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的基因组中提取生物学相关信息的能力仍然受到缺乏遗传工具的阻碍，无法鉴定其包含的许多功能未知的基因的生物学功能。我们和其他人已经将许多经典的微生物遗传学工具应用于B。burgdorferi，并已使用它们分离无效突变体及其互补衍生物，以分析这些基因对B的贡献。burgdorferi生物学和毒力。还没有人能够产生基因组饱和文库或分离该病原体的条件和条件致死突变体。本研究的长期目标是鉴定和表征B的毒力决定因子。burgdorferi。一组潜在的毒力决定因素，可能参与持久性的这种生物体在哺乳动物宿主是粘附素和粘附素调节剂介导的粘附细胞外基质中的I型胶原蛋白。我们在上一个项目期间获得的数据表明，基因表达在B。在B中，burgdorferi可以以调节的方式被调节。使用修饰的B，组成型表达泰特阻遏物的伯氏菌株。含有tetO操纵子的burgdorferi杂合启动子。该系统与非抗生素诱导剂脱水四环素（ATc）一起用于通过基因融合和反义RNA调节基因表达，以及制备和筛选反义RNA文库。我们还发现Mariner转座子系统可用于分离B中的转座突变体。burgdorferi染色体和质粒基因。在综合这些研究结果的基础上，我们提出以下具体目标。1)鉴别和表征新B。使用反义RNA文库、粘附素/粘附素调节剂的同基因突变体和微阵列的伯氏1型胶原粘附素/粘附素调节剂。2)描述B。使用涉及饱和B的全局基因组方法，在慢性感染的MyD 88敲除小鼠中与细胞外基质定殖相关的伯氏螺旋体基因。通过微阵列筛选burgdorferi基因组转座子文库。3)在B中完成ATc调节基因表达的新系统的开发。burgdorferi来研究其对ECM的粘附。公共卫生相关性：莱姆病是美国最常见的病媒传播疾病。该项目将开发新的分子遗传工具，用于研究莱姆病细菌B的基因功能。burgdorferi。这些工具将用于识别可能对这种生物的毒力负责的基因，这些基因可能是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.