Distinct Molecular Features of NleG Type 3 Secreted Effectors Allow for Different Roles during Citrobacter rodentium Infection in Mice

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1_Cr, NleG7_Cr, and NleG8_Cr-encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleG_Cr knockout strains, we show that NleG7_Cr contributes to bacterial survival during enteric infection while NleG1_Cr promotes the expression of diarrheal symptoms and NleG8_Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8_Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8_Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8_Ec) effector, which shares 60% identity with NleG8_Cr, is engaged in interactions with human GOPC. The crystal structure of the NleG8_Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8_Ec does not complement the ΔnleG8_Cr phenotype during infection, revealing functional diversification between these NleG effectors.