Uncovering host interaction partners of NleG effectors secreted by Escherichia coli

The pathogenic strategy of Escherichia coli (E. coli), particularly enterohaemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC), involves an arsenal of over 60 translocated proteins termed effectors. These effectors enable manipulation of the host cellular mechanisms to aid colonization and promote bacterial survival and infection. Among these effectors are 14 members that form the largest T3SS effector family, the NleG effector family, which can manipulate the ubiquitin pathway despite their bacterial origin. This family consists of ubiquitin E3 ligases, with two members having demonstrated ubiquitin-mediated degradation of two different host eukaryotic proteins, despite sharing nearly identical structures. How these effectors differentiate between substrates, as well as the remaining members’ targets, remains unknown. To better understand this family's prevalence and role in pathogenesis, we initially conducted an in silico analysis of all completed E. coli genomes to identify NleG family members, revealing that the majority of EHEC strains encode all 14 members. They are also found in E. albertii, S. bongori, C. braakii, and C. rodentium. Subsequently, we characterized NleG substrates using affinity purification-mass spectrometry (AP-MS) and yeast-2-hybrid (Y2H) validation, uncovering cross-species substrate redundancy for HK2 and MED, along with potential novel interactions. Finally, we aimed to identify the molecular determinants responsible for substrate specificity. Using sequence analysis, AlphaFold3-generated protein complexes, and Y2H, we pinpointed substrate-specific residues that mediate interactions between NleG2-3 and HK2, and NleG5-1 and MED15. Our findings offer further insights into the pathogenic behaviour of the NleG effector family, particularly regarding targeted substrates and specificity—potentially supporting its development as a biological tool and reducing the burden of EHEC infection.