Genetics of Coxiella burnetii

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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.