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.

我们在从受感染的细胞中获取适合微阵列分析的伯内特衣藻 RNA 方面取得了重大进展。 与我们的系统相结合，用环境稳定的 SCV 同步感染细胞，我们现在可以研究 C. burnetii 形态发育过程中发生的协调基因调控。 此外，我们还配制了一种缓冲液，可以显着改善伯内氏梭菌的细胞外代谢适应性。这将允许研究伯内特衣藻在特定条件下对不同环境刺激的转录反应。 我们已经完成了 K 型和 G 型人类心内膜炎分离株以及减毒杜格威分离株的基因组测序。广泛的生物信息分析正在进行中，以确定柯克斯体的致病决定因素、进化关系和基因组可塑性机制。 作为我们开发伯内氏衣藻遗传系统的持续努力的一部分，我们开发了一种克隆生物体的新方法，该方法涉及通过显微操作从受感染的单层细胞中切除单个载有伯内氏衣藻的液泡。 这是获得伯氏梭菌克隆的有效且可重复的程序，并且利用该技术将极大地帮助我们克隆和分析该生物体的同基因突变体的能力。 脂多糖是伯氏念珠菌唯一确定的毒力因子。 产生全长 LPS 的毒力 I 期生物体在体外重复传代后转化为无毒力 II 期生物体，合成严重截短的 LPS。使用所描述的克隆程序，我们已经克隆并正在扩展不同柯克斯体分离株的许多 II 期克隆。 这些克隆的扩展将允许通过重测序微阵列进行 SNP 分析，以确定 SNP 和与 II 期转化和无毒力相关的其他遗传多态性。 利用体外转录和翻译产生的蛋白质，我们构建了包含大约 1500 个柯克斯体蛋白（柯克斯体蛋白质组的 75%）的蛋白质微阵列。通过用一系列人类免疫血清探测该阵列，我们鉴定出了大约 50 种免疫原性蛋白质。 与 10 种免疫原性最高的蛋白质相对应的融合蛋白已被纯化，目前正在 ELISA 甲酸盐中测试这些融合蛋白是否可用作 Q 热诊断。 未来的计划包括在 Q 热小鼠模型中测试这些蛋白质的疫苗功效。