title: The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK.
abstract: Enteropathogenic Escherichia coli (EPEC) is a gastrointestinal pathogen that utilizes a type III secretion system (T3SS) to inject an array of virulence effector proteins into host enterocytes to subvert numerous cellular processes for successful colonization and dissemination. The T3SS effector NleD is a 26-kDa zinc metalloprotease that is translocated into host enterocytes, where it directly cleaves and inactivates the mitogen-activated protein kinase signaling proteins JNK and p38. Here a library of 91 random transposon-based, in-frame, linker insertion mutants of NleD were tested for their ability to cleave JNK and p38 during transient transfection of cultured epithelial cells. Immunoblot analysis of p38 and JNK cleavage showed that 7 mutant derivatives of NleD no longer cleaved p38 but maintained the ability to cleave JNK. Site-directed mutation of specific regions surrounding the insertion sites within NleD revealed that a single amino acid, R203, was essential for cleavage of p38 but not JNK in a direct in vitro cleavage assay, in transiently transfected cells, or in EPEC-infected cells. Mass spectrometry analysis narrowed the cleavage region to within residues 187 and 213 of p38. Mutation of residue R203 within NleD to a glutamate residue abolished the cleavage of p38 and impaired the ability of NleD to inhibit AP-1-dependent gene transcription of a luciferase reporter. Furthermore, the R203 mutation abrogated the ability of NleD to dampen interleukin-6 production in EPEC-infected cells. Overall, this work provides greater insight into substrate recognition and specificity by the type III effector NleD.
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, 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, 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, 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, Australia.
authors.forename: Nichollas E
authors.initials: NE
authors.lastname: Scott
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia.|Department of Molecular Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Mühlen
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia.
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Hartland
authors.affiliation: Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia jaclynp@unimelb.edu.au.
authors.forename: Jaclyn S
authors.initials: JS
authors.lastname: Pearson
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D054308:Enteropathogenic Escherichia coli / Q000502:physiology*; D004927:Escherichia coli Infections / Q000378:metabolism* / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D059748:Proteolysis; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D016207:Cytokines; D029968:Escherichia coli Proteins; C506689:Z0990 protein, E coli; D001120:Arginine; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases
keywords: EPEC; JNK; NleD; metalloprotease; mitogen-activated protein kinases; p38; type III effector proteins