Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that utilises a type III secretion system (T3SS) to inject virulence effector proteins directly into enterocytes. NleB1 is a novel glycosyltransferase effector from EPEC that catalyses the addition of a single GlcNAc moiety in an N-glycosidic linkage to arginine. NleB1 modifies Arg117 of the Fas associated death domain protein, FADD, which prevents assembly of the canonical death inducing signalling complex (DISC) and inhibits FasL-induced cell death. NleB1 also modifies the equivalent arginine residues in TRADD and RIPK1.
Apart from the DxD catalytic motif of NleB1, little is known about other functional sites in the protein and the regions required for substrate binding and specificity. Here a library of 30 random transposon-based, in-frame, linker insertion mutants of NleB1 were tested during EPEC infection of HeLa cells for their ability to block caspase-8 activation in response to FasL. Immunoblot analysis of caspase-8 cleavage products showed that 14 mutant derivatives of NleB1 no longer inhibited caspase-8 activation, including the catalytic DxD mutant. Regions of interest around the linker insertion sites were examined further with multiple or single amino acid substitutions. Co-immunoprecipitation studies of 34 site-directed mutants showed that the NleB1 derivatives, E253A, Y219A and P63ILN/A63AAA, bound but did not GlcNAcylate FADD. A further mutant derivative, P236DG/A236AA, did not bind or GlcNAcylate FADD. Further testing of these mutants with TRADD and RIPK1, showed that NleB1 bearing the mutations E253A and Y219A can still bind to RIPK1 but not to TRADD. Infection of mice with the EPEC-like mouse pathogen Citrobacter rodentium expressing NleB E253A and NleB Y219A showed that these 2 strains were attenuated, indicating the importance of Glu253 and Tyr219 in NleB1 virulence. In summary, we identified new amino acid residues critical for NleB1 activity and confirmed that FADD GlcNAcylation is critical for NleB1 function.