Abstract
The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium 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 (NleBCR in C. rodentium) that modifies conserved arginine residues in death domain-containing host proteins with N-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 in vitro, or C. rodentium infection in vivo NleB overexpression resulted in arginine GlcNAcylation of multiple host proteins. However, NleB delivery during EPEC and C. rodentium infection caused rapid and preferential modification of Arg117 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/NleBCR antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner.
Highlights
The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium during mammalian infections
As with other glycosylation events, the tendency of glycopeptides to undergo ion competition/suppression in the presence of abundant non-glycosylated peptides [24, 25] suggests that enrichment would be required for a proteome-wide investigation of arginine GlcNAcylation, similar to approaches used for other posttranslational modification (PTM) (26 –28)
This stability enabled the localization of Arg-GlcNAc to the previously reported sites for Fas-associated via death domain (FADD) (Arg117; Fig. 1C) and TNFRSF1A-associated via death domain (TRADD) (Arg235; supplemental Annotations) [17, 18] while enabling the assessment of novel arginine GlcNAcylation events, including of the charged multivesicular body protein 2a (Arg16; Fig. 1D)
Summary
The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium during mammalian infections. Defining the targets of NleB during bacterial infection degrades receptor-interacting protein homotypic interaction motif -containing proteins, thereby blocking receptor-interacting protein homotypic interaction motif-dependent inflammatory and necroptotic signaling pathways [13], and NleF, which directly inhibits caspase-4, -8, and -9 activation [14,15,16] Another nle effector, NleB1, is the prototypic member of a novel family of bacterial glycosyltransferase enzymes that mediate the glycosylation of arginine residues, an atypical posttranslational modification (PTM) not observed in eukaryotic cells [17, 18]. Using a recently developed antibody specific for ArgGlcNAc linkages [23], we established an Arg-GlcNAc-specific enrichment method coupled with mass spectrometry (MS) to provide a robust means to monitor arginine GlcNAcylation during A/E pathogen infection We applied this to identify the endogenous targets modified by NleB1/NleBCR during wild-type EPEC and C. rodentium infection. These findings expand our understanding of NleB1/NleBCR-mediated Arg GlcNAcylation as an irreversible and silent modification and highlight the promiscuous nature of NleB1 under non-wild-type infection conditions
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