SURFACE CHANGE AND VIRULENCE IN COXIELLA BURNETII

伯内特柯克斯体的表面变化和毒力

• 批准号: 3129693
• 负责人: LOUIS P MALLAVIA
• 金额: $ 27.61万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-07-01 至 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3129693
• 关键词: Escherichia coli; Q fever; SDS polyacrylamide gel electrophoresis; acute disease /disorder; bacterial proteins; chronic disease /disorder; electroporation; endocarditis; endonuclease; enzyme linked immunosorbent assay; gene expression; genetic regulation; genetic strain; guinea pigs; host organism interaction; immunofluorescence technique; laboratory mouse; laboratory rabbit; microorganism antigen; microorganism growth; molecular cloning; monoclonal antibody; nucleic acid probes; plasmids; polymerase chain reaction; southern blotting; surface antigens; tissue /cell culture; virulence; western blottings

Coxiella burnetii is the causative agent of both acute Q fever and chronic endocarditis in humans. Both plasmid and chromosomal differences correlate with the type of disease (acute or chronic) caused by an isolate suggesting that the different isolates have different virulence determinants. We have designed experiments to extend our knowledge of C. burnetii genomic and plasmid DNA and their relationship to disease. Unique plasmid regions will be analyzed by cloning, restriction enzyme, Southern hybridization and sequencing analyses. We will also identify the plasmid origin of replication for studies on transformation and mutagenesis of C. burnetii. Plasmid encoded surface proteins (possible virulence factors) will be identified by looking for protease-sensitive surface proteins produced by maxicells containing pUC/C. burnetii plasmid constructs and also by TnphoA mutagenesis. We will identify chromosomal encoded surface antigens employing both cosmid and 1 ZapII gene libraries in E. coli, by screening for colonies/plaques reacting with C. burnetii-specific antisera and monoclonal antibodies to isolates with differing disease potential. The distribution of antigens in C. burnetii isolates will be determined using surface iodination, PAGE, and western analyses. To understand the role of gene in virulence and intracellular growth of C. burnetii, we have designed experiments to evaluate gene function directly within the organism. We will use previously cloned C. burnetii chromosomal and plasmid genes as specific probes to assay their transcriptional activation at different times and under different conditions, in vitro and in vivo. Concurrently, we will examine qualitative differences in C. burnetii RNA production. Genes specifying unique or differentially expressed transcripts produced at different time points in vitro or post-infection will be cloned and sequenced. Proteins being synthesized under these conditions will also be evaluated by 1 and 2 -D PAGE. In this fashion we will develop an understanding of gene expression in C. burnetii during the host-parasite interaction. We also will examine plasmids and the chromosome for alternative virulence functions; factors that enhance intracellular survival and growth and thus pathogenesis. We will clone genes encoding key metabolic functions as well as those shown to be important for intracellular survival of other organisms. These genes will be cloned from C. burnetii employing the methods of PCR, mutant complementation and using homologous genes as probes. And because the development of genetic exchange systems is vital to an understanding of rickettsial genetic mechanisms, we will continue to develop transformation and mutagenesis systems for C. burnetii using electroporation and antibiotic selection in cell cultures.

Coxiella burnetii是急性Q发烧和慢性的原因 人类心内膜炎。 质粒和染色体差异相关 由分离物引起的疾病类型（急性或慢性） 不同的分离株具有不同的毒力决定因素。 我们有 设计了实验，以扩展我们对C. burnetii基因组和 质粒DNA及其与疾病的关系。 独特的质粒区域将 通过克隆，限制酶，南部杂交和 测序分析。 我们还将确定 复制burnetii的转化和诱变研究。 质粒编码的表面蛋白（可能的毒力因子）将是 通过寻找对蛋白酶敏感的表面蛋白的确定 含有PUC/C的Maxicell。 burnetii质粒构建体以及tnphoa 诱变。 我们将确定染色体编码的表面抗原 通过筛查在大肠杆菌中同时使用cosmid和1个Zapii基因库 用于菌落/斑块与伯内特特异性抗体抗体反应的菌落和 具有不同疾病潜力的分离株的单克隆抗体。 这 抗原在C. burnetii分离株中的分布将使用 表面碘化，页面和西方分析。 了解 burnetii的毒力和细胞内生长的基因，我们设计了 直接在生物体中评估基因功能的实验。 我们 将使用先前克隆的伯氏菌C. burnetii染色体和质粒基因作为 在不同的特定探针上测定其转录激活 时间和在不同条件下，体外和体内。 同时 我们将检查C. burnetii RNA产生的定性差异。 指定在以上产生的独特或差异表达的转录本的基因 在体外或感染后的不同时间点将被克隆，并且 测序。 在这些条件下合成的蛋白质也将是 通过1和2 -D页面进行评估。 以这种方式，我们将开发 在宿主 - 寄生虫期间对基因表达的理解 相互作用。 我们还将检查质粒和染色体 替代毒力功能；增强细胞内的因素 生存和生长，因此发病机理。 我们将克隆基因编码 关键的代谢功能以及证明对 其他生物的细胞内生存。 这些基因将从 C. burnetii采用PCR，突变体互补和使用的方法 同源基因作为探针。 而且因为遗传的发展 交换系统对于理解立克遗传至关重要 机制，我们将继续发展转化和诱变 用于使用电穿孔和抗生素选择的C. burnetii的系统 细胞培养。