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

• 批准号: 8354084
• 负责人: Troy Michael Bankhead
• 金额: $ 21.76万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8354084
• 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

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.

描述（由申请人提供）：东非复发性发烧螺旋体的免疫逃避和复发性菌血症的关键机制是vlp和VSP表面蛋白的抗原性变量。先前的研究涉及美国西部特有物种Borrelia HERMSII血清型的DNA序列分析，暗示了上游同源序列（UHS）和下游同源序列（DHS）对抗原性转换的重要性。尽管DNA序列和统计分析暗示了这些DNA元素对VLP/VSP抗原转换的重要性，但直接突变研究仍然缺乏这些元素在抗原变异中的机械作用。我们的长期目标是破译杜顿河B. duttonii中VLP/VSP抗原变异的机械细节，并将这些发现扩展到虱子 - 传播变体B. recurrentis。该应用的目的是验证抗原变异所需的duttonii的假定DN元素。我们的中心假设是，UHS和DHS位点起作用的DNA元素，这对于在VLP/VSP表达位点上有效基因转化是必需的。拟议的研究的理由是，成功完成将证明我们实验方法的实用性，这对于获得长期资助以进一步研究这种重要的免疫逃避机制是必要的。因此，拟议的研究与NIH使命的那部分有关，该研究与发展基本知识有关，这有可能有助于减轻人类疾病和残疾的负担。 在DNA序列分析和先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的来检验：1）确定UHS和DHS对有效VLP/VSP重组的重要性； 2）确定对VLP/VSP重组的DHS居民倒置重复的要求。在第一个目标下，将产生UHS和DHS元素内的突变和删除。然后，这些突变体将用于感染免疫能力的小鼠，以寻找与野生型对照相比的抗原转换损失。在第二个目标下，DHS内的倒置重复将中断，同时保持整体序列长度相同。与野生型对照相比，将在感染免疫能力小鼠后监测抗原性变异。拟议的工作具有创新性，因为它代表了任何复发性发烧伯氏菌种的抗原变异系统首次成为突变。当应用时，这些结果有望允许该系统的靶向，以显着降低该病原体持续性AD引起哺乳动物宿主疾病的能力。 公共卫生相关性：拟议的研究是发烧研究的重要领域，该研究具有潜在的适用性，可用于理解Borrelia duttonii和其他引起发烧的波雷利亚物种的免疫逃避和发病机理。拟议的研究与公共卫生相关，因为所产生的发现有可能从根本上推进Duttonii免疫逃避的领域，并且可能对其他动物和人类病原体中的抗原变异系统具有广泛的影响。因此，最终期望这些发现适用于人类的健康。