Ehrlichia chaffeensis TRP120-mediated ubiquitination and proteasomal degradation of tumor suppressor FBW7 increases oncoprotein stability and promotes infection

Abstract

Ehrlichia chaffeensis (E. chaffeensis) exploits evolutionarily conserved Notch and Wnt host cell signaling pathways to downregulate innate immune host defenses and promote infection. The multifunctional E. chaffeensis TRP120 effector which has HECT E3 ubiquitin ligase activity, interacts with the host nuclear tumor suppressor F-BOX and WD domain repeatingcontaining 7 (FBW7). FBW7 is the substrate recognition subunit of the Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex (SCF) known to negatively regulate a network of oncoproteins (Notch, cyclin E, c-Jun, MCL1 and cMYC). In this study, we demonstrate that TRP120 and FBW7 colocalize strongly in the nucleus by confocal immunofluorescent microscopy and interactions between TRP120 and FBW7 FBOX and WD40 domains were demonstrated by ectopic expression and co-immunoprecipitation. Although FBW7 gene expression increased during E. chaffeensis infection, FBW7 levels significantly decreased (>70%) by 72 h post infection. Moreover, an iRNA knockdown of FBW7 coincided with increased E. chaffeensis infection and levels of Notch intracellular domain (NICD), phosphorylated c-Jun, MCL-1 and cMYC, which are negatively regulated by FBW7. An increase in FBW7 K48 ubiquitination was detected during infection by co-IP, and FBW7 degradation was inhibited in infected cells treated with the proteasomal inhibitor bortezomib. Direct TRP120 ubiquitination of native and recombinant FBW7 was demonstrated in vitro and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. This study identifies the tumor suppressor, FBW7, as a TRP120 HECT E3 Ub ligase substrate, and demonstrates that TRP120 ligase activity promotes ehrlichial infection by degrading FBW7 to maintain stability of Notch and other oncoproteins involved in cell survival and apoptosis.

Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with In Vivo Infection Assessment

“…Outer membranes of Gram-negative bacteria contain many proteins that perform essential functions, such as for the nutrient uptake mediated by porin activities, cell adhesion, cell signaling, and waste export, as well as to support the evasion of host defense mechanisms by pathogenic bacteria ( 40 ). Several P28 outer membrane proteins (P28/OMP) encoded from a multigene locus are identified as immunodominant proteins in E. chaffeensis ( 41 – 47 ). Similarly, the 120-kDa surface protein (also known as TRP 120) is recognized as an immunodominant protein of E. chaffeensis ( 41 – 47 ).…”

“…Several P28 outer membrane proteins (P28/OMP) encoded from a multigene locus are identified as immunodominant proteins in E. chaffeensis ( 41 – 47 ). Similarly, the 120-kDa surface protein (also known as TRP 120) is recognized as an immunodominant protein of E. chaffeensis ( 41 – 47 ). Mutations reported in the current study included two P28/OMP genes (p28-1/OMP-20 and OMP-1v/OMP-6) and TRP 120.…”

“…These data suggest that disruptions in the majority of the pathogen genes can be detrimental to its persistence in vivo and that the disrupted genes causing the rapid clearance are among the many essential genes of E. chaffeensis. chaffeensis (41)(42)(43)(44)(45)(46)(47). Similarly, the 120 kDa surface protein (also known as TRP 120) is recognized as an immunodominant protein of E. chaffeensis (41)(42)(43)(44)(45)(46)(47).…”

Interferon Type I Regulates Inflammasome Activation and High Mobility Group Box 1 Translocation in Hepatocytes During Ehrlichia‐Induced Acute Liver Injury

“…(34) These receptors could be triggered by Ehrlichia tandem repeat proteins (TRPs), such as TRP75 and TRP120, that are secreted into cytosol and are known to interact with a diverse group of human proteins associated with innate responses, multiple metabolic processes, and signal transduction. (14,35,36) Alternatively, bacterial or mitochondrial DNA can bind to toll-like receptor 9 (TLR9), another upstream receptor that is involved in production of IFN-I cytokines. This possibility is based on our study showing that the TLR9-MYD88 pathway plays a role in inflammasome activation in macrophages and IOE-induced liver injury.…”

“…The exact microbial ligand that triggers caspase-11 activation in our model remains elusive. However, Ehrlichia TRPs that are secreted by the type I secretion system (14,35,36) or Ehrlichia translocated factor-2 (Etf-2) that is secreted by the type IV secretion system into cytosol (40) are likely ligands or PAMPs that trigger inflammasome activation. (41,42) Additionally, our data suggest that DAMPs, such as mitochondrial (mt) DAMPs, are potential ligands that mediate canonical and noncanonical inflammasome activation.…”