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Ehrlichia chaffeensis (E. chaffeensis) has evolved eukaryotic ligand mimicry to repurposes multiple cellular signaling pathways for immune evasion. In this investigation, we demonstrate that TRP120 has a novel repetitiveshortlinermotif (SLiM) that activates the evolutionarily conserved Hedgehog (Hh) signaling pathway to inhibit apoptosis. In silico analysis revealed that TRP120 has sequence and functional similarity with Hh ligands and a candidate Hh ligand SLiM was identified. siRNA knockdown of Hh signaling and transcriptional components significantly reduced infection. Co-immunoprecipitation and surface plasmon resonance demonstrated that rTRP120-TR interacted directly with Hh receptor Patched-2 (PTCH2). E. chaffeensis infection resulted in early activation of Hh transcription factor GLI-1 and upregulation of Hh target genes. Moreover, soluble recombinant TRP120 (rTRP120) activated Hh and induced gene expression consistent with the eukaryotic Hh ligand. The TRP120 Hh SLiM (NPEVLIKD) induced nuclear translocation of GLI-1 in THP-1 cells and primary human monocytes and induced a rapid and expansive activation of Hh pathway target genes. Furthermore, Hh activation was blocked by an α-TRP120 Hh SLiM antibody.TRP120 Hh SLiM significantly increased levels of Hh target, anti-apoptotic protein B-cell lymphoma 2 (BCL-2), and siRNA knockdown of BCL-2 dramatically inhibited infection.Blocking Hh signaling with the inhibitor Vismodegib, induced a pro-apoptotic cellular program defined by decreased mitochondria membrane potential, significant reductions in BCL-2, activation of caspase 3 and 9, and increased apoptotic cells. This study reveals a novel E. chaffeensis SLiM ligand mimetic that activates Hh signaling to maintain E. chaffeensis infection by engaging a BCL-2 anti-apoptotic cellular program.Author summaryEhrlichia chaffeensis is an obligately intracellular bacterium that preferentially infects and replicates within mononuclear phagocytes and survives intracellularly by modulating cellular signaling pathways to subvert innate immune defenses. This investigation reveals the complex and expanding role that the E. chaffeensis TRP120 moonlighting effector and SLiM ligand mimetics have on immune subversion and infection through activation and regulation of evolutionarily conserved signaling pathways. Herein, we define a TRP120 Hh SLiM mimetic that induces Hh signaling and regulates the anti-apoptotic protein BCL-2 to prevent sequential activation of caspase 9 and 3, promoting E. chaffeensis infection. This study defines a novel prokaryotic SLiM mimetic that repurposes evolutionarily conserved eukaryotic signaling pathways to promote survival of an intracellular bacterium.