Characterizing Notch Ligand Mimic Function of the Ehrlichia TRP120 Effector in Suppression of Host Cell Apoptosis

点击翻译按钮获取中文摘要

PROJECT SUMMARY/ABSTRACT Human monocytotropic ehrlichiosis (HME) is a tick-borne zoonosis caused by the intracellular gram-negative bacterium, Ehrlichia chaffeensis. Since 2000, only 200 cases of ehrlichiosis were reported, however in 2017 the number of reported cases has increased to 1,642. Notably, the number of reported cases is underestimated by 100-fold due to underreporting and underdiagnosing. HME results in patient hospitalization in 43–62% of cases and progression of the disease can result in life threatening outcomes, including respiratory failure, meningitis, renal failure, hepatic failure, and coagulopathies. This study aims to unmask the molecular mechanisms utilized by E. chaffeensis to repurpose host immune defenses through pathogen mimicry of host proteins for infection and survival. Molecular mimicry is a well-documented survival stategy utilized by pathogens. In particular, several pathogens have evolved specific interaction motifs that mimic host motifs to facilitate host-pathogen interactions for exploitation of host machinery. Recent studies by our laboratory have shown host-pathogen interactions to occur through the tandem repeat protein (TRP) effector, TRP120, which is able to directly activate Notch signaling. Notch activation has been shown to play significant roles in various other functions, including innate immune mechanisms such as autophagy and apoptosis. Recent studies have demonstrated inhibition of host cell apoptosis as a mechanism utilized by E. chaffeensis for survival. Activation of Notch has shown to assist in inhibition of apoptosis by stabilizing expression of an anti-apoptotic protein, X-Linked Inhibitor of Apoptosis (XIAP). Interestingly, TRP120 has been shown, by our laboratory, to directly interact with ADAM17, a Notch metalloprotease, and FBW7, a Notch antagonist. Further, colocalization of TRP120 with both ADAM17 and the Notch-1 receptor has been demonstrated. Using sequence homology databases, we have demonstrated sequence homology of a motif in the tandem repeat domain of TRP120 (TRP120-TR) and several Notch ligands. Moreover, we have demonstrated the TRP120-TR domain is necessary for Notch activation during E. chaffeensis and have currently identified a 35-amino acid TRP120 Notch activation motif. We investigated expression levels of XIAP during infection to determine if Notch activation is potentially leading to stability of XIAP. Results demonstrated an increase in XIAP expression at later time points of infection. In the present study, which is the next step toward achieving our main research goal, we will perform protein-protein interaction studies using surface plasmon resonance (SPR) and atomic force microscopy (AFM) with TRP120 recombinant proteins and synthetic peptides to define the precise TRP120 Notch ligand mimic motif and characterize TRP120-Notch receptor interaction. We will further use in vitro studies to determine the functional role of Notch activation during E. chaffeensis infection. The extensive training I will receive while completing this project will advance my knowledge in the fields of cell biology, pathology and infectious disease, and will provide the necessary training and resources needed to advance as an independent researcher in academia in the future.