Characterisation of a family of novel glycosyltransferases from enteropathogenic Escherichia coli and Salmonella

© 2016 Dr. Tania Wong Fok LungEnteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen of children that utilises a type III secretion system (T3SS) to inject virulence effector proteins into enterocytes during infection. 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 arginine-117 (Arg117) of the Fas associated death domain (DD) 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 the DD proteins, tumour necrosis factor receptor type 1 (TNFR1)-associated death domain (TRADD) and receptor-interacting protein kinase 1 (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 22 random transposon-based, in-frame, linker insertion mutants of NleB1 were tested for their ability to block caspase-8 activation in response to FasL during EPEC infection. 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. Coimmunoprecipitation studies of 34 site-directed mutants showed that the NleB1 derivatives with the E253A, Y219A, and PILN(63– 66)AAAA (in which the PILN motif from residues 63 to 66 was changed to AAAA) mutations bound to but did not GlcNAcylate FADD. A further mutant derivative, the PDG(236 –238)AAA mutant, did not bind to or GlcNAcylate FADD. Further testing of these mutants with TRADD and RIPK1, showed that NleB1 bearing the mutations E253A and Y219A could still bind to FADD and RIPK1 but not to TRADD. Infection of mice with the EPEC-like mouse pathogen Citrobacter rodentium expressing NleBE253A and NleBY219A showed that these 2 strains were attenuated, indicating the importance of the residues E253 and Y219 in NleB1 virulence in vivo. In summary, we identified new amino acid residues critical for NleB1 activity and confirmed that FADD GlcNAcylation was critical for NleB1 function. Close homologues of NleB1 are found in Salmonella enterica serovar Typhimurium and these are termed SseK1, SseK2 and SseK3. We hypothesized that the SseK effectors would also bind to DD proteins and inhibit apoptotic or inflammatory signalling. The SseK effectors did not appear to play a strong role in the inhibition of death receptor signaling given that we could not detect binding of the SseK effectors to the death domain proteins FADD, TRADD and RIPK1, which are targets of NleB1. A further survey of DD proteins revealed that SseK3 bound to TNFR1. However S. Typhimurium did not appear to inhibit TNF-induced IL-8 production and the biological significance of this interaction is still unknown. We conclude that the SseKs have an alternative function during S. Typhimurium infection to NleB1 in EPEC