Ehrlichia chaffeensis Activates Notch Signaling Through SLiM Mimicry to Inhibit Apoptosis

Ehrlichia chaffeensis is a small, obligately intracellular gram-negative bacterium, and the etiological agent of human monocytotropic ehrlichiosis (HME), an emerging, life threatening tick-borne zoonosis. E. chaffeensis infects mononuclear phagocytes and has evolved molecular strategies to reprogram the host cell involving secreted effectors that interact directly with the host cell targets. Recently, we have shown E. chaffeensis evasion of innate defenses of the macrophage involve activation of Wnt, Hedeghog and Notch signaling pathways. Interestingly, the E. chaffeensis tandem repeat effector, TRP120, has been shown to interact with host proteins important for activation and regulation of conserved signaling pathways including Wnt, Notch and Sonic Hedgehog. In this study, we investigated the molecular interactions and functional implications of Notch activation during E. chaffeensis infection.

The objective of this research project is to identify the SLiM ligand mimetic in E. chaffeensis TRP120 and the functional implications of TRP120 Notch activation. Two aims were originally proposed to investigate this hypothesis. Aim 1 was to elucidate the molecular interactions required for TRP120 Notch activation. Aim 2 was to investigate the role of E. chaffeensis Notch stabilization of XIAP and inhibition of caspase activation. Based on the evidence collected during my research, I have concluded that a TRP120- TR Notch SLiM memetic motif directly binds Notch-1 at a region containing the LBD to activate Notch signaling. TRP120 Notch activation results in an anti-apoptotic program involving inhibitor of apoptosis proteins (IAPs) that inhibits caspase activation for intracellular survival. We demonstrate sequence homology between TRP120 and Notch ligands and determined that the TRP120-TR shares significant identity with known Notch ligands. We determined direct interactions between TRP120 and NECD recombinant protein containing ligand interaction domain, EGFs 1-13. We further defined the TRP120-TR domain as being capable of Notch activation and have defined the TRP120 Notch SLiM memetic motif required for Notch activation. Furthermore, we determined a direct correlation between Notch activation and inhibition of apoptosis linked to an increase in XIAP expression during E. chaffeensis infection. Increased XIAP levels correlated with increased NICD levels during E. chaffeensis infection and after TRP120 Notch ligand mimetic peptide treatment. Additionally, increased XIAP expression was consistent with increased pro-caspase levels. siRNA knockdown or inhibition of XIAP with small molecule inhibitor significantly increased apoptosis and Caspase-3, -7 and -9 levels and decreased ehrlichial load. This investigation reveals a mechanism whereby E. chaffeensis repurposes Notch signaling to stabilize XIAP and inhibit apoptosis. Understanding the molecular basis of Ehrlichia-Notch ligand mimicry is important for understanding the survival strategies of intracellular pathogenesis. Defining such interactions may lead to the development of novel therapeutics that target host-pathogen protein-protein interactions. The proposed study is highly significant in revealing a molecular mechanism whereby obligately intracellular pathogens, with small genomes and limited protein effectors, have evolved moonlighting proteins and molecular mimicry to rewire conserved signaling pathways and cellular functions to ensure persistent infection and survival. The identification of a short linear motif found within a non-canonical Notch ligand gives more insight into the underlying molecular mechanisms of aberrant Notch activation and may therefore lead to therapeutic approaches for diseases by which constitutively activated Notch signaling leads to disease onset and progression. Understanding Notch activation during E. chaffeensis infection will allow for the development of agents targeting critical steps of Notch signaling to inhibit infection and survival in the macrophage.