Virulence Determinants of Borrelia burgdorferi

描述（由申请人提供）：伯氏疏螺旋体是北美莱姆病的病原体，由伊蚊属蜱传播。它是一种高度侵入性的螺旋体，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。该疾病有局部、播散和慢性分期，伯氏疏螺旋体似乎主要引起皮肤、神经系统、心血管和关节炎症状，尽管它能够侵入几乎任何组织，建立长期感染，并诱导炎症反应。这种细菌不产生已知的毒素，其发病机制在很大程度上是未知的。由于极低的转换率和质粒丢失，使用低传代感染性伯氏疏螺旋体进行遗传研究一直具有挑战性；因此，迄今为止仅对10个基因在哺乳动物-蜱感染循环中的重要性进行了研究。本项目将采用特征标记诱变的方法，对一个可转化、可感染的伯氏疏螺旋体B31克隆进行诱变，系统分析1740个蛋白编码基因在C3H/HeN小鼠和肩棘蜱感染中的作用。在Aim 1中，将构建一个包含-3,600个签名标记突变体的库并分析插入位点；该文库将提供给所有研究人员，并将作为感染性分析和体外生长所需最小基因含量估计的基础。在Aim 2中，将在Aim 1中获得的突变体组接种到小鼠体内，并在接种后2至4周分析多个器官部位是否存在生物体。传染性降低的突变体将通过质粒分析和基因互补进一步表征，以确定突变基因是否是毒力决定因素。目标3将集中于鉴定伯氏疏螺旋体在肩带伊蚊蜱中的传染性、持久性和传播所需的基因。本研究的结果将对哺乳动物-蜱感染循环所需的伯氏疏螺旋体基因有一个全面的认识，并将在未来几年对这些基因的功能作用进行详细分析。莱姆病是美国最常见的节肢动物传播疾病，由螺旋体伯氏疏螺旋体和相关生物体引起。这些细菌通过蜱虫传播，并在人体中引起长期感染，影响皮肤、神经系统、关节和心脏。由于我们对伯氏疏螺旋体如何致病知之甚少，因此很难设计出更好的方法来预防、诊断和治疗莱姆病。这项研究的目的是确定在感染过程中重要的每一个伯氏疏螺旋体基因，这样我们就可以利用得到的信息来帮助减少莱姆病对美国和世界其他地区人们的影响。

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.