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

复发性FEVLP/VSP抗原变异系统的突变分析

基本信息

批准号：
8501363
负责人：
Troy Michael Bankhead
金额：
$ 17.1万
依托单位：
WASHINGTON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8501363
关键词：
Active Sites African Animals Antigenic Switching Antigenic Variation Archives Area Attenuated Back Bacteremia Beryllium Boron Borrelia Clinical Cloning DNA DNA Fingerprinting DNA Sequence DNA Sequence Analysis Deletion Mutation Disease Elements Fever Funding Future Gene Conversion Genes Genetic Recombination Goals Health Human Immune Immune response Immunocompetent Indium Infection Knowledge Length Lice Lipoproteins Membrane Proteins Mission Monitor Mus Mutation Order Spirochaetales Outcome Pathogenesis Perinatal mortality demographics Pregnancy Prevalence Public Health Publishing Recurrence Relapse Relapsing Fever Research Role Sequence Homology Serotyping Site Spontaneous abortion Surface System Tanzania Testing Time Tropical Disease United States Variant Work base cis acting element disability experience fetal forgetting innovation mortality mutant neglect pathogen relapsing fever borrelia telomere

项目摘要

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.

描述(由申请人提供)：东非复发性热螺旋体的免疫逃避和反复菌血症的一个关键机制是VLP和VSP表面蛋白的抗原性变异。以前的研究涉及美国西部特有的赫氏疏螺旋体血清型的DNA序列分析，表明上游同源序列(UHS)和下游同源序列(DHS)对于抗原切换的重要性。虽然DNA序列和统计分析表明这些DNA元件在VLP/VSP抗原转换中的重要性，但提供这些元件在任何复发性发热疏螺旋体的抗原变化中的机制作用的直接突变研究仍然缺乏。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种--复发芽孢杆菌。本申请的目的是验证杜通氏杆菌抗原变异所需的推定的DN元件。我们的中心假设是UHS和DHS位点作为顺式作用DNA元件发挥作用，这是VLP/VSP表达位点高效基因转换所必需的。拟议研究的基本原理是，成功完成这项研究将证明我们的实验方法的实用性，这是为进一步研究这一重要的免疫逃避机制获得长期资金所必需的。因此，拟议的研究与NIH使命中与发展基础知识相关的部分相关，这可能有助于减轻人类疾病和残疾的负担。在DNA序列分析和以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验证：1)确定UHS和DHS对于有效的VLP/VSP重组的重要性；2)建立DHS驻留的反向重复序列对VLP/VSP重组的要求。在第一个目标下，将在UHS和DHS元素中产生突变和删除。然后，这些突变体将被用来感染具有免疫能力的小鼠，以寻找与野生型对照相比抗原切换的丢失。在第二个目标下，DHS内的反向重复将被中断，同时保持总序列长度相同。在感染具有免疫能力的小鼠后，将监测与野生型对照相比的抗原变化。这项拟议的工作是创新的，因为这是首次将任何复发热疏螺旋体物种的抗原变异系统作为突变目标。当应用时，这些结果有望允许该系统的靶向，以显著降低这种病原体在哺乳动物宿主中持续致病的能力。