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

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. Author summary Bacterial gut pathogens including enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC), manipulate host cell function by using a type III secretion system to inject 'effector' proteins directly into the host cell cytoplasm. We and others have shown that many of these effectors are novel enzymes, including NleB1, which transfers a single N-acetylglucosamine (GlcNAc) sugar to arginine residues, mediating Arg-GlcNAc glycosylation. Here, we found that a close homologue of NleB1 that is also present in EPEC and EHEC termed NleB2, uses a different sugar during glycosylation. We demonstrated that in contrast to NleB1, the preferred nucleotide-sugar substrate of NleB2 is UDP-glucose and we identified the amino acid residue within NleB2 that dictates this unique catalytic activity. Substitution of this residue in NleB2 and NleB1 switches the sugar donor usage of these enzymes but does not affect their ability to inhibit host cell signalling. Thus, NleB2 is the first identified bacterial arginine-glucose transferase, an activity which has previously only been described in plants and algae.

Automated summary

During infection, bacterial pathogens EPEC and EHEC use type III secretion system to inject effector proteins into host cells, directly manipulating host cell functions. NleB2, a close homologue of NleB1, is the first identified bacterial arginine-glucose transferase, inhibiting host protein function through arginine glycosylation, similar to NleB1's Arg-GlcNAc glycosylation. Substitution of specific residues in NleB2 and NleB1 switches sugar donor usage without affecting their ability to inhibit host cell signaling.