Project Summary Ehrlichia chaffeensis is a gram-negative, obligately intracellular bacterium that is the causative agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. A major knowledge gap is in our understanding of the mechanisms whereby E. chaffeensis establishes intracellular infection of the mononuclear phagocyte and avoids innate host defenses. We recently demonstrated that E. chaffeensis utilizes a type 1 secretion (T1S) system to export tandem repeat protein (TRP) effectors that interacts with host cell DNA and a functionally diverse array of host proteins involved in transcriptional and translational regulation, post translational modification, signaling, immune response, intracellular trafficking, cytoskeletal organization and apoptosis. The premise of this investigation is based on our new data that demonstrates E. chaffeensis also activates the Sonic Hedgehog (Shh) signaling pathway, and inhibition of Shh pathway results in cell death, thus limiting ehrlichial infection and replication. Shh is an evolutionary conserved pathway involved in vertebrate and invertebrate embryonic development; however, the pathway has more recently been associated with tissue homeostasis, regeneration and regulation of apoptosis. Activation of Shh involves nuclear translocation of the active GLI zinc finger transcription factor where it activates transcription of anti- apoptotic target genes. Preliminary studies that serve as the basis for this proposal demonstrate that E. chaffeensis TRP120 activates Shh signaling resulting in nuclear translocation of GLI1 and transcriptional upregulation of anti-apoptotic genes. The objective of this proposal is to demonstrate E. chaffeensis inhibits host cell apoptosis by activation of the Shh signaling pathway to modulate p53 and MCL1, and enhances activation of GLI1 targeted anti-apoptotic genes by altering the polycomb repressive complex (PRC). We hypothesize E. chaffeensis TRP120 activates Shh pathway to induce an anti-apoptotic cell survival program that involves p53/MCL1 modulation, and mediates epigenetic regulation of Shh targeted anti-apoptotic genes through PRC destabilization and JMJD3 demethylation. This central hypothesis will be examined by the following aims: (1) Demonstrate E. chaffeensis TRP120 effector activates the Shh pathway to inhibit apoptosis (2) Determine the influence of E. chaffeensis mediated destabilization of PRC1 on GLI targeted anti-apoptotic gene expression and epigenetics. This investigation will expand our knowledge regarding exploitation of highly conserved host cell pathways by E. chaffeensis effectors in order to facilitate bacterial intracellular survival. Moreover, new targets for the development of novel therapeutic approaches against E. chaffeensis infection will be identified.