Genetics of Coxiella burnetii

伯内氏柯克斯体的遗传学

• 批准号: 7592301
• 负责人: robert a heinzen
• 金额: $ 83.28万
• 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/7592301
• 关键词: Accounting; Acute; Antibiotics; Antigens; Architecture; Attenuated; Bacteria; Biogenesis; Bioinformatics; Biology; Buffers; Categories; Cells; Centers for Disease Control and Prevention (U.S.); Chimeric Proteins; Chronic; Chronic Disease; Cloning; Collection; Condition; Coxiella; Coxiella burnetii; DNA; Development; Diagnostic; Dose; Endocarditis; Enzyme-Linked Immunosorbent Assay; Excision; Fever; Formates; Future; Gene Expression Regulation; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Genetic Transformation; Genetic Variation; Genome; Genomics; Human; Immune Sera; In Vitro; Individual; Infection; Influenza; Investigation; Length; Lesion; Lipopolysaccharides; Metabolic; Methods; Microarray Analysis; Micromanipulation; Modeling; Molecular Biology; Molecular Weight; Mus; Nature; Numbers; Organism; Organism Cloning; Other Genetics; Pathogenesis; Phagolysosome; Phase; Phenotype; Procedures; Production; Protein Microchips; Proteins; Proteome; Proteomics; Q Fever; RNA; Reproduction spores; Resistance; Screening procedure; Source; Specificity; Stimulus; Stress; Structure; Subunit Vaccines; System; Techniques; Testing; Translations; Vaccination; Vaccines; Vacuole; Variant; Virulence; Virulence Factors; Virulent; Whole Organism; Zoonoses; base; biothreat; comparative; egg; extracellular; factor C; fitness; genetic manipulation; genome sequencing; high throughput screening; immunogenic; improved; in vivo; insight; killings; monolayer; mutant; novel vaccines; pathogen; response; tissue culture; tool; vaccine efficacy

We have made significant progress in acquiring C. burnetii RNA from infected cells that is suitable for microarray analysis. In conjunction with our system to synchronously infect cells with the environmentally stable SCV, we can now investigate the coordinated gene regulation occurring during C. burnetii morphological development. Moreover, we have formulated a buffer that dramatically improves extracellular C. burnetii metabolic fitness. This will allow investigation of C. burnetii transcriptional responses to diverse environmental stimuli under defined conditions. We have finished sequencing the genomes of the K and G human endocarditis isolates and the attenuated Dugway isolate. Extensive bioinformatic analysis is ongoing to define Coxiella pathogenetic determinants, evolutionary relationships, and mechanisms of genome plasticity. As part of our ongoing efforts to develop genetic systems for C. burnetii, we have developed a new method to clone the organism that involves excision of individual C. burnetii-laden vacuoles from infected cell monolayers by micromanipulation. This is an efficient and reproducible procedure to obtain C. burnetii clones, and utilization of this technique will dramatically aid our ability to clone and analyze isogenic mutants of the organism. Lipopolysaccharide is the only defined virulence factor of C. burnetii. Virulent phase I organisms, producing full-length LPS, convert to avirulent phase II organisms, synthesizing severely truncated LPS, upon repeat in vitro passages. Using the cloning procedure descried about, we have cloned and are now expanding a number of phase II clones of different isolates of Coxiella. Expansion of these clones will allow SNP analysis via re-sequencing microarrays to define SNPs and other genetic polymorphisms associated with conversion to phase II and avirulence. Using proteins generated by in vitro transcription and translation, we have constructed a protein microarray that contains approximately 1500 Coxiella proteins (75% of the Coxiella proteome). By probing this array with a collection of human immune serum, we have identified roughly 50 immunogenic proteins. Fusion proteins corresponding to the 10 most highly immunogenic proteins have been purified and these are currently being tested in an ELISA formate for there utility as a Q fever diagnostic. Future plans include testing these proteins for vaccine efficacy in a murine model of Q fever.

我们在从感染的细胞中获取了适合微阵列分析的感染细胞中取得了重大进展。 结合我们的系统，与环境稳定的SCV同步感染细胞，我们现在可以研究在C. burnetii形态发展过程中发生的协调基因调节。 此外，我们制定了一个缓冲液，该缓冲液极大地改善了细胞外C. burnetii代谢适应性。这将允许在定义的条件下研究对各种环境刺激的转录响应。 我们已经完成了K和G人心内膜炎分离株和减弱的Dugway分离株的测序。正在进行广泛的生物信息学分析，以定义Coxiella致病决定因素，进化关系和基因组可塑性的机制。 作为我们为开发C. burnetii开发遗传系统的努力的一部分，我们开发了一种新方法来克隆有机体，涉及通过微观渗透从受感染的细胞单层中切除单个含有C. burnetii的液泡的生物。 这是获得C. burnetii克隆的有效且可再现的程序，该技术的利用将极大地有助于我们克隆和分析生物体的同源性突变体的能力。 脂多糖是C. burnetii的唯一定义的毒力因子。 具有全长LP的有力的I期生物，转化为无毒的II期生物，并在重复体外传递后，将严重截断的LPS合成。使用描述的克隆程序，我们已经克隆了克隆，现在正在扩展许多不同分离菌的II期克隆。 这些克隆的扩展将允许通过重新序列微阵列来定义SNP和其他遗传多态性与转化为II和II期和气势相关的其他遗传多态性。 使用体外转录和翻译产生的蛋白质，我们构建了一个蛋白质微阵列，其中包含大约1500个Coxiella蛋白（占Coxiella蛋白质组的75％）。通过探测该阵列的人类免疫血清，我们已经确定了大约50种免疫原性蛋白。 与10种最高度免疫原性蛋白相对应的融合蛋白已被纯化，目前正在ELISA甲酸盐中测试这些蛋白，以作为Q热诊断。 未来的计划包括在Q发烧的鼠模型中测试这些蛋白质的疫苗功效。