Proteolytic Processing and Activation of <named-content content-type="genus-species">Clostridium perfringens</named-content> Epsilon Toxin by Caprine Small Intestinal Contents

ABSTRACT Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must...

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Autores principales:	John C. Freedman, Jihong Li, Francisco A. Uzal, Bruce A. McClane
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/23f4dcbff7d34740b15b94603f59458f
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spelling	oai:doaj.org-article:23f4dcbff7d34740b15b94603f59458f2021-11-15T15:45:54ZProteolytic Processing and Activation of <named-content content-type="genus-species">Clostridium perfringens</named-content> Epsilon Toxin by Caprine Small Intestinal Contents10.1128/mBio.01994-142150-7511https://doaj.org/article/23f4dcbff7d34740b15b94603f59458f2014-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01994-14https://doaj.org/toc/2150-7511ABSTRACT Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. IMPORTANCE Processing and activation by intestinal proteases is a prerequisite for ETX-induced toxicity. Previous studies had characterized the activation of ETX using only arbitrarily chosen amounts of purified trypsin and/or chymotrypsin. Therefore, the current study examined ETX activation ex vivo by natural host intestinal contents. These analyses demonstrated that (i) ETX processing in host intestinal contents occurs in an ordered, stepwise fashion, (ii) processing of prototoxin by host intestinal contents results in higher-molecular-mass material and 3 distinct ~27-kDa ETX species, and (iii) serine proteases, such as trypsin, chymotrypsin, and other proteases, including carboxypeptidases, play a role in the activation of ETX by intestinal contents. These studies provide new insights into the activation and processing of ETX and demonstrate that this process is more complicated than previously appreciated.John C. FreedmanJihong LiFrancisco A. UzalBruce A. McClaneAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 5 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 John C. Freedman Jihong Li Francisco A. Uzal Bruce A. McClane Proteolytic Processing and Activation of <named-content content-type="genus-species">Clostridium perfringens</named-content> Epsilon Toxin by Caprine Small Intestinal Contents
description	ABSTRACT Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. IMPORTANCE Processing and activation by intestinal proteases is a prerequisite for ETX-induced toxicity. Previous studies had characterized the activation of ETX using only arbitrarily chosen amounts of purified trypsin and/or chymotrypsin. Therefore, the current study examined ETX activation ex vivo by natural host intestinal contents. These analyses demonstrated that (i) ETX processing in host intestinal contents occurs in an ordered, stepwise fashion, (ii) processing of prototoxin by host intestinal contents results in higher-molecular-mass material and 3 distinct ~27-kDa ETX species, and (iii) serine proteases, such as trypsin, chymotrypsin, and other proteases, including carboxypeptidases, play a role in the activation of ETX by intestinal contents. These studies provide new insights into the activation and processing of ETX and demonstrate that this process is more complicated than previously appreciated.
format	article
author	John C. Freedman Jihong Li Francisco A. Uzal Bruce A. McClane
author_facet	John C. Freedman Jihong Li Francisco A. Uzal Bruce A. McClane
author_sort	John C. Freedman
title	Proteolytic Processing and Activation of <named-content content-type="genus-species">Clostridium perfringens</named-content> Epsilon Toxin by Caprine Small Intestinal Contents
title_short	Proteolytic Processing and Activation of <named-content content-type="genus-species">Clostridium perfringens</named-content> Epsilon Toxin by Caprine Small Intestinal Contents
title_full	Proteolytic Processing and Activation of <named-content content-type="genus-species">Clostridium perfringens</named-content> Epsilon Toxin by Caprine Small Intestinal Contents
title_fullStr	Proteolytic Processing and Activation of <named-content content-type="genus-species">Clostridium perfringens</named-content> Epsilon Toxin by Caprine Small Intestinal Contents
title_full_unstemmed	Proteolytic Processing and Activation of <named-content content-type="genus-species">Clostridium perfringens</named-content> Epsilon Toxin by Caprine Small Intestinal Contents
title_sort	proteolytic processing and activation of <named-content content-type="genus-species">clostridium perfringens</named-content> epsilon toxin by caprine small intestinal contents
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/23f4dcbff7d34740b15b94603f59458f
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Proteolytic Processing and Activation of <named-content content-type="genus-species">Clostridium perfringens</named-content> Epsilon Toxin by Caprine Small Intestinal Contents

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