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 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 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.

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

Abstract Ehrlichia chaffeensis evades innate host defenses by reprogramming the mononuclear phagocyte through mechanisms that involve exploitation of multiple evolutionarily conserved cellular signaling pathways including Notch. This immune evasion strategy is directed in part by tandem repeat protein (TRP) effectors. Specifically, the TRP120 effector activates and regulates Notch signaling through interactions with the Notch receptor and the negative regulator, F-Box and WD repeat domain-containing 7 (FBW7). However, the specific molecular interactions and motifs required for E. chaffeensis TRP120-Notch receptor interaction and activation have not been defined. To investigate the molecular basis of TRP120 Notch activation, we compared TRP120 with endogenous canonical/non-canonical Notch ligands and identified a short region of sequence homology within the tandem repeat (TR) domain. TRP120 was predicted to share biological function with Notch ligands, and a function-associated sequence in the TR domain was identified. To investigate TRP120-Notch receptor interactions, colocalization between TRP120 and endogenous Notch-1 was observed. Moreover, direct interactions between full length TRP120, the TRP120 TR domain containing the putative Notch ligand sequence, and the Notch receptor LBR were demonstrated. To molecularly define the TRP120 Notch activation motif, peptide mapping was used to identify an 11-amino acid short linear motif (SLiM) located within the TRP120 TR that activated Notch signaling and downstream gene expression. Peptide mutants of the Notch SLiM or anti-Notch SLiM antibody reduced or eliminated Notch activation and NICD nuclear translocation. This investigation reveals a novel molecularly defined pathogen encoded Notch SLiM mimetic that activates Notch signaling consistent with endogenous ligands. Importance E. chaffeensis infects and replicates in mononuclear phagocytes, but how it evades innate immune defenses of this indispensable primary innate immune cell is not well understood. This investigation reveals the molecular details of a ligand mimicry cellular reprogramming strategy that involves a short linear motif (SLiM) which enables E. chaffeensis to exploit host cell signaling to establish and maintain infection. E. chaffeensis TRP120 is a moonlighting effector that has been associated with cellular activation and other functions including ubiquitin ligase activity. Herein, we identify and demonstrate that a SLiM present within each tandem repeat of TRP120 activates Notch signaling. Notch is an evolutionarily conserved signaling pathway responsible for many cell functions including cell fate, development, and innate immunity. The proposed study is significant because it reveals the first molecularly defined pathogen encoded SLiM that appears to have evolved de novo to mimic endogenous Notch ligands. Understanding Notch activation during E. chaffeensis infection provides a model in which to study pathogen exploitation of signaling pathways and will be useful in developing molecularly-targeted countermeasures for inhibiting infection by a multitude of disease-causing pathogens that exploit cell signaling through molecular mimicry. Author Summary E. chaffeensis is a small, obligately intracellular, Gram-negative bacterium that has evolved cellular reprogramming strategies to subvert innate defenses of the mononuclear phagocyte. Ehrlichial TRP effectors interface with the host cell and are involved in pathogen-host interplay that facilitates exploitation and manipulation of cellular signaling pathways; however, the molecular interactions and functional outcomes are not well understood. This study provides molecular insight into a eukaryotic mimicry strategy whereby secreted effectors of obligately intracellular pathogens activate the evolutionarily conserved Notch signaling pathway through a short linear motif ligand mimetic to promote intracellular infection and survival. 

/pdf/i-ehrlichia-i-slim-ligand-mimetic-activates-notch-signaling-2wd9yrk6.pdf

<i>Ehrlichia</i> SLiM ligand mimetic activates Notch signaling in human monocytes

Abstract 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 repetitive s hort li ner m otif (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 summary Ehrlichia 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. 

Matt T. Trentman

Papers

<i>Ehrlichia</i> SLiM ligand mimetic activates Notch signaling in human monocytes

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