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 repetitive short liner motif (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 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 novel E. chaffeensis SLiM ligand mimetic that activates Hh signaling to maintain E. chaffeensis infection by engaging a BCL-2 anti-apoptotic cellular program.

EhrlichiaNotch signaling induction promotes XIAP stability and inhibits apoptosis

“…Pathogens have evolved various means of subverting innate immune defense of the host for survival (2,43,(55)(56)(57)(58)(59)(60). One of the most well-studied mechanisms of bacterial pathogens remix, or adapt this material for any purpose without crediting the original authors.…”

“…Infectious dense-cored E. chaffeensis with T1SS TRP120 effector on the surface binds (1) to the ligand binding region (LBR) of the Notch-1 receptor via the Notch ligand memetic motif (Patterson, Velayutham et al 2022) and activates Notch signaling. (2) NICD translocates to the nucleus and activates Notch gene transcription and (3) binds directly to XIAP prevent XIAP degradation, (4) inhibiting Caspase-3, −7 and −9 activation. (5) Active Caspase-3 may also cleave XIAP into two fragments: BIR1 and −2 and BIR3-RING.…”

“…Ehrlichia chaffeensis is an obligately intracellular Gram-negative bacterium, and the etiologic agent of human monocytotropic ehrlichiosis (HME), a life-threatening emerging tickborne zoonosis (1). E. chaffeensis preferentially infects mononuclear phagocytes and has evolved sophisticated molecular-based strategies to evade host defense mechanisms for survival (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). E. chaffeensis immune evasion strategies are mediated, in part, by tandem repeat proteins (TRPs).…”

“…TRPs are type 1 secretion system (T1SS) effectors that also elicit strong host antibody responses during infection (19–22). Notably, TRP120 decorates the surface of dense-cored ehrlichiae and has multiple moonlighting functions including as a transcription factor, HECT E3 ubiquitin ligase, and cellular signaling ligand mimetic to repurpose host cell signaling (2, 9, 13–16, 18, 23). These TRP120 functions directly impact host gene expression and chromatin epigenetics, pathogen-host interactions, and cellular signaling (2, 4–7, 9, 12–14, 18, 24, 25).…”

“…Our laboratory has recently reported that E. chaffeensis evasion of macrophage host defenses involves activation of conserved host signaling pathways, including Wnt, Notch and Hedgehog (2, 13, 14). Notably, TRP120 activates the evolutionarily conserved Notch signaling pathway using a novel molecularly defined pathogen-encoded Notch SLiM ligand mimetic found within the tandem repeat (TR) domain (13).…”