The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK.

2.50
Hdl Handle:
http://hdl.handle.net/10033/620854
Title:
The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK.
Authors:
Creuzburg, Kristina; Giogha, Cristina; Wong Fok Lung, Tania; Scott, Nichollas E; Mühlen, Sabrina; Hartland, Elizabeth L; Pearson, Jaclyn S
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.
Affiliation:
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Citation:
The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK. 2017, 85 (2) Infect. Immun.
Journal:
Infection and immunity
Issue Date:
Feb-2017
URI:
http://hdl.handle.net/10033/620854
DOI:
10.1128/IAI.00620-16
PubMed ID:
27872241
Type:
Article
Language:
en
ISSN:
1098-5522
Appears in Collections:
publications of the department of molecular Infectionbiology (MIBI)

Full metadata record

DC FieldValue Language
dc.contributor.authorCreuzburg, Kristinaen
dc.contributor.authorGiogha, Cristinaen
dc.contributor.authorWong Fok Lung, Taniaen
dc.contributor.authorScott, Nichollas Een
dc.contributor.authorMühlen, Sabrinaen
dc.contributor.authorHartland, Elizabeth Len
dc.contributor.authorPearson, Jaclyn Sen
dc.date.accessioned2017-03-10T09:34:37Z-
dc.date.available2017-03-10T09:34:37Z-
dc.date.issued2017-02-
dc.identifier.citationThe Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK. 2017, 85 (2) Infect. Immun.en
dc.identifier.issn1098-5522-
dc.identifier.pmid27872241-
dc.identifier.doi10.1128/IAI.00620-16-
dc.identifier.urihttp://hdl.handle.net/10033/620854-
dc.description.abstractEnteropathogenic 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.en
dc.language.isoenen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.titleThe Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK.en
dc.typeArticleen
dc.contributor.departmentHelmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.en
dc.identifier.journalInfection and immunityen

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