Virulence Determinants of Borrelia burgdorferi

描述(申请人提供)：伯氏疏螺旋体是北美莱姆病的病原体，由硬蜱属的硬蜱传播。它是一种高度侵袭性的螺旋体，可在人类和其他哺乳动物中引起感染和表现，持续数月至数年。该病有局部性、播散性和慢性阶段，伯氏杆菌似乎主要通过侵入几乎任何组织、建立长期感染和诱导炎症反应的能力而引起皮肤、神经、心血管和关节炎症状。该细菌不产生已知的毒素，其致病机制在很大程度上尚不清楚。由于极低的转化率和极低的质粒丢失，使用低传代、感染性伯氏杆菌进行的遗传学研究一直具有挑战性；结果，迄今为止只有10个基因被研究它们在哺乳动物-壁虱感染循环中的重要性。在本项目中，我们将利用伯氏杆菌B31的可转化感染性克隆中的签名标记突变，系统地分析1740蛋白编码基因在感染C3H/HEN小鼠和肩部硬蜱中所起的作用。在目标1中，将构建一个包含3,600个带有签名标记的突变体的文库，并对插入位点进行分析；该文库将向所有研究人员开放，并将作为传染性分析和估计体外生长所需的最低基因含量的基础。在目标2中，在目标1中获得的突变体组将被接种到小鼠体内，并在接种后2至4周分析多个器官部位是否存在有机体。感染力降低的突变株将通过质粒分析和基因互补进一步鉴定，以确定突变基因是否为毒力决定因素。目的3将集中于鉴定伯氏杆菌感染性、持久性和传播所需的基因。这项研究的结果将是对哺乳动物-壁虱感染周期所需的伯氏杆菌基因的全面了解，并将在未来几年推动对这些基因的功能作用的详细分析。莱姆病是美国最常见的节肢动物传播疾病，由螺旋形伯氏疏螺旋体细菌和相关生物引起。这些细菌通过扁虱传播，会在人体内引起长期感染，影响皮肤、神经系统、关节和心脏。由于我们对伯氏杆菌如何致病知之甚少，因此很难设计出更好的方法来预防、诊断和治疗莱姆病。这项研究的目标是确定在感染过程中重要的每一个伯氏杆菌基因，以便我们可以利用由此产生的信息来帮助减少莱姆病对美国和世界其他地区人民的影响。

DESCRIPTION (provided by applicant): Borrelia burgdorferi is the causative agent of Lyme disease in North America and is transmitted by ticks of the genus Ixodes. It is a highly invasive spirochete that can cause infection and manifestations in humans and other mammals that persist for months to years. The disease has localized, disseminated, and chronic stages, and B. burgdorferi appears to cause dermal, neurologic, cardiovascular, and arthritic symptoms primarily though the ability to invade almost any tissue, establish long-term infection, and induce inflammatory responses. The bacterium produces no known toxins, and its mechanisms of pathogenesis are largely unknown. Genetic studies using low-passage, infectious B. burgdorferi have been challenging due to exceedingly low transformation rates and plasmid loss; as a result, only 10 genes have been investigated to date with regard to their importance in the mammal-tick infectious cycle. In this project, signature-tagged mutagenesis in a transformable, infectious clone of B. burgdorferi B31 will be used to systematically analyze the roles of the 1740 protein-encoding genes in the infection of C3H/HeN mice and Ixodes scapularis ticks. In Aim 1, a library of -3,600 signature-tagged mutants will be constructed and insertion sites analyzed; this library will be made available to all investigators and will serve as a basis for infectivity analysis and the estimation of the minimal gene content required for in vitro growth. In Aim 2, groups of the mutants obtained in Aim 1 will be inoculated into mice and multiple organ sites analyzed for the presence of organisms 2 to 4 weeks after inoculation. Mutants with decreased infectivity will be characterized further by plasmid analysis and gene complementation to determine whether the mutated genes are virulence determinants. Aim 3 will be focused on the identification of genes required for the infectivity, persistence and transmission of B. burgdorferi in Ixodes scapularis ticks. The result of this study will be a comprehensive view of the B. burgdorferi genes required for mammal-tick infectious cycle, and will fuel the detailed analysis of the functional roles of these genes in future years. Lyme disease, the most common arthropod-borne disease in the United States, is caused by the spiral-shaped bacterium Borrelia burgdorferi and related organisms. These bacteria are transmitted by ticks and cause a long-term infection in people that affects the skin, nervous system, joints, and heart. Because we know so little about how B. burgdorferi causes disease, it is difficult to design better ways to prevent, diagnose, and treat Lyme disease. The goal of this study is to identify every B. burgdorferi gene that is important in the infection process, so that we can use the resulting information to help reduce the impact of Lyme disease on people in the United States and in other parts of the world.