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.

Ehrlichia chaffeensisTRP120 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 thatE. chaffeensisand 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 byE. chaffeensis, rTRP120 and Wnt5a. Similar Hippo gene expression profiles were also stimulated byE. chaffeensis, rTRP120, SLiM and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors (Yap and TEAD) significantly decreasedE. chaffeensisinfection. 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 geneSLC2A1(encodes glucose transporter 1; GLUT1) was upregulated byE. chaffeensisand corresponded to increased levels of GLUT1. Conversely, siRNA knockdown ofSLC2A1significantly 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 thatE. chaffeensisdeactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Beginning in 1997, a series of Ohio Supreme Court decisions ruled that Ohio's school foundation-based funding system was unconstitutional. Despite judicially mandated reform directives, little change was made until recently when Ohio adopted a modified Evidence-Based Model (EBM) into its statutory funding scheme. Ohio's EBM is intended to remedy many of the unconstitutional deficiencies found by the courts. However, due to artificial caps and guarantees on the funding that a district can receive, the EBM has yet to impact school funding in a major way. A review of the EBM, as enacted in Ohio, describes the changes to the system, as well as the shortcomings.

Ehrlichia chaffeensishas evolved multiple strategies to evade innate defenses of the mononuclear phagocyte. Recently, we reported theE. chaffeensisTRP120 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 determinedE. chaffeensisactivation of Notch signaling increases XIAP levels, thereby inhibiting intrinsic apoptosis. Increased NICD and XIAP levels were detected duringE. chaffeensisinfection 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 wherebyE. chaffeensisrepurposes Notch signaling to stabilize XIAP and inhibit apoptosis.

As an obligately intracellular bacterial pathogen that selectively infects the mononuclear phagocyte, Ehrlichia chaffeensis has evolved sophisticated mechanisms to subvert innate immune defenses. While the bacterium accomplishes this through a variety of mechanisms, a rapidly expanding body of evidence has revealed that E. chaffeensis has evolved survival strategies that are directed by the versatile, intrinsically disordered, 120 kDa tandem repeat protein (TRP120) effector. E. chaffeensis establishes infection by manipulating multiple evolutionarily conserved cellular signaling pathways through effector-host interactions to subvert innate immune defenses. TRP120 activates these pathways using multiple functionally distinct, repetitive, eukaryote-mimicking short linear motifs (SLiMs) located within the tandem repeat domain that have evolved in nihilo. Functionally, the best characterized TRP120 SLiMs mimic eukaryotic ligands (SLiM-icry) to engage pathway-specific host receptors and activate cellular signaling, thereby repurposing these pathways to promote infection. Moreover, E. chaffeensis TRP120 contains SLiMs that are targets of post-translational modifications such as SUMOylation in addition to many other validated SLiMs that are curated in the eukaryotic linear motif (ELM) database. This review will explore the extracellular and intracellular roles TRP120 SLiM-icry plays during infection - mediated through a variety of SLiMs - that enable E. chaffeensis to subvert mononuclear phagocyte innate defenses.