GENETIC APPROACHES TO VIRULENCE IN B. BURGDORFERI

描述：(改编自申请人的摘要)：伯氏疏螺旋体是 一种可在体外培养的细菌，是莱姆病的原因。它很小 基因组大约包含1000个染色体基因和400个质粒基因。 尽管知道伯氏杆菌基因组的完整DNA序列， 唯一体内表达的伯氏杆菌的鉴定与鉴定 毒力决定因素由于缺乏快速和 疏螺旋体的有效遗传系统。我们已经开发出一种基因系统 B.burgdorfed，由染色体外克隆载体pGKI2， 增强型绿色荧光蛋白作为潜在报告基因与抗性 以红霉素、卡那霉素等抗生素为选择性标记物。我们有 也能够证明BMP基因簇在B.B. Burgdorferi Sensu Lato菌株，该簇的基因经历了 环境调节的差异表达暗示了潜在的作用 这些基因的毒力。我们提出的实验协议基于 我们的初步工作由两个假设构成：1)高效分子 伯氏杆菌的遗传系统可以被开发，以及2)BMP的作用 伯氏杆菌生物学中的基因簇和毒力可以用 这些系统。长期目标是鉴定伯氏杆菌的毒力 决定因素和提高我们对它们在体内表达和 为此，我们提出了以下具体目标：1)持续发展和 伯氏杆菌染色体外克隆系统的改进(2) 分离和补充伯氏杆菌bmpD、bmpC和bmpa零突变体 确定这些基因在伯氏杆菌毒力中的可能作用 感染的体外和体内模型系统；以及3)表征启动子 和bmpC调控DNA序列的转录融合 增强型绿色荧光蛋白与荧光激活细胞 分类(FACS)。我们预计这些实验将允许延长 分子Koch对唯一AUD特异性表征的假设 伯氏杆菌的分子毒力决定因素。

DESCRIPTION: (adapted from the applicant's abstract): Borrelia burgdorferi is an in vitro culturable bacterium that is the cause of Lyme disease. Its small genome contain approxnnately 1,000 chromosomal genes and 400 plasmid genes. Despite knowledge of the complete DNA sequence of the B. burgdorferi genome, identification and characterization of unique in vivo expressed B. burgdorferi virulence determinants has been delayed by the lack of expeditious and efficacious genetic systems in Borrelia. We have developed a genetic system in B. burgdorfed that consists of the extrachromosomal cloning vector, pGKI2, enhanced green fluorescent protein as a potential reporter gene, and resistance to erythromycin, kanamycin, and other antibiotics as selective markers. We have also been able to show that the bmp gene cluster is highly conserved among B. burgdorferi sensu lato strains, and that the genes of this cluster undergo environmentally modulated differential expression suggesting a potential role in virulence for these genes. The experimental protocol we propose is based on our preliminary work and framed by two hypotheses: 1) efficient molecular genetic systems can be developed for B. burgdorferi, and 2) the role of the bmp gene cluster in B.burgdorferi biology and virulence can be ascertained using these systems. With the long-term aim of identifying B.burgdorferi virulence determinants and improving our understanding of their in vivo expression and regulation, we propose the following Specific Aims: 1) Continue development and improvement of an extrachromosomal cloning system for B. burgdorferi, 2) isolate and complement B. burgdorferi bmpD, bmpC and bmpA null mutants to determine the possible role of these genes in B. burgdorferi virulence in in vitro and in vivo model systems of infection; and 3) characterize promoters and regulatory DNA sequences of bmpC using transcriptional fusions with enhanced green fluorescent protein (EGFP) and fluorescence-activated cell sorting (FACS). We expect these experiments will permit the extension of the molecular Koch's postulates to the characterization of unique aud specific molecular virulence determinants of B. burgdorferi.