Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory mod...

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Autores principales:	Carlos Medina, Eva María Camacho, Amando Flores, Beatriz Mesa-Pereira, Eduardo Santero
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/43d1ef00410c40338b132c2bf21812e9
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spelling	oai:doaj.org-article:43d1ef00410c40338b132c2bf21812e92021-11-18T06:48:53ZImproved expression systems for regulated expression in Salmonella infecting eukaryotic cells.1932-620310.1371/journal.pone.0023055https://doaj.org/article/43d1ef00410c40338b132c2bf21812e92011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21829692/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.Carlos MedinaEva María CamachoAmando FloresBeatriz Mesa-PereiraEduardo SanteroPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 8, p e23055 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Carlos Medina Eva María Camacho Amando Flores Beatriz Mesa-Pereira Eduardo Santero Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.
description	In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m) promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal) system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.
format	article
author	Carlos Medina Eva María Camacho Amando Flores Beatriz Mesa-Pereira Eduardo Santero
author_facet	Carlos Medina Eva María Camacho Amando Flores Beatriz Mesa-Pereira Eduardo Santero
author_sort	Carlos Medina
title	Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.
title_short	Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.
title_full	Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.
title_fullStr	Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.
title_full_unstemmed	Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.
title_sort	improved expression systems for regulated expression in salmonella infecting eukaryotic cells.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/43d1ef00410c40338b132c2bf21812e9
work_keys_str_mv	AT carlosmedina improvedexpressionsystemsforregulatedexpressioninsalmonellainfectingeukaryoticcells AT evamariacamacho improvedexpressionsystemsforregulatedexpressioninsalmonellainfectingeukaryoticcells AT amandoflores improvedexpressionsystemsforregulatedexpressioninsalmonellainfectingeukaryoticcells AT beatrizmesapereira improvedexpressionsystemsforregulatedexpressioninsalmonellainfectingeukaryoticcells AT eduardosantero improvedexpressionsystemsforregulatedexpressioninsalmonellainfectingeukaryoticcells
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Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

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