A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.

Autores
Garimano, Nicolás Ezequiel; Amaral, María Marta; Ibarra, Cristina Adriana
Año de publicación
2020
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Shiga toxin-2 (Stx2) is produced and released by E. coli O157:H7 (O157:H7) into the intestinal lumen after colonization, and is able to translocate to the circulatory system and reach target cells causing hemolytic uremic syndrome. Our aim was to elucidate which pathways were involved in Stx2 endocytosis and translocation across intestinal cells infected with STEC. HCT-8 cells grown on 96-well plates were preincubated with specific endocytosis inhibitors such as Eliglustat (EG), Dynasore (DY), MßCD or Amiloride (AM). Then, cells were washed and incubated for 4 h with 100 ng/ml Stx2 alone or in the presence of O157:H7 mutant lacking stx2 gene (O157:H7Ðstx2). Stx2 uptake was measured by flow cytometry and its cytotoxic effect by neutral red uptake assay. Translocation of Stx2 was evaluated by inhibitor preincubation of HCT-8, grown as monolayers on Millicell inserts, and incubated with O157:H7 Ðstx2+ Stx2. Then Stx2 cytotoxicity was quantified in lower chamber media by neutral red uptake, To analyze inhibitors effect on bacteria attachment, bacterial adherence assays were performed on HCT-8 monolayers cultured on 24- wells plates. EG caused the maximum decrease of Stx2 cytotoxic activity, followed by MßCD. AM and DY significantly neutralized Stx2 cytotoxicity but only in presence of O157:H7Ðstx2.Furthermore, Stx2 uptake was reduced when cells were preincubated with EG or MßCD, compared to DY or AM (p<0.05), indicating that Stx2 uptake may depend on Gb3 receptor, and, to a lesser extent, on cholesterol, which is consistent with a necessary interaction between Stx2 and its receptor to cause cytotoxicity. Moreover, both dynamin-dependent endocytosis and Gb3-independent macropinocytosis became relevant only when bacteria were present, suggesting that these mechanisms are sensible to bacterial infection. Taken together, our study suggests that the mechanisms responsible for enhanced cytotoxicity and transcytosis during infection may have the same endocytic origin
Fil: Garimano, Nicolás Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina
Fil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina
Fil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina
LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología y IX Reunión Anual de la Asociación Argentina de Nanomedicinas
Mar del Plata
Argentina
Sociedad Argentina de Investigación Clínica
Asociación Argentina de Farmacología Experimental
Sociedad Argentina de Biología
Sociedad Argentina de Protozoología
Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio
Asociación Argentina de Nanomedicinas
Materia
E. coli
SUH
Stx2
Transcytosis
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:11336/203627
Acceder
id CONICETDig_0f12423be4e12efe9f4c2fc77abb2198
oai_identifier_str oai:ri.conicet.gov.ar:11336/203627
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.Garimano, Nicolás EzequielAmaral, María MartaIbarra, Cristina AdrianaE. coliSUHStx2Transcytosishttps://purl.org/becyt/ford/3.3https://purl.org/becyt/ford/3Shiga toxin-2 (Stx2) is produced and released by E. coli O157:H7 (O157:H7) into the intestinal lumen after colonization, and is able to translocate to the circulatory system and reach target cells causing hemolytic uremic syndrome. Our aim was to elucidate which pathways were involved in Stx2 endocytosis and translocation across intestinal cells infected with STEC. HCT-8 cells grown on 96-well plates were preincubated with specific endocytosis inhibitors such as Eliglustat (EG), Dynasore (DY), MßCD or Amiloride (AM). Then, cells were washed and incubated for 4 h with 100 ng/ml Stx2 alone or in the presence of O157:H7 mutant lacking stx2 gene (O157:H7Ðstx2). Stx2 uptake was measured by flow cytometry and its cytotoxic effect by neutral red uptake assay. Translocation of Stx2 was evaluated by inhibitor preincubation of HCT-8, grown as monolayers on Millicell inserts, and incubated with O157:H7 Ðstx2+ Stx2. Then Stx2 cytotoxicity was quantified in lower chamber media by neutral red uptake, To analyze inhibitors effect on bacteria attachment, bacterial adherence assays were performed on HCT-8 monolayers cultured on 24- wells plates. EG caused the maximum decrease of Stx2 cytotoxic activity, followed by MßCD. AM and DY significantly neutralized Stx2 cytotoxicity but only in presence of O157:H7Ðstx2.Furthermore, Stx2 uptake was reduced when cells were preincubated with EG or MßCD, compared to DY or AM (p<0.05), indicating that Stx2 uptake may depend on Gb3 receptor, and, to a lesser extent, on cholesterol, which is consistent with a necessary interaction between Stx2 and its receptor to cause cytotoxicity. Moreover, both dynamin-dependent endocytosis and Gb3-independent macropinocytosis became relevant only when bacteria were present, suggesting that these mechanisms are sensible to bacterial infection. Taken together, our study suggests that the mechanisms responsible for enhanced cytotoxicity and transcytosis during infection may have the same endocytic originFil: Garimano, Nicolás Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaLXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología y IX Reunión Anual de la Asociación Argentina de NanomedicinasMar del PlataArgentinaSociedad Argentina de Investigación ClínicaAsociación Argentina de Farmacología ExperimentalSociedad Argentina de BiologíaSociedad Argentina de ProtozoologíaAsociación Argentina de Ciencia y Tecnología de Animales de LaboratorioAsociación Argentina de NanomedicinasFundacion Revista Medicina2020info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/203627A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología y IX Reunión Anual de la Asociación Argentina de Nanomedicinas; Mar del Plata; Argentina; 2019; 1-5CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.medicinabuenosaires.com/indices-de-2010-a-2019/Nacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:05:17Zoai:ri.conicet.gov.ar:11336/203627instacron: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:34982025-10-22 11:05:18.175CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.
title A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.
spellingShingle A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.
Garimano, Nicolás Ezequiel
E. coli
SUH
Stx2
Transcytosis
title_short A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.
title_full A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.
title_fullStr A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.
title_full_unstemmed A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.
title_sort A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.
dc.creator.none.fl_str_mv Garimano, Nicolás Ezequiel
Amaral, María Marta
Ibarra, Cristina Adriana
author Garimano, Nicolás Ezequiel
author_facet Garimano, Nicolás Ezequiel
Amaral, María Marta
Ibarra, Cristina Adriana
author_role author
author2 Amaral, María Marta
Ibarra, Cristina Adriana
author2_role author
author
dc.subject.none.fl_str_mv E. coli
SUH
Stx2
Transcytosis
topic E. coli
SUH
Stx2
Transcytosis
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.3
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Shiga toxin-2 (Stx2) is produced and released by E. coli O157:H7 (O157:H7) into the intestinal lumen after colonization, and is able to translocate to the circulatory system and reach target cells causing hemolytic uremic syndrome. Our aim was to elucidate which pathways were involved in Stx2 endocytosis and translocation across intestinal cells infected with STEC. HCT-8 cells grown on 96-well plates were preincubated with specific endocytosis inhibitors such as Eliglustat (EG), Dynasore (DY), MßCD or Amiloride (AM). Then, cells were washed and incubated for 4 h with 100 ng/ml Stx2 alone or in the presence of O157:H7 mutant lacking stx2 gene (O157:H7Ðstx2). Stx2 uptake was measured by flow cytometry and its cytotoxic effect by neutral red uptake assay. Translocation of Stx2 was evaluated by inhibitor preincubation of HCT-8, grown as monolayers on Millicell inserts, and incubated with O157:H7 Ðstx2+ Stx2. Then Stx2 cytotoxicity was quantified in lower chamber media by neutral red uptake, To analyze inhibitors effect on bacteria attachment, bacterial adherence assays were performed on HCT-8 monolayers cultured on 24- wells plates. EG caused the maximum decrease of Stx2 cytotoxic activity, followed by MßCD. AM and DY significantly neutralized Stx2 cytotoxicity but only in presence of O157:H7Ðstx2.Furthermore, Stx2 uptake was reduced when cells were preincubated with EG or MßCD, compared to DY or AM (p<0.05), indicating that Stx2 uptake may depend on Gb3 receptor, and, to a lesser extent, on cholesterol, which is consistent with a necessary interaction between Stx2 and its receptor to cause cytotoxicity. Moreover, both dynamin-dependent endocytosis and Gb3-independent macropinocytosis became relevant only when bacteria were present, suggesting that these mechanisms are sensible to bacterial infection. Taken together, our study suggests that the mechanisms responsible for enhanced cytotoxicity and transcytosis during infection may have the same endocytic origin
Fil: Garimano, Nicolás Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina
Fil: Amaral, María Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina
Fil: Ibarra, Cristina Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina
LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología y IX Reunión Anual de la Asociación Argentina de Nanomedicinas
Mar del Plata
Argentina
Sociedad Argentina de Investigación Clínica
Asociación Argentina de Farmacología Experimental
Sociedad Argentina de Biología
Sociedad Argentina de Protozoología
Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio
Asociación Argentina de Nanomedicinas
description Shiga toxin-2 (Stx2) is produced and released by E. coli O157:H7 (O157:H7) into the intestinal lumen after colonization, and is able to translocate to the circulatory system and reach target cells causing hemolytic uremic syndrome. Our aim was to elucidate which pathways were involved in Stx2 endocytosis and translocation across intestinal cells infected with STEC. HCT-8 cells grown on 96-well plates were preincubated with specific endocytosis inhibitors such as Eliglustat (EG), Dynasore (DY), MßCD or Amiloride (AM). Then, cells were washed and incubated for 4 h with 100 ng/ml Stx2 alone or in the presence of O157:H7 mutant lacking stx2 gene (O157:H7Ðstx2). Stx2 uptake was measured by flow cytometry and its cytotoxic effect by neutral red uptake assay. Translocation of Stx2 was evaluated by inhibitor preincubation of HCT-8, grown as monolayers on Millicell inserts, and incubated with O157:H7 Ðstx2+ Stx2. Then Stx2 cytotoxicity was quantified in lower chamber media by neutral red uptake, To analyze inhibitors effect on bacteria attachment, bacterial adherence assays were performed on HCT-8 monolayers cultured on 24- wells plates. EG caused the maximum decrease of Stx2 cytotoxic activity, followed by MßCD. AM and DY significantly neutralized Stx2 cytotoxicity but only in presence of O157:H7Ðstx2.Furthermore, Stx2 uptake was reduced when cells were preincubated with EG or MßCD, compared to DY or AM (p<0.05), indicating that Stx2 uptake may depend on Gb3 receptor, and, to a lesser extent, on cholesterol, which is consistent with a necessary interaction between Stx2 and its receptor to cause cytotoxicity. Moreover, both dynamin-dependent endocytosis and Gb3-independent macropinocytosis became relevant only when bacteria were present, suggesting that these mechanisms are sensible to bacterial infection. Taken together, our study suggests that the mechanisms responsible for enhanced cytotoxicity and transcytosis during infection may have the same endocytic origin
publishDate 2020
dc.date.none.fl_str_mv 2020
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
Reunión
Journal
http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/203627
A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología y IX Reunión Anual de la Asociación Argentina de Nanomedicinas; Mar del Plata; Argentina; 2019; 1-5
CONICET Digital
CONICET
url http://hdl.handle.net/11336/203627
identifier_str_mv A transcellular Gb3 dependent pathway is mainly responsible for Shiga toxin-2 cytotoxicity and translocation across human intestinal epithelial cells infected with E. coli O157:H7.; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología y IX Reunión Anual de la Asociación Argentina de Nanomedicinas; Mar del Plata; Argentina; 2019; 1-5
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.medicinabuenosaires.com/indices-de-2010-a-2019/
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
dc.coverage.none.fl_str_mv Nacional
dc.publisher.none.fl_str_mv Fundacion Revista Medicina
publisher.none.fl_str_mv Fundacion Revista Medicina
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
_version_ 1846781329215062016
score 12.982451

Publicaciones similares