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.

Bioinformatic discovery of a toxin family in Chryseobacterium piperi with sequence similarity to botulinum neurotoxins

“…These sequences possess partial similarity only to the BoNT translocation domain (17.3% maximum sequence identity, PSI-BLAST E -value = 7 × 10 −40 ). Group II is formed by M91 family peptidases such as the Escherichia coli type III effector toxin NleD, which cleaves host JNK and p38 32 . These sequences possess remote detectable homology to the BoNT-LC with 14.9% maximum sequence identity and a PSI-BLAST E -value of 4 × 10 −5 (see Methods).

Figure 1Bioinformatic detection of BoNT-related genes in microbial genomes.

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Enteropathogenic Escherichia coli—A Summary of the Literature

“…They also activate the host cell-signaling pathways, causing the alteration of the cytoskeleton, leading to the loss of the microvilli. After tyrosine-protein kinase and protein kinase A modifies Tir, it is inserted into the host cell membrane [43,44,[46][47][48][49][50][51][52][53][54].…”

Attaching and Effacing Pathogens: The Effector ABC of Immune Subversion

“…Baruch et al showed that NleD cleaves JNK1 and JNK2 within their respective activation loops, which reduces the level of phosphorylated c-Jun [12], a subunit of the transcription factor AP-1. This, in turn, inhibits AP-1-mediated transcription of pro-inflammatory and pro-apoptotic genes [30]. The cleavage site in p38 was narrowed down to a region between amino acid residues W187 and M213 [30], which is close to the cleavage site published for JNK2, which corresponds to amino acid P182 in p38.…”