Ehrlichia Glycoprotein Modulation of Macrophage Gene Expression

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DESCRIPTION (provided by applicant): The long term goal of this research project is to understand the role of nuclear translocated proteins in ehrlichial pathobiology. Ehrlichia chaffeensis, an obligate intracellular bacterium, is the agent of human monocytotropic ehrlichiosis (HME), a life-threatening emerging tick-borne zoonosis. During E. chaffeensis infection, transcription levels of numerous monocyte genes are altered including those involved in innate and cell-mediated immune responses, apoptosis, membrane trafficking, signal transduction and cell cycle. The E. chaffeensis gp200, the largest ehrlichial glycoprotein characterized in our laboratory, has been identified within the nuclei of Ehrlichia-infected monocytes. Although macrophage gene expression appears to be altered to favor ehrlichial survival, the role played in gene regulation by nuclear translocated proteins of ehrlichial origin is unknown. This gap in scientific knowledge regarding the role of gp200 in the macrophage nucleus is an impediment towards understanding ehrlichial pathobiology and manipulating these medically important bacteria. The objective of this proposal is to determine the mechanism involved in gp200 nuclear translocation and to identify gp200 nuclear targets. We hypothesize that E. chaffeensis gp200 is translocated to the nucleus via a glycan-dependant nuclear import pathway, where it acts as a transcriptional repressor, activator and/or regulator to promote intracellular ehrlichial survival. We propose the following specific aims: 1) determine the host cell nuclear import pathway involved in the translocation of gp200 to the monocyte nucleus, and 2) identify the monocyte nuclear proteins and DNA motifs that interact with gp200 during infection. The nuclear import pathway will be mapped by observing the nuclear translocation of fluorescent gp200 in the presence of numerous pathway inhibitors. A protein/DNA array and SELEX analysis will be performed to identify the host genomic sequences bound by gp200, and the nuclear protein binding partners of gp200 will be identified with a protein/protein array and co- immunoprecipitation. Together, these results will further our understanding of pathogenic bacterial glycoprotein function and identify a role for protein glycosylation in ehrlichial virulence. HME is an emerging public health concern, and this research will facilitate development of new therapeutic interventions against one of the most prevalent life-threatening tick-borne diseases in North America.