Mechanistic determinants of vls antigenic variation in the Lyme disease spirochet

莱姆病螺旋体vls抗原变异的机制决定因素

• 批准号: 8075608
• 负责人: Troy Michael Bankhead
• 金额: $ 7.4万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8075608
• 关键词: Animals; Antigenic Switching; Antigenic Variation; Area; Arthritis; Base Pairing; Beryllium; Binding Sites; Biological Assay; Borrelia; Borrelia burgdorferi; Carditis; Cloning; DNA; DNA Sequence Analysis; Direct Repeats; Drug Delivery Systems; Elements; Environment; Escherichia coli; Event; Gene Conversion; Gene Mutation; Gene Rearrangement; Genes; Genetic Recombination; Genomics; Goals; Health; Human; Immune; Immune response; Infection; Infection prevention; Integration Host Factors; Intercistronic Region; Knock-out; Knowledge; Lead; Length; Location; Lyme Disease; Membrane Proteins; Mission; Mus; Mutation; Nature; Neurologic; North America; Order Spirochaetales; Outcome; Pathogenesis; Pharmaceutical Preparations; Plague; Plasmid Cloning Vector; Plasmids; Positioning Attribute; Process; Proteins; Public Health; Publishing; Reaction; Research; Research Personnel; Role; Site; Structure; System; Testing; Therapeutic; Tick-Borne Diseases; Vaccines; Work; base; cis acting element; disability; genome sequencing; innovation; mutant; pathogen; prevent; public health relevance; recombinase; reconstitution; research study; telomere; vector; working group

DESCRIPTION (provided by applicant): A key mechanism to immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is recombination at the vls locus. Recently, a targeted-deletion mutant of vls was generated that demonstrated its absolute requirement for persistence in mice. Despite this advance, there remains a fundamental gap in our understanding of the mechanism behind vlsE antigenic variation. Our long-term goal is to identify and characterize the Borrelia protein(s) responsible for antigenic variation of vlsE, as well as the mammalian host factor(s) involved in activating vlsE recombination. The objective of this application is to identify cis-acting DNA elements required for vls switching, and to establish the importance of the structure and position of the vls locus for vlsE recombination. The central hypothesis is that the 51 bp inverted repeat and 17 bp direct repeats of the B. burgdorferi vls locus act as cis-acting DNA elements that are essential for vls switching. Furthermore, we hypothesize that the position of the vls locus adjacent to the telomere end is necessary for efficient vlsE recombination, and that antigenic switching requires a cis organization of the locus. The rationale for the proposed research is that, once the key cis-acting elements and important structural components are defined, they will provide critical clues to the protein(s) involved with vlsE recombination. 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 the published B. burgdorferi genome sequence and cited work by other groups, this hypothesis will be tested by pursuing two specific aims: 1) Identify the cis-acting DNA elements required for vlsE recombination; and 2) Establish the importance of vls locus structure and positioning for vlsE recombination. Under the first aim, mutations of these DNA repeat regions within the vls locus will be generated and cloned into an E. coli plasmid vector. These vls mutant plasmids will then be transformed into a vls-deficient strain of B. burgdorferi in order to reconstitute the vls locus, and transformants will be used to infect both immunologically-competent and -deficient mice. Under the second aim, DNA constructs with increased distances between the vls locus and the telomere end will be assayed for the ability to carry out vlsE recombination. In addition, spirochetes harboring the vlsE gene and silent cassettes on two separate linear DNA molecules will be examined for vls switching after being passaged through a mouse host. The proposed work is innovative, because it involves replacement of genetically manipulated versions of the complete locus into the vls-knockout clone of B. burgdorferi. When applied, the results from the proposed studies are expected to allow the targeting of this system in order to significantly reduce the ability of this pathogen to establish a persistent infection in the mammalian host. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important area of Lyme disease research that has potential applicability to understanding immune evasion and pathogenesis by Borrelia burgdorferi. The proposed research has relevance to public health because the resulting discoveries have the potential to fundamentally advance the field of B. burgdorferi 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（申请人提供）：莱姆病螺旋体（伯氏疏螺旋体）免疫逃避和持续感染的关键机制是vls基因座的重组。最近，产生了vls的靶向缺失突变体，证明了其在小鼠中持续存在的绝对要求。尽管取得了这一进展，但我们对vlsE抗原变异背后的机制的理解仍然存在根本性的差距。我们的长期目标是鉴定和表征负责vlsE抗原变异的疏螺旋体蛋白，以及参与激活vlsE重组的哺乳动物宿主因子。本申请的目的是鉴定vls转换所需的顺式作用DNA元件，并确定vls基因座的结构和位置对vlsE重组的重要性。中心假设是B的51 bp反向重复和17 bp正向重复。Burgdorferi vls基因座作为vls转换所必需的顺式作用DNA元件。此外，我们假设VLS位点邻近端粒末端的位置对于有效的VLS E重组是必要的，并且抗原转换需要该位点的顺式组织。这项研究的基本原理是，一旦确定了关键的顺式作用元件和重要的结构组分，它们将为参与vlsE重组的蛋白质提供关键线索。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。以出版的B为指导。根据burgdorferi基因组序列和其他小组的引用工作，该假设将通过追求两个特定目标来检验：1）鉴定vlsE重组所需的顺式作用DNA元件;和2）建立vls基因座结构和定位对于vlsE重组的重要性。在第一个目标下，将产生vls基因座内这些DNA重复区的突变并克隆到E. coli质粒载体。然后将这些vls突变质粒转化到B的vls缺陷型菌株中。Burgdorferi以重建VLS基因座，并且转化体将用于感染免疫活性小鼠和免疫缺陷小鼠。在第二个目标下，将测定vls基因座和端粒末端之间距离增加的DNA构建体进行vlsE重组的能力。此外，将检查在两个单独的线性DNA分子上携带vlsE基因和沉默盒的螺旋体在通过小鼠宿主传代后的vls转换。所提出的工作是创新的，因为它涉及将完整基因座的遗传操作版本替换为B的vls敲除克隆。burgdorferi。当应用时，预期拟定研究的结果将允许靶向该系统，以显著降低该病原体在哺乳动物宿主中建立持续感染的能力。 公共卫生关系：拟议的研究是莱姆病研究的一个重要领域，对了解伯氏疏螺旋体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。Burgdorferi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。