Coxiella burnetii is an obligate intracellular bacterium which causes both acute Q fever and chronic endocarditis in humans. While information has accumulated concerning aspects of C. burnetii-host interaction, virtually nothing is known about virulence mechanisms, requirements for intracellular growth, or gene expression during intracellular growth. To develop an understanding of the nature and role of genes involved in the growth and virulence of C. burnetii the following experiments are proposed. 1) Characterize potential virulence factors with regard to their role in entry and survival in the host cell phagolysosome. Their subcellular location will be determined by iodination, PAGE, Western analyses and immunoelectron microscopy. Studies will determine if these factors effect host signal transduction pathways or have immunomodulatory properties. 2) To understand the factors necessary for C. burnetii to survive and replicate in the phagolysosome, experiments have been designed to characterize its development cycle. This will be done by characterizing the two stage-specific variants comprising the developmental cycle of C. burnetii. The variants will be purified by centrifugation and protein profiles characterized by 1D and 2D PAGE. Genes encoding proteins consistently unique to a variant form will be cloned by reverse genetics. N-terminal amino acid sequences will be used to clone the genes using oligonucleotide probes or by using degenerate primers and PCR to amplify C. burnetii DNA. 3) C. burnetii small cell variants (SCV) will be used to establish infections in cell cultures to examine gene expression by monitoring protein synthesis in vivo using radiolabeled amino acids. Initial stages of infection will be examined in vitro using acid activated C. burnetii SCV. Proteins expressed during infection and/or in vitro acid activation will be compared to the two cell variants. 4) Recently reported transformation procedures will be used to develop methods for gene inactivation and complementation in C. burnetii. Suicide vectors containing fragments of internal regions of C. burnetii genes plus antibiotic resistance genes will be introduced into C. burnetii to inactivate the target gene via homologous recombination. Shuttle vectors containing the plasmid origin of replication from C. burnetii plasmids will be introduced in C. burnetii mutants to carry out complementation studies. These experiments will allow the P.I. to directly study virulence factors and determine their role in the ability of these obligate intracellular bacteria to cause disease and persist in the body for extended periods of time,
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