Proteomic and systems biology analysis of the response of monocytes to infection by Coxiella burnetii and exposure to innate immune adjuvants
Shipman, Matthew Richard.
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Coxiella burnetii is an obligate intracellular pathogen that infects human monocytes, specifically inhabiting the phagolysosome. C. burnetii is a potential bioterror agent and is classified by the National Institute for Allergies and Infectious Diseases (NIAID) as a category B pathogen. This bacterium is remarkably infectious, requiring as little as one bacterium to cause infection. We used phase II C. burnetii, an avirulent laboratory strain that acts as a model for wild type phase I strains. Our research was directed towards a deeper understanding of the monocyte proteome in response to a) infection by phase II C. burnetii, and b) exposure to immune adjuvants known to increase monocyte resistance to infection by C. burnetii. Monomac I cells were infected with phase II C. burnetii and aliquots were taken at 24, 48, and 96 hours postinfection. Experiments with immune adjuvants that increase monocyte killing of C. burnetii, involved Monomac I cells treated with Securinine, E. coli lipopolysaccharide (LPS), and monophosphoryl lipid A (MPL). Securinine is a GABA A receptor antagonist that is being developed at Montana State University for biodefense purposes, and triggers an innate immune response that differs from classic Toll-like receptor (TLR) stimulation of innate immunity represented by LPS and MPL. We employed multiplex 2D gel electrophoresis (m2DE) using ZDyes, a new generation of covalent fluorescent protein dyes being developed at Montana State University, coupled with MS/MS analysis and bioinformatics to determine the proteome changes in Monomac I cells in response to the conditions described above, and to develop a preliminary mechanistic model using a systems biology approach to account for the observed changes and propose multiple testable hypotheses to focus downstream research efforts. We also tested the effects on Monomac I cells infected with phase II C. burnetii +/- Securinine. We observed a high proportion of cell death in the + Securinine samples, using a dosage of Securinine higher than the optimal effective dosage. The information derived from this experiment will be useful in monitoring the tendency towards cell death in Securinine treated samples both from C. burnetii infected monocytes and other cell types (e.g. neurons) that contain GABA A receptors.