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.

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…134 As mimics of the eukaryotic RING/U-box E3 ligases, NleG family effectors are also identified in other pathogenic E. coli such as EPEC and Salmonella. 135 Both EHEC and EPEC deliver the effectors NleE and Tir to subvert the NF-κB pathway. NleE directly methylates the cysteine residue in the ZnF of TAB2/3 to hinder their ubiquitination, whereas Tir facilitates the binding of host protein tyrosine phosphatases SHP-1 and SHP-2 to TRAF6, which prevents the ubiquitination of TRAF6.…”

Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase

“…Kaposi's sarcoma-associated herpesvirus possesses two ubiquitin ligases, MIR-1 and -2, that belong to the RING-like family of E3 ligases and function to down-regulate several cell membrane proteins, including MHC-1, required for antigen presentation (25). Enterohemmorrhagic Escherichia coli contains a family of NleG effectors that possess conserved U-Box ligase domain architecture (26). The Shigella-like Pseudomonas syringiae plant pathogen also introduces effectors through a type III secretion apparatus, one of which (AvrPtoB) possesses a plant homeodomain related to eukaryotic RING domain ligases and inhibits apoptosis of infected cells (27).…”

Distribution of Pathogenicity Islands OI-122, OI-43/48, and OI-57 and a High-Pathogenicity Island in Shiga Toxin-Producing Escherichia coli

“…NleG proteins are E3 ubiquitin ligases analogous to RING finger and U-box enzymes in eukaryotes. Although the exact functions of NleG2-3, NleG6-2, and NleG 5-2 are still unclear, similar proteins have been identified as effectors that suppress immune response from the host (15).…”