Coxiella burnetii is the etiologic bacterial agent of the disease Q fever, an acute, flu-like illness that can progress to endocarditis in immunocompromised individuals. In contrast to most pathogens, C. burnetii thrives in an acidic, lyosome-like compartment called the parasitophorous vacuole (PV) within alveolar macrophages. The pathogen secretes bacterial proteins into the host cytosol via a type IV secretion system (T4SS) to control host signaling events required for PV generation and prevention of apoptosis. Importantly, host kinase pathways play a major role in C. burnetii infection, as protein kinase C, myosin light chain kinase, and others are required for PV generation. Additionally, Akt and Erk1/2 are activated during infection and required for C. burnetii prevention of host cell apoptosis. In this dissertation, I show that cAMP-dependent protein kinase (PKA) plays a role in two critical aspects of C. burnetii infection of macrophages. PKA is a versatile host kinase involved in over 100 signaling events including transcriptional regulation, cytokine signaling, survival signaling, and regulation of the cytoskeleton. We demonstrate PKA's requirement for PV formation and show that PKA activity increases during infection. Several downstream PKA substrates are differentially phosphorylated, including the immune regulatory protein p105 and the pro-apoptotic protein Bad, suggesting that C. burnetii directs specific signaling events through PKA. Additionally, PKA is required for C. burnetii prevention of macrophage apoptosis in a mechanism that involves phosphorylation and inactivation of the pro-apoptotic protein Bad. During infection, Bad is phosphorylated at Ser155 by PKA and is phosphorylated at Ser136, likely by Akt. Phosphorylation results in binding to the adaptor protein 14-3-3β, and we show that 14-3-3β and Bad are recruited to the C. burnetii PV. Interestingly, the proteins remain localized at the PV during induction of apoptosis; however, PKA inhibition prevents Bad recruitment to the PV. We also discovered that PKA subunits localize to the PV in a T4SS-dependent manner, and this recruitment correlates with the time of PKA activation during infection. Lastly, we identified the actin modulatory protein, vasodilator-stimulated phosphoprotein (VASP), as a candidate PKA-regulated protein involved in PV formation and determined that PKA inhibition disrupts the actin cytoskeleton. Importantly, actin polymerization is required for PV generation, and disruption of the actin network likely alters PV generation. Collectively, these results support a model in which C. burnetii recruits PKA to the pathogen's replication vacuole to direct specific signaling events required for PV generation and maintenance and apoptosis inhibition.
|Advisor:||Voth, Daniel E.|
|Commitee:||Blevins, Jon S., MacNicol, Angus M., Morrison, Richard P., Ponnappan, Usha|
|School:||University of Arkansas for Medical Sciences|
|Department:||Microbiology and Immunology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 74/09(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Microbiology, Immunology|
|Keywords:||Apoptosis, Coxiella burnetii, Intracellular pathogen, Macrophages, Parasitophorous vacuole, cAMP dependent protein kinase|
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