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: 2003277
• 负责人: LOUIS P MALLAVIA
• 金额: $ 20.71万
• 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 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2003277
• 关键词: Escherichia coli; Q fever; Rickettsiales; SDS polyacrylamide gel electrophoresis; bacterial genetics; bacterial proteins; cell transformation; endocarditis; gene expression; genetic strain; host organism interaction; immunoelectron microscopy; microorganism growth; molecular cloning; polymerase chain reaction; surface antigens; tissue /cell culture; transfection /expression vector; virulence; western blottings

DESCRIPTION (Adapted from the applicant's abstract): Coxiella burnetii is an obligate intracellular bacterium which causes both acute Q fever and chronic endocarditis in humans. While information has accumulated concerning aspects of C. burnetii-host interaction, virtually nothing is known about virulence mechanisms, requirements for intracellular growth, or gene expression during intracellular growth. To develop an understanding of the nature and role of genes involved in the growth and virulence of C. burnetii the following experiments are proposed. 1) Characterize potential virulence factors with regard to their role in entry and survival in the host cell phagolysosome. Their subcellular location will be determined by iodination, PAGE, Western analyses and immunoelectron microscopy. Studies will determine if these factors effect host signal transduction pathways or have immunomodulatory properties. 2) To understand the factors necessary for C. burnetii to survive and replicate in the phagolysosome, experiments have been designed to characterize its development cycle. This will be done by characterizing the two stage-specific variants comprising the developmental cycle of C. burnetii. The variants will be purified by centrifugation and protein profiles characterized by 1D and 2D PAGE. Genes encoding proteins consistently unique to a variant form will be cloned by reverse genetics. N-terminal amino acid sequences will be used to clone the genes using oligonucleotide probes or by using degenerate primers and PCR to amplify C. burnetii DNA. 3) C. burnetii small cell variants (SCV) will be used to establish infections in cell cultures to examine gene expression by monitoring protein synthesis in vivo using radiolabeled amino acids. Initial stages of infection will be examined in vitro using acid activated C. burnetii SCV. Proteins expressed during infection and/or in vitro acid activation will be compared to the two cell variants. 4) Recently reported transformation procedures will be used to develop methods for gene inactivation and complementation in C. burnetii. Suicide vectors containing fragments of internal regions of C. burnetii genes plus antibiotic resistance genes will be introduced into C. burnetii to inactivate the target gene via homologous recombination. Shuttle vectors containing the plasmid origin of replication from C. burnetii plasmids will be introduced in C. burnetii mutants to carry out complementation studies. These experiments will allow the P.I. to directly study virulence factors and determine their role in the ability of these obligate intracellular bacteria to cause disease and persist in the body for extended periods of time,

描述（改编自申请人的摘要）：Coxiella burnetii是 强制性细胞细菌，既引起急性Q发烧和 人类慢性心内膜炎。 虽然信息积累了 关于C. burnetii-host互动的各个方面，几乎没有什么是 知道毒力机制，细胞内生长的要求或 细胞内生长过程中的基因表达。 建立对 与C的生长和毒力有关的基因的性质和作用。 提出了以下实验。 1）表征潜力 毒力因素在其进入和生存中的作用 宿主细胞吞噬物体。 它们的亚细胞位置将由 碘化，页面，西方分析和免疫电子显微镜。 研究 将确定这些因素是否影响宿主信号转导途径或 具有免疫调节特性。 2）了解必要的因素 为了使C. burnetii在吞噬体中生存和复制，实验 旨在表征其开发周期。 这将完成 通过表征包括两个阶段的变体 C. burnetii的发育周期。 变体将被纯化 以1D和2D页为特征的离心和蛋白质曲线。 基因 编码蛋白质始终是变体形式独特的蛋白质。 反向遗传学。 N末端氨基酸序列将用于克隆 使用寡核苷酸探针或使用退化引物和PCR到TO的基因 放大C. burnetii DNA。 3）C。burnetii小细胞变体（SCV）将是 用于在细胞培养物中建立感染以检查基因表达 使用放射性标记的氨基酸在体内监测蛋白质合成。 使用酸激活，将在体外检查感染的初始阶段 C. Burnetii SCV。 在感染和/或体外酸中表达的蛋白质 将激活与两个细胞变体进行比较。 4）最近报道 转化程序将用于开发基因的方法 C. burnetii中的失活和补充。 含有自杀媒介 C. burnetii基因的内部区域的碎片以及抗生素 抗性基因将被引入C. burnetii中，以使其失活 通过同源重组靶基因。 穿梭媒介包含 将引入来自C. burnetii质粒复制的质粒起源 在C. burnetii突变体中进行互补研究。 这些 实验将允许P.I.直接研究毒力因素和 确定它们在这些强制性细胞内细菌的能力中的作用 为了引起疾病并持续长时间的体内，