Mutational analysis of the vlp/vsp antigenic variation system of the relapsing fe

描述（由申请人提供）：东非回归热螺旋体杜托疏螺旋体（Borrelia duttonii）免疫逃避和复发性菌血症的关键机制是 Vlp 和 Vsp 表面蛋白的抗原变异。先前涉及美国西部特有种赫氏疏螺旋体血清型 DNA 序列分析的研究表明上游同源序列 (UHS) 和下游同源序列 (DHS) 对于抗原转换的重要性。尽管 DNA 序列和统计分析表明这些 DNA 元件对 vlp/vsp 抗原转换的重要性，但仍缺乏直接突变研究，为这些元件在任何回归热伯氏疏螺旋体物种的抗原变异中提供机制作用。我们的长期目标是破译达托氏芽孢杆菌中 vlp/vsp 抗原变异的机制细节，并将这些发现扩展到虱传变种复发芽孢杆菌。本应用的目的是验证抗原变异所需的杜托氏博氏菌的假定 DN 元件。我们的中心假设是 UHS 和 DHS 位点作为顺式作用 DNA 元件，是 vlp/vsp 表达位点有效基因转换所必需的。拟议研究的基本原理是，成功完成将证明我们实验方法的实用性，这对于获得长期资金以进一步研究这一重要的免疫逃避机制是必要的。因此，拟议的研究与 NIH 使命的一部分相关，该使命涉及发展基础知识，这将有可能有助于减轻人类疾病和残疾的负担。 在 DNA 序列分析和之前发表的工作的指导下，我们的假设将通过追求两个具体目标来检验：1）确定 UHS 和 DHS 对于有效 vlp/vsp 重组的重要性； 2) 确定 DHS 驻留反向重复序列对 vlp/vsp 重组的要求。第一个目标是在 UHS 和 DHS 元件内产生突变和缺失。然后，这些突变体将被用来感染免疫活性小鼠，以寻找与野生型对照相比抗原转换的损失。在第二个目标下，DHS 内的反向重复将被中断，同时保持整体序列长度相同。感染免疫活性小鼠后，将监测与野生型对照相比的抗原变异。这项工作具有创新性，因为它代表了首次针对任何回归热疏螺旋体属物种的抗原变异系统进行突变。当应用时，这些结果预计将允许该系统的靶向，以显着降低该病原体在哺乳动物宿主中持续存在并引起疾病的能力。 公共卫生相关性：拟议的研究是回归热研究的一个重要领域，对于了解达氏疏螺旋体和其他引起回归热的疏螺旋体物种的免疫逃避和发病机制具有潜在的适用性。拟议的研究与公共卫生相关，因为由此产生的发现有可能从根本上推进达托氏芽胞杆菌免疫逃避领域的发展，并可能对其他动物和人类病原体的抗原变异系统产生广泛的影响。因此，这些发现最终有望适用于人类的健康。

DESCRIPTION (provided by applicant): A key mechanism for immune evasion and recurrent bacteremia by the East African relapsing fever spirochete, Borrelia duttonii, is antigenic variatio of the Vlp and Vsp surface proteins. Previous studies involving DNA sequence analysis of Borrelia hermsii serotypes, the species endemic to the western United States, have implicated the importance of an upstream homology sequence (UHS) and downstream homology sequence (DHS) for antigenic switching. Although DNA sequence and statistical analysis has implicated the importance of these DNA elements for vlp/vsp antigenic switching, direct mutational studies providing a mechanistic role for these elements in antigenic variation by any relapsing fever Borrelia species is still lacking. Our long-term goals are to decipher the mechanistic details of vlp/vsp antigenic variation in B. duttonii, and to expand these findings to the louse-borne variant, B. recurrentis. The objective of this application is to verify putative DN elements of B. duttonii that are required for antigenic variation. Our central hypothesis is that UHS and DHS sites function as cis-acting DNA elements that are necessary for efficient gene conversion at the vlp/vsp expression site. The rationale for the proposed research is that successful completion will demonstrate the practicality of our experimental approach, which is necessary in order to obtain long-term funding for further research on this important immune evasion mechanism. 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. Guided by DNA sequence analysis and previously published work, our hypothesis will be tested by pursuing two specific aims: 1) Establish the importance of the UHS and DHS for efficient vlp/vsp recombination; and 2) Establish the requirement of a DHS-resident inverted repeat for vlp/vsp recombination. Under the first aim, mutations and deletions within the UHS and DHS elements will be generated. These mutants will then be used to infect immunocompetent mice to look for a loss of antigenic switching compared to wild-type controls. Under the second aim, the inverted repeat within the DHS will be interrupted while keeping the overall sequence length the same. Antigenic variation compared to wild-type controls will be monitored after infecting immunocompetent mice. The proposed work is innovative, because it represents the first time that an antigenic variation system of any relapsing fever Borrelia species has been targeted for mutation. When applied, these results are expected to allow the targeting of this system in order to significantly reduce the ability of this pathogen to persist ad cause disease in the mammalian host. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important area of relapsing fever research that has potential applicability to understanding immune evasion and pathogenesis by Borrelia duttonii and other relapsing fever-causing Borrelia species. The proposed research has relevance to public health because the resulting discoveries have the potential to fundamentally advance the field of B. duttonii immune evasion, and may have broad implications for antigenic variation systems in other animal and human pathogens. Thus, the findings are ultimately expected to be applicable to the health of human beings.