Molecular Genetic Basis Of The Infectious Cycle Of Borre

莱姆病的病原体伯氏疏螺旋体通过野生哺乳动物和蜱之间的传染循环在自然界中维持。像许多细菌病原体一样，B。伯氏菌必须科普一系列不断变化的环境条件，以便成功地持续存在、增殖和在宿主之间传播。B。burgdorferi含有丰富的线性和环状质粒，越来越多的证据表明这些质粒携带对适应至关重要的基因。研究特定质粒和基因产物在B感染周期中功能的主要障碍。Burgdorferi氏菌不能对致病菌株进行遗传操作。为了解决这个问题，我们已经开发了在B31-MI（感染性B）中进行定点诱变的条件。其基因组最近被测序。我们灭活了B31-MI中的几个质粒和染色体基因，并确定携带这些突变的克隆对小鼠没有感染性。然而，我们发现广泛的异质性克隆和突变体来自B31-MI的基础上的菌落表型，生长速率，质粒含量和蛋白质组成。B31-MI内的这种高度克隆多态性使突变体的评估复杂化，并表明它不是用于传染性B的遗传研究的适当菌株背景。伯格多费里。为了建立一个明确的等基因克隆的靶向诱变研究，我们从B31-MI衍生了一个野生型克隆，它对小鼠具有感染性并可转化。我们的研究还表明，B的不稳定性。Burgdorferi基因组要求仔细监测衍生突变体的质粒含量和失活基因的互补，以便用该病原体进行遗传研究。我们已经灭活了感染性克隆中的几个质粒编码基因，并证明这些克隆和互补对照保留了所有质粒。因此，我们终于成功地对致病性B进行了基因操作。伯氏克隆这允许比较突变体和同基因野生型克隆，以鉴定特定基因产物的功能。在与一个校外研究者联盟的合作下，我们开发了一系列所有的B。burgdorferi基因，并使用该阵列比较不同生长条件下以及突变体和野生型克隆之间的基因表达的全球模式。我们已经建立了一个实验性的感染循环，在这个循环中，我们正在评估缺乏特定基因或质粒的突变克隆在蜱虫载体和哺乳动物宿主中的感染性以及它们之间的传播能力。我们正在进一步描述特定B的功能。通过对同基因突变体和野生型克隆进行生化分析，获得了burgdorferi基因产物。

Borrelia burgdorferi, the cauative 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 a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi contains abundant linear and circular plasmids and there is growing evidence that these plasmids carry genes critical for adaptation. A major obstacle to studying the functions of specific plasmids and gene products in the infectious cycle of B. burgdorferi has been an inability to genetically manipulate pathogenic strains. To address this issue, we have developed conditions for site-directed mutagenesis in B31-MI, the infectious B. burgdorferi strain whose genome was recently sequenced. We inactivated several plasmid and chromosomal genes in B31-MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31-MI based on colony phenotype, growth rate, plasmid content and protein composition. This high degree of clonal polymorphism within B31-MI complicates the assessment of mutants and indicates that it is not an appropriate strain background for genetic studies of infectious B. burgdorferi. In order to establish a well-defined isogenic clone for targeted mutagenesis studies, we derived a wild type clone from B31-MI that was infectious for mice and transformable. Our studies also demonstrate that the instability of the B. burgdorferi genome mandates careful monitoring of plasmid content of derived mutants and complementation of inactivated genes in order to perform genetic studies with this pathogen. We have inactivated several plasmid-encoded genes in an infectious clone and demonstrated that these clones and the complemented controls have retained all plasmids. Thus we have finally succeeded in genetically manipulating a pathogenic B. burgdorferi clone. This allows comparisons of mutant and isogenic wild type clones in order to identify the functions of specific gene products. In collaboration with a consortium of extramural investigators, we have developed an array of all B. burgdorferi genes and are using this array to compare global patterns of gene expression under different growth conditions and between mutant and wild type clones. We have established an experimental infectious cycle in which we are assessing the competence of mutant clones lacking specific genes or plasmids for infectivity in both the tick vector and mammalian host, and for transmission between them. We are further characterizing the functions of specific B. burgdorferi gene products through biochemical analyses of isogenic mutant and wild type clones.