PATHOGENESIS OF INFECTION WITH THE LYME DISEASE SPIROCHETE, BORRELIA BURGDORFERI

我们正在开发一个系统的基本组件，通过它可以 莱姆病发病机制及基础生物学研究 病原体伯氏疏螺旋体。灵敏而准确的检测 生物体是必要的，以便将它们的存在(或不存在)与 疾病症状，并便于准确诊断感染。 生物体的关键结构和调控元件的特征 是确定潜在致病机制所必需的。最终， 关键蛋白质可以通过基因操作和重新引入，而 关于感染和疾病评估的结果。 1.检测：建立了灵敏、特异的聚合酶链式反应方法 染色体编码的伯氏杆菌DNA序列。序列比较 允许设计具有广泛反应性但具有伯氏杆菌特异性的底物。一个 改良式套式引物法提高了灵敏度和重复性 在存在过多真核DNA的情况下的特异性。在进行测试时 31个不同的伯氏杆菌地理和生物学分离株 来源，都被检测到了。 2.分子特征：伯氏杆菌的几个特征 地址。根据对不同组的细菌的反应性对菌株进行分类 聚合酶链式反应将伯氏杆菌菌株分为两组。分析和 编码两种主要外表面蛋白的基因测序 确定了这些基因表达变化的潜在机制 免疫优势成分。提供了一种电镀方案的修改 一种更快速、更有效的生物克隆方法，并使 变异体的克隆分析。分离和研究其表达的基因 在温度上升后增加(热休克基因)将提供 关于它们在细胞生长、致病机制、质粒中的作用的信息 复制和适应压力。 3.基因转移：为了能够对伯氏杆菌进行遗传分析，两个 基因转移到疏螺旋体的方法正在开发中。向量有 被构建为允许转移的DNA整合或转座到 染色体。使用电穿孔进行转化的参数如下 正在接受测试。替代电穿孔、交配的条件 与大肠杆菌的关系正在建立中。

We are developing the essential components of a system with which to investigate the pathogenesis of Lyme disease and the basic biology of the causative agent, Borrelia burgdorferi. Sensitive and accurate detection of organisms is necessary in order to compare their presence (or absence) with disease symptoms and to facilitate an accurate diagnosis of infection. Characterization of key structural and regulatory elements of an organism is requisite to identifying potential pathogenic mechanisms. Ultimately, critical proteins can be genetically manipulated and reintroduced and the outcome on infection and disease assessed. 1. Detection: A sensitive and specific PCR assay has been developed from a chromosomally encoded B. burgdorferi DNA sequence. Sequence comparisons permit the design of broadly reactive but burgdorferi-specific primers. A modified protocol using 'nested' primers has increased the sensitivity and specificity in the presence of excess eukaryotic DNA. When tested against 31 different B. burgdorferi isolates of broad geographic and biological origins, all were detected. 2. Molecular Characterization: Several features of B. burgdorferi are being addressed. Classification of isolates by reactivity with different sets of PCR primers subdivides burgdorferi strains into 2 groups. Analysis and sequencing of the genes encoding 2 major outer surface proteins has identified potential mechanisms for variation in expression of these immunodominant components. Modification of a plating protocol has provided a more rapid and efficient means of cloning organisms and has permitted the clonal analysis of variants. Isolating and studying genes whose expression is increased after a temperature upshift (heat shock genes) will provide information about their roles in cell growth, pathogenesis, plasmid replication and adaptation to stress. 3. Gene Transfer: In order to allow genetic analysis of B. burgdorferi, two methods for gene transfer into Borrelia are being developed. Vectors have been constructed to allow transferred DNA to integrate or transpose into the chromosome. Parameters for transformation using electroporation are being tested. Conditions for the alternative to electroporation, mating with E. coli, are being established.