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The Type III Effector NleD from Enteropathogenic Escherichia coli Differentiates between Host Substrates p38 and JNK.
- Source :
-
Infection and immunity [Infect Immun] 2017 Jan 26; Vol. 85 (2). Date of Electronic Publication: 2017 Jan 26 (Print Publication: 2017). - Publication Year :
- 2017
-
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.<br /> (Copyright © 2017 American Society for Microbiology.)
- Subjects :
- Amino Acid Sequence
Arginine metabolism
Cell Line
Cytokines biosynthesis
Escherichia coli Proteins chemistry
Escherichia coli Proteins genetics
Humans
Mutagenesis, Insertional
Proteolysis
Signal Transduction
Enteropathogenic Escherichia coli physiology
Escherichia coli Infections metabolism
Escherichia coli Infections microbiology
Escherichia coli Proteins metabolism
Host-Pathogen Interactions
JNK Mitogen-Activated Protein Kinases metabolism
p38 Mitogen-Activated Protein Kinases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1098-5522
- Volume :
- 85
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Infection and immunity
- Publication Type :
- Academic Journal
- Accession number :
- 27872241
- Full Text :
- https://doi.org/10.1128/IAI.00620-16