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

Joshuap.M Newson,Nichollase Scott,Ivy Yeuk Wah Chung,Tania Wong Fok Lung,Cristina Giogha,Jiyao Gan,Nancy Wang,Richard A Strugnell,Nathaniel F Brown,Miroslaw Cygler,Jaclyn S Pearson,Elizabeth L Hartland

doi:10.1074/mcp.ra118.001093

Abstract

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.

Highlights

Salmonella Effectors SseK1 and SseK3 Target Death Domain Proteins in the TNF and TRAIL Signaling Pathways*□S
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, whereas SseK3 modified the signaling receptors TNFR1 and TRAILR
We confirmed the importance of this conserved glutamic acid to the biochemical activity of SseK1 and SseK3 by infecting RAW264.7 cells with a Salmonella Typhimurium SL1344 triple ⌬sseK123 deletion mutant [6] complemented with either native SseK1, -2 or -3, catalytic triad mutants, or mutants lacking the conserved glutamic acid (SseK1E255A, SseK2E271A, and SseK3E258A)

Summary

Introduction

Salmonella Effectors SseK1 and SseK3 Target Death Domain Proteins in the TNF and TRAIL Signaling Pathways*□S. Pathogenic serovars of Salmonella utilize two type three secretion systems (T3SS), 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 [1, 2] 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 [3, 4]). SseK family members show high sequence similarity to NleB1, a T3SS effector protein from enteropathogenic Escherichia coli (EPEC), which functions as an arginine glycosyltransferase and catalyzes the addition of N-acetylglucosamine (GlcNAc) to arginine residues of the mammalian signaling adaptors FADD and TRADD, a modification termed Arg-GlcNAcylation [7, 8]. These studies describing catalytically important regions of the glycosyltransferases provide opportunities to better understand the function of these novel enzymes [10]

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Results

Conclusion