Regulated expression of B. burgdorferi virulence genes

描述（由申请人提供）：从莱姆病的病因伯氏疏螺旋体（Borrelia burgdorferi）的基因组中提取生物学相关信息的能力继续受到缺乏遗传工具的阻碍，无法确定其包含的许多功能未知的基因的生物学功能。为了分析这些基因对伯氏疏螺旋体生物学和毒力的贡献，我们和其他人采用了一些经典的微生物遗传学工具来分离伯氏疏螺旋体的零突变体及其互补衍生物。还没有人能够产生基因组饱和文库或分离这种病原体的条件和条件致死突变体。我们研究的长期目标是鉴定和表征伯氏疏螺旋体的毒力决定因素。一组潜在的毒力决定因素可能参与了这种生物体在哺乳动物宿主中的持续存在，即粘附素和粘附素调节剂，它们介导其与细胞外基质中的I型胶原的粘附。我们在之前项目期间获得的数据表明，使用含有tetO操作符的修饰的伯氏疏螺旋体杂交启动子，可以以一种调控的方式调节伯氏疏螺旋体的基因表达，该菌株组成表达Tet抑制因子。该系统与非抗生素诱导剂无氢四环素（ATc）结合，通过基因融合和反义RNA调控基因表达，制备和筛选反义RNA文库。我们还发现mariner转座子系统可用于分离伯氏疏螺旋体染色体和质粒基因的转座子突变体。基于这些发现，我们提出以下具体目标：1)利用反义RNA文库、黏附素/黏附素调节剂的等基因突变体和微阵列鉴定和表征新型伯氏疏螺旋体1型胶原黏附素/黏附素调节剂。2)利用芯片筛选的饱和伯氏疏螺旋体基因组转座子文库，利用全球基因组方法表征慢性感染MyD88敲除小鼠细胞外基质定植相关的伯氏疏螺旋体基因。3)在伯氏疏螺旋体中完成ATc调控基因表达的新系统，研究其与ECM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工具，用于研究莱姆病细菌伯氏疏螺旋体基因的功能。这些工具将用于识别可能导致这种生物体毒性的基因，这些基因可能成为针对莱姆病的新疫苗和抗生素的目标。

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.