Ehrlichia chaffeensis selectively infects mononuclear phagocytes and resides in early-endosome-like compartments, forming membrane-bound microcolonies called morulae. The mechanisms by which E. chaffeensis is internalized, establishes intracellular infection and avoids innate host defenses are not understood, but appear to occur through functionally relevant host-pathogen interactions associated with newly described type 1 secretion (T1S) tandem repeat protein (TRP) effectors. We have determined that TRP120 is translocated into the host cell nucleus, contains a novel TR DNA binding domain, binds a GC-rich DNA motif, and is a substrate of host ubiquitin (Ub) and small ubiquitin-like modifier (SUMO) post translational modification (PTM) pathways, which are known to extensively expand interactive and functional capability of eukaryotic proteins. The goal of this study is to demonstrate that TRP120 is a dual-function transcription factor that has interplay with the Ub/SUMO pathways to mediate nuclear translocation, subnuclear associations and regulate host defense gene expression. Through this investigation we will define the regulatory mechanisms mediating TRP120 trafficking to the nucleus, characterize subnuclear localization and interactions, determine the molecular basis of TRP120-DNA binding, and role of TRP120 and PTMs in modulating host gene expression using state-of-the-art molecular and cellular approaches. In the first aim, we will use TRP120 mutants and small molecule inhibitors to determine the role of eukaroytic PTMs in TRP120 nuclear localization and define subnuclear localization and molecular interactions. In the second aim, TRP120-DNA interactions, transcription factor function, and role of TRP120 PTMs will be investigated using molecular approaches including one-yeast hybrid, in vitro transcription, and mammalian reporter gene assays. The capacity of TRP120 to modulate host target gene expression and epigenetic patterns in a cellular context will be examined in aim three using TRP120 target gene expression arrays and chromatin immunoprecipitation to determine DNA and histone modifications as well as functional assays to examine defects in cellular defense mechanisms. As the important role of microbial nuclear effectors in pathobiology is emerging, the molecularly characterized Ehrlichia TRP effectors offer a relevant and well defined model for investigating mechanisms of direct transcriptional modulation of host genes by this molecular strategy of pathogen-directed manipulation of the phagocyte. Characterization of the host cell pathways modulated by ehrlichial effectors and the molecular mechanisms through which these are mediated will expand our understanding of the cell biology of infection and survival by intracellular microbes. This is necessary to identify novel host targets for therapeutics based on mechanistically defined host-pathogen interactions potentially utilized by a wide variety of pathogens.
Ehrlichia chaffeensis is the etiologic agent of the life-threatening disease human monocytotropic ehrlichiosis. E chaffeensis selectively invades mammalian mononuclear phagocytes and establishes intracellular infection through effector-mediated mechanisms that manipulate host cellular functions and evade innate immune responses. Molecular characterization of these processes provides insight into mechanisms through which microbes exploit host cellular functions, and identifies novel pharmacological targets in the host organism for antimicrobial therapeutics.
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