Pittner, N. | Search bioRxiv

bioRxiv2023

Ehrlichia Wnt short linear motif ligand mimetic deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis

Ehrlichia chaffeensis TRP120 effector has evolved short linear motif (SLiM) ligand mimicry to repurpose multiple evolutionarily conserved cellular signaling pathways including Wnt, Notch and Hedgehog. In this investigation, we demonstrate that E. chaffeensis and recombinant TRP120 deactivate Hippo signaling resulting in activation of Hippo transcription coactivator Yap and target gene expression. Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and {beta}-catenin activation induced by E. chaffeensis, rTRP120 and Wnt5a. Similar Hippo gene expression profiles were also stimulated by E. chaffeensis, rTRP120, SLiM and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors (Yap and TEAD) significantly decreased E. chaffeensis infection. Yap activation was abolished in THP-1 Wnt Frizzled-5 (Fzd5) receptor knockout cells (KO), demonstrating Fzd5 receptor dependence. In addition, TRP120 Wnt-SLiM antibody blocked Hippo deactivation (Yap activation). Expression of anti-apoptotic Hippo target gene SLC2A1 (encodes glucose transporter 1; GLUT1) was upregulated by E. chaffeensis and corresponded to increased levels of GLUT1. Conversely, siRNA knockdown of SLC2A1 significantly inhibited infection. Higher GLUT1 levels correlated with increased BCL-xL and decreased Bax levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in levels of GLUT1 and BCL-xL, and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimetic and demonstrates that E. chaffeensis deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

microbiology↗

bioRxiv2023

Ehrlichia Notch signaling induction promotes XIAP stability and inhibits apoptosis

Ehrlichia chaffeensis has evolved multiple strategies to evade innate defenses of the mononuclear phagocyte. Recently, we reported the E. chaffeensis TRP120 effector functions as a Notch ligand mimetic and a ubiquitin ligase that degrades the nuclear tumor suppressor, F-box and WD repeat domain-containing 7 (FBW7), a negative regulator of Notch. The Notch receptor intracellular domain (NICD) is known to inhibit apoptosis primarily by interacting with X-linked inhibitor of apoptosis protein (XIAP) to prevent degradation. In this study, we determined E. chaffeensis activation of Notch signaling increases XIAP levels, thereby inhibiting intrinsic apoptosis. Increased NICD and XIAP levels were detected during E. chaffeensis infection and after TRP120 Notch ligand mimetic peptide treatment. Conversely, XIAP levels were reduced in the presence of Notch inhibitor DAPT. Cytoplasmic colocalization of NICD and XIAP was observed during infection and a direct interaction was confirmed by co-immunoprecipitation. Procaspase levels increased temporally during infection, consistent with increased XIAP levels; however, knockdown of XIAP during infection significantly increased apoptosis and Caspase-3, -7 and -9 levels. Further, treatment with SM-164, a second mitochondrial activator of caspases (Smac/DIABLO) antagonist, resulted in decreased procaspase levels and increased caspase activation, induced apoptosis, and significantly decreased infection. In addition, iRNA knockdown of XIAP also decreased infection and significantly increased apoptosis. Moreover, ectopic expression of TRP120 HECT Ub ligase catalytically defective mutant in HeLa cells decreased NICD and XIAP levels and increased caspase activation compared to WT. This investigation reveals a mechanism whereby E. chaffeensis repurposes Notch signaling to stabilize XIAP and inhibit apoptosis. Author SummaryEhrlichia chaffeensis is a tick-borne, obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes. E. chaffeensis survives by mobilizing various molecular strategies to promote cell survival, including modulation of apoptosis. This investigation reveals an E. chaffeensis initiated, Notch signaling regulated, antiapoptotic mechanism involving inhibitor of apoptosis proteins (IAPs). Herein, we demonstrate that E. chaffeensis induced Notch activation results in Notch intracellular domain stabilization of X-linked inhibitor of apoptosis protein (XIAP) to inhibit intrinsic apoptosis. This study highlights a novel mechanistic strategy whereby intracellular pathogens repurpose evolutionarily conserved eukaryotic signaling pathways to engage an antiapoptotic program for intracellular survival.

microbiology↗