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Analysis of the C. burnetii Type 4B Secretion System homolog DotA During Infection and In Vitro Growth

Analysis of the C. burnetii Type 4B Secretion System homolog DotA During Infection and In Vitro Growth

Alicia Aguilar

Microbiology and Molecular Genetics

Coxiella burnetii is a globally distributed gram negative, obligate intracellular pathogen that is the causative agent of Q fever in humans. After infection, C. burnetii is known to traverse the endocytic pathway and replicate within a phago-lysosomal vacuole termed the Parasitophorous Vacuole (PV) while manipulating many host cell functions. The C. burnetii genome encodes for an effective virulence tool called the Type 4B Secretion System (T4BSS) which it uses to deliver bacterial effector proteins into the host cell cytosol. The T4BSS is typically composed of multiple proteins that form a syringe-like structure. One of the C. burnetii proteins hypothesized to play an important role in the development of the T4BSS is DotA. Data from previous studies show that DotA is released into the media during in vitro growth and that it colocalizes on the PV membrane during host cell infection. In order to identify how DotA is interacting with the host cell and other C. burnetii T4BSS components, we propose to develop a GST-DotA chimeric protein and perform pull down assays. Using Invitrogen’s Gateway technology we will clone a fragment of dotA into a GST vector. Once verified, we will express the GST-tagged DotA in E.coli and perform pull down assays against host cell lysates and axenically grown C. burnetii. We anticipate that DotA will interact with other proteins of the C. burnetii T4BSS as well as host cell proteins thereby highlighting its role in the structural formation of T4BSS as well as other unknown functions. The discoveries of DotA protein-protein interactions will help us understand the molecular mechanisms of C. burnetii pathogenesis and possibly identify key interactions that promote host cell infection.