Bui, D. C. | bioRxiv Science

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↗

bioRxiv2022

Ehrlichia SLiM ligand mimetic activates Notch signaling in human monocytes

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. ImportanceE. 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 SummaryE. 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.

pathology↗