MOLECULAR MECHANISMS OF VARIATION AND ADAPTATION IN BORRELIA BURGDORFERI

伯氏疏螺旋体是莱姆病的病原体，在自然界中是通过野生哺乳动物和扁虱之间的感染循环维持的。像许多细菌病原体一样，伯氏杆菌必须应对一系列不断变化的环境条件，才能成功地持续、增殖并在宿主之间传播。细菌外表面是与宿主相互作用的主要部位。伯氏杆菌外表面蛋白(OSPs)的排列已被证明随着感染周期的不同而变化。这些不同的OSP很可能赋予了螺旋体与其必须生存的不同环境相关的独特性质。我们的主要目标是用遗传学方法阐明伯氏杆菌适应的分子机制及其在感染循环中的作用。我们以前已经证明，温度代表一个重要的环境变量，在培养温度上升后，许多OSP的合成都会增加。蛋白质水平与转录水平平行，与基因表达水平的调控一致。伯氏杆菌的全基因组序列确定了3个转录因子的同源物：sigma 70、sigma 54和rpos。我们感兴趣的是rpos和sigma 54在温度诱导和OSP基因转录调控中的作用。我们已经克隆了伯氏杆菌Sigma 54和rpos基因，并证明了一株福氏志贺氏菌rpos突变体与伯氏杆菌rpos基因的部分互补。我们已经灭活了伯氏杆菌rpos基因，并证明该突变株在稳定期表现出较低的渗透阻力。通过二维凝胶对细菌总蛋白的比较，揭示了野生型和rpos突变螺旋体之间的一些差异。正在进行的研究旨在鉴定这些蛋白质。伯氏杆菌的基因组序列还鉴定了二糖壳二糖转运体(Cel)的同源基因。几丁质(硬壳层的一种成分)被降解为壳二糖，这可能是伯氏假丝虫体内碳水化合物的重要来源。我们已经开始了对Cel基因的遗传分析，以确定它们在伯氏杆菌中的作用和功能。我们已经灭活了其中一个基因CelB，并表明该突变株在复杂的生长介质中生长得与野生型伯氏杆菌一样好。初步结果表明，在缺乏N-乙酰氨基葡萄糖(NAG)的培养条件下，CelB突变体的生长受到抑制，提示通过CEL转运蛋白对壳二糖的摄取可能满足NAG的需要。-伯氏疏螺旋体、螺旋体、莱姆病、分子遗传学、壁虱传播、适应性反应

Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with an array of changing environmental conditions to successfully persist, proliferate and be transmitted between hosts. The bacterial outer surface represents the primary site for interactions with the host. The array of B. burgdorferi outer surface proteins (Osps) has been shown to vary with the infectious cycle. It is likely that these different Osps endow the spirochete with distinct properties relevant to the disparate environments in which it must survive. Our broad objective is to use a genetic approach to elucidate the molecular mechanisms of adaptation in B. burgdorferi and their roles in the infectious cycle. We have previously demonstrated that temperature represents an important environmental variable, and that synthesis of a number of Osps is increased after a rise in culture temperature. Protein levels parallel transcript levels, consistent with regulation at the level of gene expression. The complete genomic sequence of B. burgdorferi identified homologs of three transcription factors, sigma 70, sigma 54, and RpoS. We are interested in the roles of RpoS and sigma 54 in the temperature induction and transcriptional regulation of osp genes. We have cloned the borrelial sigma 54 and rpoS genes and demonstrated partial complementation of a Shigella flexneri rpoS mutant with the B. burgdorferi rpoS gene. We have inactivated the B. burgdorferi rpoS gene and demonstrated that this mutant exhibits decreased osmotic resistance during stationary phase. A comparison of total bacterial proteins by two-dimensional gels revealed several differences between wild type and rpoS mutant spirochetes. Ongoing studies are aimed at the identification of these proteins. The genomic sequence of B. burgdorferi also identified a homolog of a transporter for the disaccharide chitobiose (cel). The degradation of chitin (a component of tick cuticle) to chitobiose could provide an important source of carbohydrate for B. burgdorferi in the tick. We have begun a genetic analysis of the cel genes to determine their role and function in B. burgdorferi. We have inactivated one of these genes, celB, and shown that this mutant grows as well as wild type B. burgdorferi in complex growth medium. Preliminary results indicate that growth of the celB mutant is impaired relative to wild type B. burgdorferi in medium lacking N-acetyl glucosamine (NAG), suggesting that uptake of chitobiose via the Cel transporter may fulfill the requirement for NAG. - Borrelia burgdorferi, spirochete, Lyme disease, molecular genetics, tick transmission, adaptive response