BIOCHEMICAL AND GENETIC MECHANISMS OF OBLIGATE INTRACELLULAR PARASITISM

专性细胞内寄生的生化和遗传机制

• 批准号: 3960467
• 负责人: J C WILLIAMS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3960467
• 关键词: Escherichia coli; Q fever; autoradiography; bacterial DNA; bacterial genetics; bacterial polysaccharides; biological polymorphism; chromatography; communicable disease diagnosis; communicable diseases; electron microscopy; gene expression; genetic library; genetic manipulation; genetic strain; histochemistry /cytochemistry; host organism interaction; intracellular parasitism; lipopolysaccharides; microorganism culture; molecular cloning; nucleic acid sequence; nucleic acid structure; plasmids; silver impregnation; ultracentrifugation; virulence

Mechanisms of phase variation in Coxiella burnetii, the etiological agent of Q-fever, were studied using variously virulent strains with different lipopolysacharide structures to obtain possible molecular correlates of attentuation. A. Genetic heterogeneity. Chromosomal DNA from the Nine Mile phase I strain of C. burnetii (CB9MIC7) cloned into the cosmid vector pHC79 was used as a probe to show a HAE III fragment present in the parent strain but absent from a spontaneously derived Nine Mile phase II strain (CB9MIIC4). An 18 kb deletion in the chromosomal DNA of CB9MIIC4 was identified. Another intrastrain spontaneous derivative, CB9M1514, also lacked the sentinel Hae III fragment. This strain carried a deletion which was approximately 29 kb, and both deletions appeared to share a common terminus within the limits of resolution. In all other strains investigated, both phase I and phase II, the DNA represented by the insert seemed grossly intact. B. Lipopolysaccharide. LPSs extracted from nine strains of C. burnetii were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and lethal toxicities in galatosamine sensitized mice. The structure of a unique disaccharide prepared from the CB9MIC7 strain was determined using negative ion extraction fast atom bombardment mass spectrometry of the N-acetylated disaccharide, direct chemical ionization mass spectrometry of the N-acetylated then permethylated disaccharide, and DCI and FAB mass spectrometry of reduced permethylated and peracetylated derivatives. The chemical structure was described as galactosaminuronyl-yield(1-6)-glucosamine [GalNU-yield(1-6)-GlcN], (C12H22N2)10] and Mr of 354. Using GalnU-yield(1-6)-G1cN and two recently described sugars, virenose and dihydrohydroxystreptose, as biochemical markers of truncated LPSs, we were able to relate Mr of selected molecular species of LPSs to truncation of LSP of C. burnetii intra- and inter- specific strains. Smooth-type LPS contained all three compounds, semi rough LPS did not contain virenose, and rough LPS was deficient in all three components. All of the LPSs were toxic in galactosamine sensitized mice albeit they were 100- to 1000-fold less toxic than Escherichia coli and Salmonella typhimurium endotoxins. Significance: Identification of chromosomal and plasmid DNA participating in virulence expression and LPS biosynthesis will facilitate our understanding of phase variation and endotoxin activities of C. burnettii.

Coxiella burnetii（病因学剂）相变的机理 使用不同的毒气菌株对Q-fever进行了研究 脂吞乳化物结构以获得可能的分子相关性 注意力。 A.遗传异质性。 九英里的染色体DNA C. burnetii（CB9MIC7）的第一阶段菌株被克隆到Cosmid Vector PHC79中 被用作探针，以显示父母菌株中存在的HAE III片段 但是自发得出的九英里II期应变不存在 （CB9MIIC4）。 CB9MIIC4的染色体DNA中的18 kb缺失为 确定。 另一个自发衍生物CB9M1514也 缺少哨兵Hae III碎片。 这种菌株带来了删除 大约是29 kb，两个删除似乎共享一个常见 末端在分辨率的范围内。 在所有其他压力中 研究了I期和II期，DNA由插入物表示 似乎非常完整。 B.脂多糖。 从九个提取的LPS 分析了burnetii的菌株的菌株分子分子的化学成分 十二烷基硫酸钠聚丙烯酰胺凝胶电泳的异质性， 葡萄醛胺敏化小鼠的致命毒性。 一个结构 使用CB9MIC7菌株制备的独特二糖 负离子提取快速原子轰击质谱 N-乙酰化二糖，直接化学电离质谱法 N-乙酰化，然后二苄二糖，DCI和Fab质量 氯化和丙酰化衍生物还原的光谱法。 这 化学结构被描述为 半乳糖氨基硝基元（1-6） - 葡萄糖胺[Galnu-Yield（1-6）-Glcn]， （C12H22N2）10]和354的MR。使用Galnu-Yield（1-6）-G1CN和两个 描述的糖，毒素和二氢羟化蛋白糖是生化的 截短的LPS的标记，我们能够关联选定分子的MR LPS的种类，可截断Burnetii C. burnetii C. c. 特定菌株。 平滑型LP包含所有三种化合物，半 Rough LPS不含病毒烯糖，而Rough LPS则不足 三个组成部分。 所有的LPS都在半乳糖胺中有毒 小鼠尽管它们的毒性比大肠杆菌低100至1000倍 和沙门氏菌鼠内毒素。 意义：识别 参与毒力表达和LPS的染色体和质粒DNA 生物合成将有助于我们对相位变化和 C. burnettii的内毒素活性。