A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System

Autores
Salazar, Mario Oscar; Viarengo, Gastón; Sciara, Mariela Ines; Kieffer, Pablo M.; Garcia Vescovi, Eleonora; Furlan, Ricardo Luis Eugenio
Año de publicación
2013
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Introduction The PhoP–PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. Objective To develop a TLC bioautography to detect inhibitors of the PhoP–PhoQ regulatory system in complex matrixes. Methods The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. Results A bioautographic assay suitable for the localisation of inhibitors of the PhoP–PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP–PhoQ system. Conclusion A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP–PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP–PhoQ regulatory system from a hydrolysed extract.
Fil: Salazar, Mario Oscar. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
Fil: Viarengo, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Sciara, Mariela Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Kieffer, Pablo M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Garcia Vescovi, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Furlan, Ricardo Luis Eugenio. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
Materia
Bioautography
Linoleic Acid
Linolenic Acid
Phop-Phoq
Salmonella
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/7468
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/7468
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory SystemSalazar, Mario OscarViarengo, GastónSciara, Mariela InesKieffer, Pablo M.Garcia Vescovi, EleonoraFurlan, Ricardo Luis EugenioBioautographyLinoleic AcidLinolenic AcidPhop-PhoqSalmonellahttps://purl.org/becyt/ford/1.4https://purl.org/becyt/ford/1Introduction The PhoP–PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. Objective To develop a TLC bioautography to detect inhibitors of the PhoP–PhoQ regulatory system in complex matrixes. Methods The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. Results A bioautographic assay suitable for the localisation of inhibitors of the PhoP–PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP–PhoQ system. Conclusion A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP–PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP–PhoQ regulatory system from a hydrolysed extract.Fil: Salazar, Mario Oscar. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Viarengo, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Sciara, Mariela Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Kieffer, Pablo M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Garcia Vescovi, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Furlan, Ricardo Luis Eugenio. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaWiley2013-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/7468Salazar, Mario Oscar; Viarengo, Gastón; Sciara, Mariela Ines; Kieffer, Pablo M.; Garcia Vescovi, Eleonora; et al.; A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System; Wiley; Phytochemical Analysis; 25; 2; 12-2013; 155-1600958-0344enginfo:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1002/pca.2482/abstractinfo:eu-repo/semantics/altIdentifier/doi/10.1002/pca.2482info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:56:27Zoai:ri.conicet.gov.ar:11336/7468instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 09:56:27.692CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System
title A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System
spellingShingle A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System
Salazar, Mario Oscar
Bioautography
Linoleic Acid
Linolenic Acid
Phop-Phoq
Salmonella
title_short A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System
title_full A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System
title_fullStr A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System
title_full_unstemmed A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System
title_sort A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System
dc.creator.none.fl_str_mv Salazar, Mario Oscar
Viarengo, Gastón
Sciara, Mariela Ines
Kieffer, Pablo M.
Garcia Vescovi, Eleonora
Furlan, Ricardo Luis Eugenio
author Salazar, Mario Oscar
author_facet Salazar, Mario Oscar
Viarengo, Gastón
Sciara, Mariela Ines
Kieffer, Pablo M.
Garcia Vescovi, Eleonora
Furlan, Ricardo Luis Eugenio
author_role author
author2 Viarengo, Gastón
Sciara, Mariela Ines
Kieffer, Pablo M.
Garcia Vescovi, Eleonora
Furlan, Ricardo Luis Eugenio
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Bioautography
Linoleic Acid
Linolenic Acid
Phop-Phoq
Salmonella
topic Bioautography
Linoleic Acid
Linolenic Acid
Phop-Phoq
Salmonella
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.4
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Introduction The PhoP–PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. Objective To develop a TLC bioautography to detect inhibitors of the PhoP–PhoQ regulatory system in complex matrixes. Methods The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. Results A bioautographic assay suitable for the localisation of inhibitors of the PhoP–PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP–PhoQ system. Conclusion A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP–PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP–PhoQ regulatory system from a hydrolysed extract.
Fil: Salazar, Mario Oscar. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
Fil: Viarengo, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Sciara, Mariela Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Kieffer, Pablo M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Garcia Vescovi, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Furlan, Ricardo Luis Eugenio. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina
description Introduction The PhoP–PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. Objective To develop a TLC bioautography to detect inhibitors of the PhoP–PhoQ regulatory system in complex matrixes. Methods The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. Results A bioautographic assay suitable for the localisation of inhibitors of the PhoP–PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP–PhoQ system. Conclusion A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP–PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP–PhoQ regulatory system from a hydrolysed extract.
publishDate 2013
dc.date.none.fl_str_mv 2013-12
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/7468
Salazar, Mario Oscar; Viarengo, Gastón; Sciara, Mariela Ines; Kieffer, Pablo M.; Garcia Vescovi, Eleonora; et al.; A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System; Wiley; Phytochemical Analysis; 25; 2; 12-2013; 155-160
0958-0344
url http://hdl.handle.net/11336/7468
identifier_str_mv Salazar, Mario Oscar; Viarengo, Gastón; Sciara, Mariela Ines; Kieffer, Pablo M.; Garcia Vescovi, Eleonora; et al.; A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP-PhoQ Regulatory System; Wiley; Phytochemical Analysis; 25; 2; 12-2013; 155-160
0958-0344
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://onlinelibrary.wiley.com/doi/10.1002/pca.2482/abstract
info:eu-repo/semantics/altIdentifier/doi/10.1002/pca.2482
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Wiley
publisher.none.fl_str_mv Wiley
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 13.13397

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