Next Article in Journal
Microcystin Content in Phytoplankton and in Small Fish from Eutrophic Nyanza Gulf, Lake Victoria, Kenya
Previous Article in Journal
Effect of Suspended Particulate Matter on the Accumulation of Dissolved Diarrhetic Shellfish Toxins by Mussels (Mytilus galloprovincialis) under Laboratory Conditions
Article Menu

Export Article

Open AccessArticle
Toxins 2018, 10(7), 274; https://doi.org/10.3390/toxins10070274

Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins

1
Centre for Applied Biotechnology, Uni Research Environment, 5006 Bergen, Norway
2
Department of Biological Sciences, University of Bergen, 5006 Bergen, Norway
3
Centre for International Health, University of Bergen, 5006 Bergen, Norway
These authors contributed equally to the work.
*
Author to whom correspondence should be addressed.
Received: 1 June 2018 / Revised: 26 June 2018 / Accepted: 28 June 2018 / Published: 3 July 2018
(This article belongs to the Special Issue Heat-Resistant Toxins of Animal, Plant and Microbial Origins)
View Full-Text   |   Download PDF [1722 KB, uploaded 3 July 2018]   |  

Abstract

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. View Full-Text
Keywords: Enterotoxigenic Escherichia coli; ETEC; diarrhea; enterotoxin; heat-stable toxin; ST; disulfide isomerase; DsbC Enterotoxigenic Escherichia coli; ETEC; diarrhea; enterotoxin; heat-stable toxin; ST; disulfide isomerase; DsbC
Figures

Figure 1

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

Supplementary material

SciFeed

Share & Cite This Article

MDPI and ACS Style

Govasli, M.L.; Diaz, Y.; Zegeye, E.D.; Darbakk, C.; Taxt, A.M.; Puntervoll, P. Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins. Toxins 2018, 10, 274.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Toxins EISSN 2072-6651 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top