Genetics of Coxiella burnetii

我们在从感染细胞中获取适合于微阵列分析的伯氏梭菌RNA方面取得了重大进展。结合我们的系统同步感染环境稳定的SCV细胞，我们现在可以研究发生在伯氏梭菌形态发育过程中的协调基因调控。此外，我们已经制定了一种缓冲液，显著改善了伯氏梭菌胞外代谢的适合性。这将使研究在特定条件下对不同环境刺激的转录反应成为可能。 我们已经完成了K和G人类心内膜炎分离株以及减毒Dugway分离株的基因组测序。广泛的生物信息学分析正在进行中，以确定柯克斯体致病决定因素、进化关系和基因组可塑性的机制。 作为我们正在进行的开发伯氏梭菌遗传系统的努力的一部分，我们开发了一种新的克隆有机体的方法，该方法包括通过显微操作从感染的细胞单层中切除单个携带伯氏弧菌的空泡。这是一种获得伯氏梭菌克隆的有效和可重复性的方法，利用这一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。 脂多糖是唯一确定的伯氏卡氏杆菌毒力因子。产生全长内毒素的毒力I相生物，在重复体外传代后，转化为无毒II期生物，合成严重截短的内毒素。使用前面描述的克隆程序，我们已经克隆了一些柯克斯体不同分离株的II期克隆，并正在进行扩展。这些克隆的扩展将允许通过重新测序微阵列进行SNP分析，以定义SNPs和其他与转换到II期和无毒相关的遗传多态。 利用体外转录和翻译产生的蛋白质，我们已经构建了一个包含大约1500个柯克斯体蛋白质(占柯克斯体蛋白质组的75%)的蛋白质微阵列。通过用一组人类免疫血清探测这个阵列，我们已经识别了大约50个免疫原性蛋白。与免疫原性最强的10种蛋白相对应的融合蛋白已被提纯，目前正在一种用于Q热诊断的ELISA甲酸盐中进行测试。未来的计划包括在Q热小鼠模型上测试这些蛋白质的疫苗效力。

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.