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Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography
Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera to...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386978/ https://www.ncbi.nlm.nih.gov/pubmed/30736336 http://dx.doi.org/10.3390/ijms20030703 |
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author | Heggelund, Julie Elisabeth Heim, Joel Benjamin Bajc, Gregor Hodnik, Vesna Anderluh, Gregor Krengel, Ute |
author_facet | Heggelund, Julie Elisabeth Heim, Joel Benjamin Bajc, Gregor Hodnik, Vesna Anderluh, Gregor Krengel, Ute |
author_sort | Heggelund, Julie Elisabeth |
collection | PubMed |
description | Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity. |
format | Online Article Text |
id | pubmed-6386978 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-63869782019-02-27 Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography Heggelund, Julie Elisabeth Heim, Joel Benjamin Bajc, Gregor Hodnik, Vesna Anderluh, Gregor Krengel, Ute Int J Mol Sci Article Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity. MDPI 2019-02-06 /pmc/articles/PMC6386978/ /pubmed/30736336 http://dx.doi.org/10.3390/ijms20030703 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Heggelund, Julie Elisabeth Heim, Joel Benjamin Bajc, Gregor Hodnik, Vesna Anderluh, Gregor Krengel, Ute Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography |
title | Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography |
title_full | Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography |
title_fullStr | Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography |
title_full_unstemmed | Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography |
title_short | Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography |
title_sort | specificity of escherichia coli heat-labile enterotoxin investigated by single-site mutagenesis and crystallography |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386978/ https://www.ncbi.nlm.nih.gov/pubmed/30736336 http://dx.doi.org/10.3390/ijms20030703 |
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