Coxiella burnetii is the causative agent of both acute Q fever and chronic endocarditis in humans. Both plasmid and chromosomal differences correlate with the type of disease (acute or chronic) caused by an isolate suggesting that the different isolates have different virulence determinants. We have designed experiments to extend our knowledge of C. burnetii genomic and plasmid DNA and their relationship to disease. Unique plasmid regions will be analyzed by cloning, restriction enzyme, Southern hybridization and sequencing analyses. We will also identify the plasmid origin of replication for studies on transformation and mutagenesis of C. burnetii. Plasmid encoded surface proteins (possible virulence factors) will be identified by looking for protease-sensitive surface proteins produced by maxicells containing pUC/C. burnetii plasmid constructs and also by TnphoA mutagenesis. We will identify chromosomal encoded surface antigens employing both cosmid and 1 ZapII gene libraries in E. coli, by screening for colonies/plaques reacting with C. burnetii-specific antisera and monoclonal antibodies to isolates with differing disease potential. The distribution of antigens in C. burnetii isolates will be determined using surface iodination, PAGE, and western analyses. To understand the role of gene in virulence and intracellular growth of C. burnetii, we have designed experiments to evaluate gene function directly within the organism. We will use previously cloned C. burnetii chromosomal and plasmid genes as specific probes to assay their transcriptional activation at different times and under different conditions, in vitro and in vivo. Concurrently, we will examine qualitative differences in C. burnetii RNA production. Genes specifying unique or differentially expressed transcripts produced at different time points in vitro or post-infection will be cloned and sequenced. Proteins being synthesized under these conditions will also be evaluated by 1 and 2 -D PAGE. In this fashion we will develop an understanding of gene expression in C. burnetii during the host-parasite interaction. We also will examine plasmids and the chromosome for alternative virulence functions; factors that enhance intracellular survival and growth and thus pathogenesis. We will clone genes encoding key metabolic functions as well as those shown to be important for intracellular survival of other organisms. These genes will be cloned from C. burnetii employing the methods of PCR, mutant complementation and using homologous genes as probes. And because the development of genetic exchange systems is vital to an understanding of rickettsial genetic mechanisms, we will continue to develop transformation and mutagenesis systems for C. burnetii using electroporation and antibiotic selection in cell cultures.
Showing the most recent 10 out of 37 publications
