Ehrlichia SLiM ligand mimetic activates Hedgehog signaling to engage a BCL-2 anti-apoptotic cellular program

Abstract

Ehrlichia chaffeensis(E.chaffeensis) has evolved eukaryotic ligand mimicry to repurpose multiple cellular signaling pathways for immune evasion. In this investigation, we demonstrate that TRP120 has a novel repetitiveshortlinearmotif (SLiM) that activates the evolutionarily conserved Hedgehog (Hh) signaling pathway to inhibit apoptosis.In silicoanalysis 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.chaffeensisinfection resulted in early upregulation of Hh transcription factor GLI-1 and regulation 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 novelE.chaffeensisSLiM ligand mimetic that activates Hh signaling to maintainE.chaffeensisinfection by engaging a BCL-2 anti-apoptotic cellular program.