Blocking Death Receptors-mediated Host Cell Death By Bacteria-catalyzed Arginine GlcNAcylation Of Death Domain

The TNF family plays critical roles in immune homeostasis, cell death and inflammation. These cytokines are recognized by the TNF receptor (TNFR) family of death receptors (DRs), including TNFR1/2, FAS and TRAIL receptors, which form a death inducing signaling complex (DISC). DISC formation critically involves death domain (DD)-mediated homotypic/heterotypic interactions among the death receptor and its downstream adaptors such as TNF receptor1-associated DD protein (TRADD) and FAS associated DD protein (FADD). Here we discover that DDs in several DD-containing proteins, including TRADD, FADD, RIPK1and TNFR1, were directly inactivated by NleB, an enteropathogenic Escherichia coli (EPEC) type Ⅲ secretion system effector previously shown to harbor NF-κB-inhibiting activity.At first, we found that NleB targeting of TRADD DD disrupted multiple TNF signaling including NF-κB, apoptosis and necroptosis in transfected or EPEC-infected host cells. NleB harbored an unprecedented GlcNAc transferase activity that specifically modified Arg-235in TRADD DD both in vitro and during EPEC infection. Arginine GlcNAcylation of TRADD DD blocked homotypic/heterotypic DD interactions and impaired assembly of the oligomeric TNFR complex, thereby disrupting multiple TNF signaling in EPEC-infected cells, including NF-κB, apoptosis and necroptosis.After extensive Blast of protein sequence, we found that Arg-235was conserved in one-third of more than30DD-containing proteins in human[1], including FADD, TNFR1, RIPK1, FAS and DR3/4/5(death receptor-3/4/5) that function in DR signaling. According to our biochemical and MS data, NleB also GlcNAcylated and inactivated other DD-containing proteins involved in death receptor signaling, including FADD, RIP1, TNFR1and FAS, serving as a general DD inhibitor. Besides blocking TNFR1complex assembling, Type Ⅲ-delivered NleB blocked FAS ligand and TRAIL-induced cell death by preventing formation of FADD-mediated death inducing signaling complex (DISC). Moreover, we showed that the arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection.The mechanism of action of NleB represents not only a new paradigm in bacterial counteracting host defenses but also a previously unappreciated posttranslational modification in eukaryotic signaling regulation.