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（由申请方提供）：东非回归热螺旋体（杜氏疏螺旋体）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。以前的研究涉及DNA序列分析的疏螺旋体hermsii血清型，物种特有的美国西部，暗示了上游同源序列（UHS）和下游同源序列（DHS）的抗原转换的重要性。虽然DNA序列和统计分析暗示了这些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，这些突变研究为这些元件在任何回归热疏螺旋体属物种的抗原变异中提供了机制作用。我们的长期目标是破译B中vlp/vsp抗原变异的机制细节。duttonii，并将这些发现扩展到虱传播的变体，B。复发。本申请的目的是验证B的推定DN元素。duttonii的抗原变异所必需的。我们的中心假设是，UHS和DHS网站的功能作为顺式作用的DNA元件，是必要的有效的基因转换在vlp/vsp表达位点。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，这是必要的，以便获得长期资金，进一步研究这一重要的免疫逃避机制。因此，拟议中的研究与NIH的使命的一部分有关，该使命涉及开发可能有助于减轻人类疾病和残疾负担的基础知识。 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特定目标来测试：1）建立UHS和DHS对于有效vlp/vsp重组的重要性;和2）建立DHS驻留反向重复对于vlp/vsp重组的要求。在第一个目标下，将产生UHS和DHS元件内的突变和缺失。然后将这些突变体用于感染免疫活性小鼠，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，同时保持总序列长度相同。在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。拟议的工作是创新的，因为它代表了第一次，任何回归热疏螺旋体物种的抗原变异系统已针对突变。当应用时，这些结果预计将允许靶向该系统，以显着降低该病原体在哺乳动物宿主中持续存在并导致疾病的能力。 公共卫生关系：拟议的研究是回归热研究的一个重要领域，对了解杜氏疏螺旋体和其他引起回归热的疏螺旋体属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有关，因为由此产生的发现有可能从根本上推动B领域的发展。duttonii免疫逃避，并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，这些发现最终有望适用于人类的健康。