp抗原变异和病原体的连续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，从vmp基因座上游的区域可能参与调节Vmp 表达，并且vmp表达缺陷的突变克隆无法进行vmp重组。 此外，缺乏位于下游同源区的反向DNA重复序列的突变克隆不 表现出Vmp抗原变异。尽管取得了这些进展，顺式作用基因的确切身份和作用仍不清楚。 在VMP调节的表达和重组中的元件仍然未知。我们申请的目的是 鉴定vmp基因座的顺式作用DNA区域，其对于vmp调节表达是重要的， 重组根据已发表的和初步的研究结果，我们的中心假设是， 重复序列参与vmp的调节表达，vmp转录是vmp基因表达所必需的。 基因转换过程此外，我们假设它是DHS的二级发夹结构- 对于vmp重组必需的常驻反向重复序列。解决这些问题的基本原理 知识差距是，由此产生的结果将为旨在 破坏支持疾病发病机制的抗原变异系统。因此，拟议的 研究与国家卫生研究院的使命有关，即发展基础知识， 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进行测试：1）确定串联的作用 2）确定vmp表达对于调节vmp表达的重要性， 基因转换，以及3）建立vmp的DHS驻留反向重复结构的要求 重组在第一个目标下，将使用我们的端粒介导的 缺失/互补技术。来自突变克隆或同基因野生型的vmp的转录和翻译 将分别通过qRT-PCR和Western印迹分析确定类型对照。此外，mRNA 在不同的时间间隔从感染小鼠获得的血液样品中定量转录物水平， 与来自野生型对照的那些相比。在第二个目标下，突变将在vmp内产生 启动子区，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中断或DNA序列改变，同时保留 整个序列长度。感染后，将监测与野生型对照相比的抗原变异 免疫活性小鼠。总的来说，我们的实验方法将允许直接突变分析推定的 顺式作用元件以获得对B的这种免疫逃避系统的进一步理解。赫姆斯。

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.