Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

Death receptor signaling is critical for cell death, inflammation, and immune homeostasis. Hijacking death receptors and their corresponding adaptors through type III secretion system (T3SS) effectors has been evolved to be a bacterial evasion strategy. NleB from enteropathogenic Escherichia coli (EPEC) and SseK1/2/3 from Salmonella enterica serovar Typhimurium (S. Typhimurium) can modify some death domains involved in death receptor signaling through arginine-GlcNAcylation. This study applied a limited substrate screen from 12 death domain proteins with conserved arginines during EPEC and Salmonella infection and found that NleB from EPEC hijacked death receptor signaling tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein (TRADD), FAS-associated death domain protein (FADD), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1), whereas SseK1 and SseK3 disturbed TNFR signaling through the modification of TRADD Arg235/245 and TNFR1 Arg376, respectively. SseK1 and SseK3 delivered by Salmonella inhibited TNF-α- but not TNF-related apoptosis-inducing ligand (TRAIL)-induced cell death, which was consistent with their host substrate recognition specificity. Taking advantage of the substrate specificity of SseK effectors, we found that only SseK1 fully rescued the bacteria colonization deficiency contributed by NleBc in Citrobacter rodentium infection animal model, indicating that TRADD was likely to be the preferred in vivo substrate corresponding to NleB/SseK1-induced bacterial virulence. Furthermore, novel auto-arginine-GlcNAcylation was observed in NleB and SseK1/3, which promoted the enzyme activity. These findings suggest that arginine-GlcNAcylation in death domains and auto-arginine-GlcNAcylation catalyzed by type III-translocated bacterial effector proteins NleB/SseKs are crucial for bacteria pathogenesis in regulating nuclear factor-κB (NF-κB) and death receptor signaling pathways. This study provides an insight into the mechanism by which EPEC and Salmonella manipulate death receptor signaling and evade host immune defense through T3SS effectors.Author SummaryEnteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are important food-borne pathogens infecting the intestine. They deliver type III secretion system effector NleB/SseKs to modify host death domain proteins by arginine GlcNAcylation. We screened the modification of 12 death domains containing conserved arginine in human genome by NleB, SseK1, SseK2, and SseK3 through ectopic co-expression and bacterial infection. Unlike multiple death receptor signaling inhibition by NleB, we found that SseK1 and SseK3 specifically hijacked tumor necrosis factor receptor 1 (TNFR1)-mediated death signaling through targeting TNFR1-associated death domain protein (TRADD) and receptor TNFR1, respectively. We identified the modification sites and suggested that TRADD was the in vivo target of NleB in mice infection model by utilizing the substrate specificity of SseK1 and SseK3, which highlighted anti-bacterial infection role of TRADD in death receptor signaling and non-death receptor signaling. In addition to the modification on host death domain substrates, we firstly elucidated the effect of auto-modification of the arginine GlcNAc transferases on the enzymatic activity, which widened our understanding of the newly discovered post translational modification in the process of pathogen-host interaction.