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NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector

During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzy...

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Autores principales: Giogha, Cristina, Scott, Nichollas E., Wong Fok Lung, Tania, Pollock, Georgina L., Harper, Marina, Goddard-Borger, Ethan D., Pearson, Jaclyn S., Hartland, Elizabeth L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238200/
https://www.ncbi.nlm.nih.gov/pubmed/34133469
http://dx.doi.org/10.1371/journal.ppat.1009658
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author Giogha, Cristina
Scott, Nichollas E.
Wong Fok Lung, Tania
Pollock, Georgina L.
Harper, Marina
Goddard-Borger, Ethan D.
Pearson, Jaclyn S.
Hartland, Elizabeth L.
author_facet Giogha, Cristina
Scott, Nichollas E.
Wong Fok Lung, Tania
Pollock, Georgina L.
Harper, Marina
Goddard-Borger, Ethan D.
Pearson, Jaclyn S.
Hartland, Elizabeth L.
author_sort Giogha, Cristina
collection PubMed
description During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation.
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spelling pubmed-82382002021-07-09 NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector Giogha, Cristina Scott, Nichollas E. Wong Fok Lung, Tania Pollock, Georgina L. Harper, Marina Goddard-Borger, Ethan D. Pearson, Jaclyn S. Hartland, Elizabeth L. PLoS Pathog Research Article During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation. Public Library of Science 2021-06-16 /pmc/articles/PMC8238200/ /pubmed/34133469 http://dx.doi.org/10.1371/journal.ppat.1009658 Text en © 2021 Giogha et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Giogha, Cristina
Scott, Nichollas E.
Wong Fok Lung, Tania
Pollock, Georgina L.
Harper, Marina
Goddard-Borger, Ethan D.
Pearson, Jaclyn S.
Hartland, Elizabeth L.
NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector
title NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector
title_full NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector
title_fullStr NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector
title_full_unstemmed NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector
title_short NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector
title_sort nleb2 from enteropathogenic escherichia coli is a novel arginine-glucose transferase effector
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8238200/
https://www.ncbi.nlm.nih.gov/pubmed/34133469
http://dx.doi.org/10.1371/journal.ppat.1009658
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