NleG Type 3 Effectors from Enterohaemorrhagic Escherichia coli Are U-Box E3 Ubiquitin Ligases

Abstract

NleG homologues constitute the largest family of type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non- LEE- encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known function. The C-terminal domain of NleG2-3 (residues 90 to 191) is the most conserved region in NleG proteins and was solved by NMR. Structural analysis of this structure revealed the presence of a RING finger/U-box motif. Functional assays demonstrated that NleG2-3 as well as NleG5-1, NleG6-2 and NleG9′ family members exhibited a strong autoubiquitination activity in vitro; a characteristic usually expressed by eukaryotic ubiquitin E3 ligases. When screened for activity against a panel of 30 human E2 enzymes, the NleG2-3 and NleG5-1 homologues showed an identical profile with only UBE2E2, UBE2E3 and UBE2D2 enzymes supporting NleG activity. Fluorescence polarization analysis yielded a binding affinity constant of 56±2 µM for the UBE2D2/NleG5-1 interaction, a value comparable with previous studies on E2/E3 affinities. The UBE2D2 interaction interface on NleG2-3 defined by NMR chemical shift perturbation and mutagenesis was shown to be generally similar to that characterised for human RING finger ubiquitin ligases. The alanine substitutions of UBE2D2 residues Arg5 and Lys63, critical for activation of eukaryotic E3 ligases, also significantly decreased both NleG binding and autoubiquitination activity. These results demonstrate that bacteria-encoded NleG effectors are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes.

Author Summary

Many bacterial pathogens utilize a multiprotein ‘‘injection needle’’ termed the type III secretion system to deliver a set of proteins called effectors into the host cell. These effectors then manipulate host signalling pathways to the advantage of the pathogen, often mimicking eukaryote-specific activities. We present a study of an uncharacterised family of effectors called NleG, secreted primarily by enterohaemorrhagic E. coli (EHEC) O157:H7, a causative agent of human gastroenteritis. We solved the solution structure of a conserved C-terminal region of an NleG family member by NMR. Structural analysis demonstrated that the NleG structure is similar to the RING finger/U-box domain found primarily in eukaryotic ubiquitin ligases. The activity of these domains in eukaryotes is an essential part of the ubiquitination signalling system. Due to its central role in cell metabolism and the host immune response, the ubiquitination system has emerged as a primary target for bacterial effectors. Our biochemical analysis demonstrated that NleG proteins selectively interact with human E2 ubiquitin conjugating enzymes and exhibit in vitro activity typical of eukaryotic E3 ligases. Our data reveal that NleG effectors structurally and functionally mimic host U-box/RING E3 ubiquitin ligases. Future research will focus on determining targets of NleG ubiquitin ligase activity and the role in E. coli pathogenesis.