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顿疏螺旋体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。以往对美国西部特有的疏螺旋体（Borrelia hermsii）血清型的DNA序列分析表明，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序列和统计分析已经暗示了这些DNA元件对vlp/vsp抗原转换的重要性，但仍然缺乏直接的突变研究，提供这些元件在任何回归热伯氏疏螺旋体物种抗原变异中的机制作用。我们的长期目标是破译duttonii中vlp/vsp抗原变异的机制细节，并将这些发现扩展到虱子传播的变体，B. recurtis。本应用程序的目的是验证duttonii的假定DN元素，这是抗原性变异所必需的。我们的中心假设是UHS和DHS位点作为顺式作用的DNA元件，在vlp/vsp表达位点进行有效的基因转化是必要的。拟议研究的理由是，成功完成将证明我们的实验方法的实用性，这对于获得长期资助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的使命有关，即发展基础知识，这可能有助于减轻人类疾病和残疾的负担。

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.