Regulated expression of B. burgdorferi virulence genes

描述(由申请人提供)：从引起莱姆病的伯氏疏螺旋体基因组中提取生物相关信息的能力继续受到缺乏遗传工具来确定其所包含的许多未知功能基因的生物学功能的阻碍。我们和其他人已经使一些经典的微生物遗传学工具适应于伯氏杆菌，并利用它们来分离零突变体及其互补的衍生物，以分析这些基因对伯氏杆菌生物学和毒力的贡献。到目前为止，还没有人能够建立基因组饱和文库，也没有人能够分离出这种病原体的条件和条件致死突变。我们研究的长期目标是鉴定和表征伯氏杆菌的毒力决定因素。一组潜在的毒力决定因素可能参与了这种微生物在哺乳动物宿主中的持久性，即黏附素和黏附素调节因子，它们介导了这种细菌与细胞外基质中的I型胶原的黏附。我们在前一个项目期间获得的数据表明，使用含有Teto操纵子的改良伯氏杆菌杂交启动子，可以在结构性表达Tet抑制子的伯氏杆菌菌株中以调节的方式调节基因的表达。该系统与非抗生素诱导剂脱水四环素(ATC)一起，通过基因融合和反义RNA调节基因表达，并用于反义RNA文库的制备和筛选。我们还发现，Mariner转座子系统可以用于分离伯氏杆菌染色体和质粒基因的转座突变体。在综合这些研究结果的基础上，我们提出以下具体目标。1)利用反义RNA文库、粘附素/粘附素调节剂的等基因突变体和微阵列，鉴定和鉴定新型伯氏杆菌1型胶原粘附素/粘附素调节剂。2)利用基因芯片筛选饱和的伯氏杆菌基因组转座子文库，采用全球基因组方法鉴定与慢性感染MyD88基因敲除小鼠细胞外基质定植相关的伯氏杆菌基因。3)在伯氏假单胞菌中建立ATC调控基因表达的新系统，研究其与细胞外基质的粘附性。公共卫生相关性：莱姆病是美国最常见的媒介传播疾病。该项目将开发新的分子遗传学工具来研究莱姆病细菌伯氏杆菌的基因功能。这些工具将被用来识别可能导致这种生物毒力的基因，并可能成为治疗莱姆病的新疫苗和抗生素的靶标。

DESCRIPTION (provided by applicant): The ability to extract biologically relevant information from the genome of Borrelia burgdorferi, the cause of Lyme disease, continues to be hampered by the lack of genetic tools to identify the biological functions of the many genes of unknown functions that it contains. We and others have adapted a number of classical microbial genetics tools to B. burgdorferi and have used them to isolate null mutants and their complemented derivatives in order to analyze the contribution of these genes to B. burgdorferi biology and virulence. No one yet has been able to produce genomic saturated libraries or to isolate conditional and conditional lethal mutants of this pathogen. The long-term goal of our research is the identification and characterization of virulence determinants of B. burgdorferi. One group of potential virulence determinants that may be involved in persistence of this organism in mammalian hosts is the adhesins and adhesin regulators mediating its adhesion to Type I collagen in the extracellular matrix. Data obtained by us during the previous project period indicated that gene expression in B. burgdorferi could be modulated in a regulated fashion in a B. burgdorferi strain constitutively expressing the Tet repressor using modified B. burgdorferi hybrid promoters containing tetO operators. This system was used with the non-antibiotic inducer anhydrotetracycline (ATc) to regulate gene expression by gene fusions and antisense RNA, and in preparing and screening antisense RNA libraries. We also found that the mariner transposon system could be used to isolate transposition mutants in B. burgdorferi chromosomal and plasmid genes. On the basis of a combination of these findings, we propose the following Specific Aims. 1) Identify and characterize novel B. burgdorferi Type 1 collagen adhesins/adhesin regulators using antisense RNA libraries, isogenic mutants for adhesins/adhesins regulators, and microarrays. 2) Characterize B. burgdorferi genes relevant to colonization of the extracellular matrix in chronically infected MyD88 knockout mice using a global genomic approach involving saturated B. burgdorferi genomic transposon libraries screened by microarrays. 3) Complete development of novel systems of ATc - regulated gene expression in B. burgdorferi to study its adhesion to the ECM. PUBLIC HEALTH RELEVANCE: Lyme disease is the most common vector-borne disease in the United States. This project will develop new molecular genetic tools for studying the function of the genes of the Lyme disease bacterium, B. burgdorferi. These tools will be used to identify genes that are potentially responsible for this organism's virulence and which could be targets for new vaccines and antibiotics against Lyme disease.