NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector.

Tania Wong Fok Lung,Nichollas E. Scott,Elizabeth L. Hartland,Marina Harper,Cristina Giogha,Ethan D. Goddard-Borger,Jaclyn S. Pearson,Georgina L. Pollock

doi:10.1371/journal.ppat.1009658

Abstract

During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation.

Highlights

Diarrhoeagenic Escherichia coli including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC), cause nearly 200,000 deaths annually worldwide [1]
We found that a close homologue of NleB1 that is present in EPEC and EHEC termed NleB2, uses a different sugar during glycosylation
Given that NleB1 targets the death domains of FADD, TRADD, RIPK1 and TNFR1 and shares 84% similarity with NleB2 at the amino acid level, we tested whether NleB2 interacted with death domain containing proteins

Summary

Introduction

Diarrhoeagenic Escherichia coli including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC), cause nearly 200,000 deaths annually worldwide [1]. EPEC and EHEC remain extracellular during infection and use a type III secretion system (T3SS) to translocate ‘effector’ proteins into the host cell cytoplasm [2]. These effectors orchestrate control over host cell physiology to facilitate infection and allow the bacteria to evade innate immune defence mechanisms [3]. Recent work has revealed that several EPEC and EHEC effectors are novel enzymes that target and interfere with the function of host cell signalling proteins. The addition of a single GlcNAc moiety to Arg residues in death domain proteins blocks the formation of critical immune signalling complexes, resulting in inhibition of inflammation and cell death during infection

Methods

Results

Discussion

Conclusion