The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems

The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by <i>Bacillus cereus</i>. Previous studies have described the formation of the Hbl complex and aimed to identify the t...

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Autores principales:	Franziska Ramm, Marlitt Stech, Anne Zemella, Hendrik Frentzel, Stefan Kubick
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	eukaryotic cell-free system tripartite enterotoxin hemolytic enterotoxin hemolysin BL <i>Bacillus cereus</i> membrane perforation Medicine R
Acceso en línea:	https://doaj.org/article/9720420952e24d429823e8e18ab6ef6a
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spelling	oai:doaj.org-article:9720420952e24d429823e8e18ab6ef6a2021-11-25T19:08:59ZThe Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems10.3390/toxins131108072072-6651https://doaj.org/article/9720420952e24d429823e8e18ab6ef6a2021-11-01T00:00:00Zhttps://www.mdpi.com/2072-6651/13/11/807https://doaj.org/toc/2072-6651The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by <i>Bacillus cereus</i>. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin’s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L<sub>1</sub>, L<sub>2</sub> and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl’s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L<sub>1</sub>-L<sub>2</sub> showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.Franziska RammMarlitt StechAnne ZemellaHendrik FrentzelStefan KubickMDPI AGarticleeukaryotic cell-free systemtripartite enterotoxinhemolytic enterotoxinhemolysin BL<i>Bacillus cereus</i>membrane perforationMedicineRENToxins, Vol 13, Iss 807, p 807 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	eukaryotic cell-free system tripartite enterotoxin hemolytic enterotoxin hemolysin BL <i>Bacillus cereus</i> membrane perforation Medicine R
spellingShingle	eukaryotic cell-free system tripartite enterotoxin hemolytic enterotoxin hemolysin BL <i>Bacillus cereus</i> membrane perforation Medicine R Franziska Ramm Marlitt Stech Anne Zemella Hendrik Frentzel Stefan Kubick The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems
description	The tripartite enterotoxin Hemolysin BL (Hbl) has been widely characterized as a hemolytic and cytotoxic virulence factor involved in foodborne diarrheal illness caused by <i>Bacillus cereus</i>. Previous studies have described the formation of the Hbl complex and aimed to identify the toxin’s mode of action. In this study, we analyzed the assembly of Hbl out of its three individual subunits L<sub>1</sub>, L<sub>2</sub> and B in a soluble as well as a putative membrane bound composition using a Chinese hamster ovary (CHO) cell-free system. Subunits were either coexpressed or synthesized individually in separate cell-free reactions and mixed together afterwards. Hemolytic activity of cell-free synthesized subunits was demonstrated on 5% sheep blood agar and identified both synthesis procedures, coexpression as well as individual synthesis of each subunit, as functional for the synthesis of an active Hbl complex. Hbl’s ability to perforate cell membranes was evaluated using a propidium iodide uptake assay. These data suggested that coexpressed Hbl subunits augmented cytotoxic activity with increasing concentrations. Further, a pre-pore-complex of L<sub>1</sub>-L<sub>2</sub> showed cytotoxic effects suggesting the possibility of an interaction between the cell membrane and the pre-pore-complex. Overall, this study shows that cell-free protein synthesis is a fast and efficient way to study the assembly of multiple protein subunits in soluble as well as vesicular fractions.
format	article
author	Franziska Ramm Marlitt Stech Anne Zemella Hendrik Frentzel Stefan Kubick
author_facet	Franziska Ramm Marlitt Stech Anne Zemella Hendrik Frentzel Stefan Kubick
author_sort	Franziska Ramm
title	The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems
title_short	The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems
title_full	The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems
title_fullStr	The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems
title_full_unstemmed	The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems
title_sort	pore-forming hemolysin bl enterotoxin from <i>bacillus cereus</i>: subunit interactions in cell-free systems
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/9720420952e24d429823e8e18ab6ef6a
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The Pore-Forming Hemolysin BL Enterotoxin from <i>Bacillus cereus</i>: Subunit Interactions in Cell-Free Systems

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