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Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins
Enterotoxigenic Escherichia coli (ETEC), which secretes the heat-stable toxin (ST) is among the four most important enteropathogens that cause moderate-to-severe diarrhea in children in low- and middle-income countries. ST is an intestinal molecular antagonist causing diarrhea and hence an attractiv...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071264/ https://www.ncbi.nlm.nih.gov/pubmed/29970812 http://dx.doi.org/10.3390/toxins10070274 |
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author | Govasli, Morten L. Diaz, Yuleima Zegeye, Ephrem Debebe Darbakk, Christine Taxt, Arne M. Puntervoll, Pål |
author_facet | Govasli, Morten L. Diaz, Yuleima Zegeye, Ephrem Debebe Darbakk, Christine Taxt, Arne M. Puntervoll, Pål |
author_sort | Govasli, Morten L. |
collection | PubMed |
description | Enterotoxigenic Escherichia coli (ETEC), which secretes the heat-stable toxin (ST) is among the four most important enteropathogens that cause moderate-to-severe diarrhea in children in low- and middle-income countries. ST is an intestinal molecular antagonist causing diarrhea and hence an attractive vaccine target. A non-toxic and safe ST vaccine should include one or more detoxifying mutations, and rigorous characterization of such mutants requires structurally intact peptides. To this end, we established a system for purification of ST and ST mutants by fusing the sequence encoding the mature ST peptide to the disulfide isomerase DsbC. A Tobacco Etch Virus protease cleavage site facilitates the proteolytic release of free ST with no additional residues. The purified ST peptides have the expected molecular masses, the correct number of disulfide bridges, and have biological activities and antigenic properties comparable to ST isolated from ETEC. We also show that free DsbC can assist in refolding denatured and misfolded ST in vitro. Finally, we demonstrate that the purification system can be used to produce ST mutants with an intact neutralizing epitope, that two single mutations, L9S and A14T, reduce toxicity more than 100-fold, and that the L9S/A14T double mutant has no measurable residual toxicity. |
format | Online Article Text |
id | pubmed-6071264 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-60712642018-08-09 Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins Govasli, Morten L. Diaz, Yuleima Zegeye, Ephrem Debebe Darbakk, Christine Taxt, Arne M. Puntervoll, Pål Toxins (Basel) Article Enterotoxigenic Escherichia coli (ETEC), which secretes the heat-stable toxin (ST) is among the four most important enteropathogens that cause moderate-to-severe diarrhea in children in low- and middle-income countries. ST is an intestinal molecular antagonist causing diarrhea and hence an attractive vaccine target. A non-toxic and safe ST vaccine should include one or more detoxifying mutations, and rigorous characterization of such mutants requires structurally intact peptides. To this end, we established a system for purification of ST and ST mutants by fusing the sequence encoding the mature ST peptide to the disulfide isomerase DsbC. A Tobacco Etch Virus protease cleavage site facilitates the proteolytic release of free ST with no additional residues. The purified ST peptides have the expected molecular masses, the correct number of disulfide bridges, and have biological activities and antigenic properties comparable to ST isolated from ETEC. We also show that free DsbC can assist in refolding denatured and misfolded ST in vitro. Finally, we demonstrate that the purification system can be used to produce ST mutants with an intact neutralizing epitope, that two single mutations, L9S and A14T, reduce toxicity more than 100-fold, and that the L9S/A14T double mutant has no measurable residual toxicity. MDPI 2018-07-03 /pmc/articles/PMC6071264/ /pubmed/29970812 http://dx.doi.org/10.3390/toxins10070274 Text en © 2018 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 Govasli, Morten L. Diaz, Yuleima Zegeye, Ephrem Debebe Darbakk, Christine Taxt, Arne M. Puntervoll, Pål Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins |
title | Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins |
title_full | Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins |
title_fullStr | Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins |
title_full_unstemmed | Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins |
title_short | Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins |
title_sort | purification and characterization of native and vaccine candidate mutant enterotoxigenic escherichia coli heat-stable toxins |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071264/ https://www.ncbi.nlm.nih.gov/pubmed/29970812 http://dx.doi.org/10.3390/toxins10070274 |
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