Introduction- Coxiella burnetii is the etiologic agent of Q fever, a potential bioweapon and select agent, andone of the most infectious pathogens known. However, there are few reports concerning this obligateintracellular agent's molecular pathogenesis or developmental cycle, and lack of a system for site-directedgenetic manipulation has severely hampered research progress. To address this dearth of information and toimprove the current state of molecular biology technology, we propose to: 1) examine the expression profilesof small cell variants (SCVs) and large cell variants (LCVs) of the developmental cycle to identify genes andproteins that allow Coxiella to survive for extended periods of time in the environment (SCV stage) and tosubsequently grow in the inhospitable confines of the host cell phagolysosome (LCV stage); 2) analyze andcompare expression profiles of in vitro versus in vivo-cultivated Coxiella and phase I versus phase IIorganisms to identify potential virulence determinants, and 3) develop a system of site-directed mutagenesisfor this organism to allow for the routine genetic manipulation and future development of an attenuatedvaccine strain. These data are expected to provide valuable information on the life cycle of Coxiella and therespective genes and proteins involved, and should lead to strategies for disrupting cellular development tocontrol Q fever. Finally, a system for routine genetic manipulation of Coxiella is an essential molecularbiology tool for examining suspected virulence genes and performing molecular Koch's postulates in vivo.Project Interactions- Project 3 (Harmsen) will interact with Project 1 (this study) as it relates to in vivocultivatedCoxiella from the mouse model. Projects 4 (Pascual) and 2 (Jutila) will benefit from Project 1 dataon stage-specific gene products; SCV-specific genes / antigens are hypothesized to be involved intransmission / early infection and LCV-specific genes / antigens are involved in intracellular replication.
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