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: 7862053
• 负责人: Troy Michael Bankhead
• 金额: $ 7.48万
• 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 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7862053
• 关键词: Animals; Antigenic Switching; Antigenic Variation; Area; Arthritis; Base Pairing; 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（由申请人提供）：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位点的重组。最近，一种vls的靶向缺失突变体被产生，证明了它在小鼠中对持久性的绝对要求。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抗原变异的伯氏疏螺旋体蛋白，以及参与激活vlsE重组的哺乳动物宿主因子。本应用程序的目的是鉴定vls转换所需的顺式作用DNA元件，并确定vls位点的结构和位置对vlsE重组的重要性。核心假设是伯氏疏螺旋体vls位点的51 bp反向重复和17 bp直接重复作为顺式作用的DNA元件，是vls切换所必需的。此外，我们假设vls位点靠近端粒末端的位置对于有效的vlsE重组是必要的，并且抗原转换需要位点的顺式组织。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成分，它们将为研究vlsE重组所涉及的蛋白质提供关键线索。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。在已发表的伯氏疏螺旋体基因组序列和其他团队引用的工作的指导下，这一假设将通过追求两个特定目标来验证：1)确定vlsE重组所需的顺式作用DNA元件；2)确立vls基因座结构和定位对vlsE重组的重要性。在第一个目标下，将产生vls位点内这些DNA重复区域的突变，并将其克隆到大肠杆菌质粒载体中。然后将这些vls突变质粒转化为vls缺陷的伯氏疏螺旋体菌株，以重建vls位点，并将转化物用于感染免疫正常和免疫缺陷的小鼠。在第二个目标下，将分析vls位点与端粒端之间距离增加的DNA构建物进行vlsE重组的能力。此外，在两个单独的线性DNA分子上携带vlsE基因和沉默磁带的螺旋体将在通过小鼠宿主后进行vls开关检查。这项提议的工作是创新的，因为它涉及到将基因操纵的完整位点替换为vls敲除的伯氏疏螺旋体克隆。当应用时，拟议研究的结果有望允许靶向该系统，以显着降低该病原体在哺乳动物宿主中建立持续感染的能力。