title: Mutagenesis and Functional Analysis of the Bacterial Arginine Glycosyltransferase Effector NleB1 from Enteropathogenic Escherichia coli.
abstract: Enteropathogenic Escherichia coli (EPEC) interferes with host cell signaling 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 the assembly of the canonical death-inducing signaling 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. In the present study, members of 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, including the catalytic DXD mutant, did not inhibit caspase-8 activation. Regions of interest around the insertion sites with multiple or single amino acid substitutions were examined further. 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. Infection of mice with the EPEC-like mouse pathogen Citrobacter rodentium expressing NleBE253A and NleBY219A showed that these strains were attenuated, indicating the importance of 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 are required for the virulence function of NleB1.
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
authors.forename: Tania
authors.initials: T
authors.lastname: Wong Fok Lung
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
authors.forename: Cristina
authors.initials: C
authors.lastname: Giogha
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
authors.forename: Kristina
authors.initials: K
authors.lastname: Creuzburg
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
authors.forename: Sze Ying
authors.initials: SY
authors.lastname: Ong
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
authors.forename: Georgina L
authors.initials: GL
authors.lastname: Pollock
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
authors.forename: Ka Yee
authors.initials: KY
authors.lastname: Fung
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
authors.forename: Jaclyn S
authors.initials: JS
authors.lastname: Pearson
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia hartland@unimelb.edu.au.
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Hartland
mesh_terms: D000818:Animals; D017209:Apoptosis; D001120:Arginine / Q000378:metabolism; D044082:Citrobacter rodentium / Q000235:genetics / Q000472:pathogenicity; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements; D004756:Enterobacteriaceae Infections / Q000382:microbiology / Q000473:pathology; D054308:Enteropathogenic Escherichia coli / Q000201:enzymology* / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D053222:Fas Ligand Protein / Q000378:metabolism; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D005260:Female; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; C505533:FADD protein, human; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; C553814:NleB protein, E coli; D037521:Virulence Factors; D001120:Arginine; D016695:Glycosyltransferases