Mutagenesis and functional analysis of the bacterial arginine glycosyltransferase effector NleB1 from enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) interferes with host cell signalling by injecting virulence effector proteins into enterocytes via a type III secretion system (T3SS). NleB1 is a novel T3SS glycosyltransferase effector from EPEC that transfers a single N -acetylglucosamine (GlcNAc) moiety in an N -glycosidic linkage to Arg 117 of the Fas associated death domain protein (FADD). GlcNAcylation of FADD prevents assembly of the canonical death inducing signalling complex and inhibits Fas ligand (FasL) induced cell death. Apart from the DxD catalytic motif of NleB1, little is known about other functional sites in the enzyme. 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 showed that 17 mutant derivatives of NleB1 no longer inhibited caspase-8 activation, including the catalytic DxD mutant. Regions of interest around the 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 PILN63-66AAAA , bound but did not GlcNAcylate FADD. A further mutant derivative, PDG236-238AAA , did not bind or GlcNAcylate FADD. Infection of mice with the EPEC-like mouse pathogen Citrobacter rodentium expressing NleB E253A and NleB Y219A showed that these 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 these were required for the virulence function of NleB1.