EhrlichiaNotch signaling induction promotes XIAP stability and inhibits apoptosis

Abstract

AbstractEhrlichia 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.Author SummaryEhrlichia chaffeensisis a tick-borne, obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes.E. chaffeensissurvives by mobilizing various molecular strategies to promote cell survival, including modulation of apoptosis. This investigation reveals anE. chaffeensisinitiated, Notch signaling regulated, antiapoptotic mechanism involving inhibitor of apoptosis proteins (IAPs). Herein, we demonstrate thatE. chaffeensisinduced 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.