The bacterial arginine glycosyltransferase effector NleB preferentially modifies Fas-associated death domain protein (FADD)

Abstract

The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic (EPEC) and during mammalian infections. To subvert these pathways and suppress the antimicrobial response, attaching and effacing pathogens use type III secretion systems to introduce effectors targeting key signaling pathways in host cells. One such effector is the arginine glycosyltransferase NleB1 (NleB in ) that modifies conserved arginine residues in death domain-containing host proteins with-acetylglucosamine (GlcNAc), thereby blocking extrinsic apoptosis signaling. Ectopically expressed NleB1 modifies the host proteins Fas-associated via death domain (FADD), TNFRSF1A-associated via death domain (TRADD), and receptor-interacting serine/threonine protein kinase 1 (RIPK1). However, the full repertoire of arginine GlcNAcylation induced by pathogen-delivered NleB1 is unknown. Using an affinity proteomic approach for measuring arginine-GlcNAcylated glycopeptides, we assessed the global profile of arginine GlcNAcylation during ectopic expression of NleB1, EPEC infection , or infection NleB overexpression resulted in arginine GlcNAcylation of multiple host proteins. However, NleB delivery during EPEC and infection caused rapid and preferential modification of Arg in FADD. This FADD modification was extremely stable and insensitive to physiological temperatures, glycosidases, or host cell degradation. Despite its stability and effect on the inhibition of apoptosis, arginine GlcNAcylation did not elicit any proteomic changes, even in response to prolonged NleB1 expression. We conclude that, at normal levels of expression during bacterial infection, NleB1/NleB antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner.

Salmonellaeffectors SseK1 and SseK3 target death domain proteins in the TNF and TRAIL signaling pathways

“…This observation, in conjunction with increased levels of Arg-GlcNAcylation seen during overexpression, suggested that many substrates may be non-authentically glycosylated when the effectors are overexpressed. These findings are similar to a previous report that suggested overexpression of NleB1 also results in Arg-GlcNAcylation of a broader range of substrates [13]. We note in particular the numerous two-component response regulators that were Arg-GlcNAcylated during overexpression of SseK1 (Fig 2A, Supplementary Table 1), perhaps suggesting a mechanism for effector-mediated regulation of two-component signaling outcomes.…”

“…While these studies have demonstrated the ability of SseK1 to GlcNAcylate a given target, the approach is substrate specific and there requires knowledge of the potential targets. To identify the full range of host Arg-GlcNAcylated substrates, we developed a quantitative approach using mass spectrometry (MS) to enrich for arginine glycosylated peptides [13]. Using peptides derived from host cells infected with S. Typhimurium ΔsseK123 over-expressing either SseK1 or inactive SseK1 E255A , label-free MS based quantification revealed a broad range of substrates that were only modified in the presence of active SseK1 (Fig.…”

“…We have previously shown that over-expression of NleB1 leads to enhanced levels of cellular arginine glycosylation [13]. Given that the overexpression of SseK1 and SseK3 also appeared to increase the range of substrates relative to endogenous wild type levels of expression (Fig 1A and B), we explored the activity of endogenous SseK1 during S. Typhimurium infection of RAW264.7 cells using our strategy for enrichment of Arg-GlcNAcylated peptides [13]. Here we compared Arg-GlcNAcylation during infection with a ΔsseK23 double deletion mutant versus a ΔsseK123 triple deletion mutant [6].…”

“…SseK3 on the other hand was reported to bind but not modify the E3-ubiquitin ligase TRIM32 [12], and was shown to weakly modify TRADD but not FADD [9]. While these studies provide useful insights, the true role of these effectors is better interrogated through non-biased screens conducted under conditions that reflect endogenous levels of the effectors and host proteins [13].…”

“…Here, we explored the endogenous Arg-GlcNAc glycosyltransferase activity of SseK1 and SseK3 during Salmonella infection. Using a mass spectrometry-based approach to enrich arginine GlcNAcylated peptides from infected host cells [13], we found that SseK1 modified the signaling adaptor TRADD, while SseK3 modified the signaling receptors TNFR1 and TRAILR. In addition, we performed structural studies on SseK3 and showed that the enzyme displays a classic GT-A glycosyltransferase fold and binds UDP-GlcNAc in a narrow and deep cleft with the GlcNAc facing the surface.…”