Mechanistic determinants of vls antigenic variation in the Lyme disease spirochet

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一个关键机制是在VLS位点进行重组。最近，一个靶向缺失的VLS突变体被产生，这证明了它在小鼠身上绝对需要持久性。尽管取得了这些进展，但我们对VlsE抗原变异背后的机制的理解仍然存在着根本的差距。我们的长期目标是鉴定和鉴定导致VlsE抗原变异的疏螺旋体蛋白(S)以及参与激活VlsE重组的哺乳动物宿主因子(S)。本应用的目的是鉴定VLS开关所需的顺式作用DNA元件，并确定VLS基因座的结构和位置对VlsE重组的重要性。中心假说是伯氏杆菌VLS基因座的51个碱基反向重复序列和17个碱基直接重复序列作为顺式作用的DNA元件，对VLS开关是必不可少的。此外，我们假设VLS基因座与端粒末端相邻的位置是有效重组VlsE所必需的，而抗原切换需要基因座的顺式组织。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，它们将为参与VlsE重组的蛋白质(S)提供关键线索。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。在已发表的伯氏杆菌基因组序列的指导下，并引用其他研究小组的工作，这一假说将通过追求两个具体目标来检验：1)确定VlsE重组所需的顺式作用DNA元件；2)确定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基因内这些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缺陷伯氏杆菌菌株，以重建VLS基因座，转化子将用于感染免疫能力和缺陷小鼠。在第二个目标下，将检测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，携带VlsE基因的螺旋体和两个独立的线性DNA分子上的无声盒将在通过小鼠宿主后检查VLS开关。这项拟议的工作是创新的，因为它涉及将完整基因座的基因操纵版本替换为伯氏杆菌的VLS基因敲除克隆。当应用时，拟议的研究结果有望允许靶向该系统，以便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。 公共卫生相关性：拟议的研究是莱姆病研究的一个重要领域，对理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。这项拟议的研究与公共卫生相关，因为由此产生的发现有可能从根本上推进伯氏杆菌免疫逃避领域，并可能对其他动物和人类病原体的抗原变异系统产生广泛影响。因此，这些发现最终有望适用于人类的健康。

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.