Salmonella Effectors SseK1 and SseK3 Target Death Domain Proteins in the TNF and TRAIL Signaling Pathways*

Strains of Salmonella utilize two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 during Salmonella infection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, whereas ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg376 and Arg293 respectively. Structural studies on SseK3 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. Together our data suggest that salmonellae carrying sseK1 and sseK3 employ the glycosyltransferase effectors to antagonise different components of death receptor signaling. Strains of Salmonella utilize two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 during Salmonella infection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, whereas ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg376 and Arg293 respectively. Structural studies on SseK3 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. Together our data suggest that salmonellae carrying sseK1 and sseK3 employ the glycosyltransferase effectors to antagonise different components of death receptor signaling. Pathogenic serovars of Salmonella utilize two type three secretion systems (T3SS) 1The abbreviations used are: T3SStype 3 secretion systemsEPECenteropathogenic Escherichia coliGlcNAcN acetylglucosaminePRMparallel reaction monitoring. 1The abbreviations used are: T3SStype 3 secretion systemsEPECenteropathogenic Escherichia coliGlcNAcN acetylglucosaminePRMparallel reaction monitoring., encoded by Salmonella pathogenicity island-1 and -2 (SPI-1 and SPI-2) to deliver distinct cohorts of effector proteins into host cells during infection (1Galan J.E. Curtiss 3rd., R. Cloning and molecular characterization of genes whose products allow Salmonella typhimurium to penetrate tissue culture cells.Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 6383-6387Crossref PubMed Scopus (759) Google Scholar, 2Shea J.E. Hensel M. Gleeson C. Holden D.W. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 2593-2597Crossref PubMed Scopus (639) Google Scholar). These effector proteins subvert normal cellular processes and collectively enable the bacteria to invade and persist within host cells, partially through the manipulation of inflammatory cell signaling and programmed cell death (reviewed in (3Jennings E. Thurston T.L.M. Holden D.W. 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