Ehrlichia Wnt SLiM ligand mimic deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis

Abstract

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 the activation of Hippo transcription coactivator Yes-associated protein (Yap). Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and β-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 transcriptional enhanced associate domain (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, the 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 B cell lymphoma-extra large (BCL-xL) and decreased BCL2-associated X, apoptosis regulator (Bax) levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in GLUT1 and BCL-xL levels and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimic and demonstrates that E. chaffeensis deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Ehrlichia chaffeensis TRP120 ubiquitinates tumor suppressor APC to modulate Hippo and Wnt signaling

“…However, ubiquitinated APC was undetectable during mid to late infection when Ehrlichia replication is occurring. One explanation for this observation, is that E. chaffeensis is targeting APC before replication begins, to prevent host-cell apoptosis and is consistent with previous findings that demonstrate E. chaffeensis activates Yap to prevent apoptosis ( Byerly et al, 2023a ). Collectively, these results demonstrate that TRP120 ubiquitinates APC to stabilize active Yap and β-catenin levels in the nucleus to promote infection.…”

“…E. chaffeensis exploits evolutionarily conserved host cell signaling pathways, including Hippo, Wnt, Notch and Hedgehog to prevent apoptosis and other innate immune defenses for intracellular survival ( Rogan et al, 2021 ; Byerly et al, 2022 ; Patterson et al, 2022 ; Byerly et al, 2023a ). Both Hippo and Wnt are known for their role in regulating cellular proliferation and cell fate ( He et al, 2015 ; Wang et al, 2017 ; Qian et al, 2021 ), and we identified TRP120 as a novel Wnt ligand mimic that activates Yap and β-catenin, preventing apoptosis for ehrlichial survival ( Rogan et al, 2021 ; Byerly et al, 2023a ). Yap and β-catenin activate gene transcription under two conditions; when Hippo signaling is inactivated and APC is degraded ( Cai et al, 2015 ; Park et al, 2015 ; Wang et al, 2017 ; Lee et al, 2018 ; Jiang et al, 2020 ).…”

“…E. chaffeensis is an obligate intracellular, Gram-negative bacterium and the etiologic agent of the prevalent and life-threatening zoonosis, human monocytotropic ehrlichiosis (HME) ( Paddock et al, 1997 ; Paddock and Childs, 2003 ). E. chaffeensis exhibits tropism for mononuclear phagocytes and activates conserved cellular signaling pathways via tandem repeat protein (TRP) effectors to promote intracellular survival ( Rogan et al, 2021 ; Byerly et al, 2022 ; Patterson et al, 2022 ; Byerly et al, 2023a ). TRP effectors are multifunctional proteins secreted by the type-1 secretion system that interact with a diverse group of host cell proteins involved in essential cellular processes ( Wakeel et al, 2009 ; Luo et al, 2011 ; Wakeel et al, 2011 ; Luo and McBride, 2012 ; Lina et al, 2016 ; Luo et al, 2017 ; Luo et al, 2018 ; Byerly et al, 2021 ).…”

“…Notably, the 120 kDa tandem repeat protein (TRP120) has emerged as a model moonlighting effector that functions as a cell signaling pathway ligand mimic, ubiquitin ligase and nucleomodulin. More recently, TRP120 has been shown to activate evolutionarily conserved signaling pathways such as Hippo, Wnt, Notch, and Hedgehog through short linear motif (SLiM) ligand mimicry ( Rogan et al, 2021 ; Byerly et al, 2022 ; Patterson et al, 2022 ; Byerly et al, 2023b ; Rogan et al, 2021 ; Byerly et al, 2022 ; Patterson et al, 2022 ; Byerly et al, 2023a ).…”

“…TRP120 contains a novel Wnt SLiM within each tandem repeat that interacts with Wnt Fzd receptors to activate Hippo and Wnt transcription factors, Yap and β-catenin, to inhibit intrinsic host cell apoptosis ( Rogan et al, 2021 ; Byerly et al, 2023a ). Hippo and Wnt signaling are regulated by the tumor suppressor, adenomatous polyposis coli (APC), which targets and shuttles Yap and β-catenin out of the nucleus to a destruction complex within the cytoplasm ( Henderson, 2000 ; Azzolin et al, 2014 ; Cai et al, 2015 ).…”