Outer membrane vesicles and soluble factors released by probiotic Escherichia coli Nissle 1917 and commensal ECOR63 enhance barrier function by regulating expression of tight junction proteins in intestinal epithelial cells

The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional regulation of claudin-2. Thus, in addition to OMVs and TcpC, other active factors released by these microbiota strains contribute to the reinforcement of the epithelial barrier. Keywords: probiotics, gut microbes, Escherichia coli, phylogenetic group B2, membrane vesicles, tight junctions, intestinal barrier, Tcp

Extracellular vesicles and soluble factors secreted by Escherichia coli Nissle 1917 and ECOR63 protect against enteropathogenic E. coli-induced intestinal epithelial barrier dysfunction

Background: Enteric pathogens have developed mechanisms to disrupt tight junctions and increase gut permeability. Many studies have analysed the ability of live probiotics to protect intestinal epithelial cells against tight junction damage caused by bacterial pathogens. Escherichia coli Nissle 1917 (EcN) is among the probiotics that positively modulates the intestinal epithelial barrier by regulating expression and distribution of tight junction proteins. We previously reported that regulation of ZO-1, claudin-14 and claudin-2 is mediated by EcN secreted factors, either free-released or associated with outer membrane vesicles (OMVs). Factors secreted by commensal ECOR63 elicited comparable effects in intact epithelial T-84 and Caco-2 cell monolayers. Results:Here we analyse the ability of OMVs and soluble secreted factors to protect epithelial barrier function in polarized T-84 and Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). Transepithelial electrical resistance, paracellular permeability, mRNA levels and subcellular distribution of tight junction proteins were monitored in the absence or presence of EcN and ECOR63 extracellular fractions. EPEC downregulated expression of ZO-1 ZO-2, occludin and claudin-14 and altered the subcellular localization of ZO-1, occludin and F-actin cytoskeleton. OMVs and soluble factors secreted by EcN and ECOR63 counteracted EPEC- altered transepithelial resistance and paracellular permeability, preserved occludin and claudin-14 mRNA levels, retained ZO-1 and occludin at tight junctions in the cell boundaries and ameliorated F-actin disorganization. Redistribution of ZO-1 was not accompanied by changes at mRNA level. Conclusions: This study provides new insights on the role of microbiota secreted factors on the modulation of intestinal tight junctions, expanding their barrier- protective effects against pathogen-induced disruption

An overview on the modulation of the intestinal barrier and immune response by membrane vesicles secreted by the probiotic Escherichia coli Nissle 1917

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/128014Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut and its efficacy in the inflammatory process undergone in ulcerative colitis has been demonstrated. The probiotic action is mainly through the modulation of intestinal epithelial tight junctions and immune system. Here we review the role of outer membrane vesicles (OMVs) released by this probiotic strain on the modulation of intestinal homeostasis. EcN OMVs enter into host epithelial cells via clathrin-mediated endocytosis and are sorted to lysosomes via endocytic compartments. In cellular models of intestinal barrier, EcN OMVs stimulate the underlying immune system through the intestinal epithelium, triggering immune and defense responses. Thus, the use of probiotic derived OMVs could be a safe probiotic-derived strategy targeting intestinal inflammatory processes

The secreted autotransporter toxin (Sat) does not act as a virulence factor in the probiotic Escherichia coli strain Nissle 1917

BACKGROUND: Escherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells. RESULTS: Here we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections. CONCLUSIONS: Sat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype

Modulación de la barrera epitelial intestinal por vesículas de membrana y factores solubles secretados por cepas probióticas y comensales de Escherichia coli

[spa] El epitelio gastrointestinal forma una barrera física y bioquímica que mantiene la segregación entre el huésped y la microbiota intestinal. La integridad de esta barrera es crítica en el mantenimiento de la homeostasis intestinal y su disfunción está relacionada con una gran variedad de enfermedades. La microbiota intestinal, y en particular las bacterias probióticas, modulan la integridad de la barrera reduciendo la permeabilidad intestinal y reforzando las uniones estrechas (TJ) entre las células adyacentes del epitelio intestinal. El probiótico Escherichia coli Nissle 1917 (EcN) es un buen colonizador del intestino humano con probada eficacia terapéutica en la remisión de la colitis ulcerosa en humanos. EcN modula positivamente la barrera epitelial intestinal a través de la regulación positiva y redistribución de las proteínas TJ ZO-1, ZO-2 y claudina-14. La regulación positiva de claudina-14 se ha atribuido a la proteína secretada TcpC. Todavía se desconoce si la regulación de ZO-1 y ZO-2 está mediada por factores secretados por EcN. El objetivo de este estudio fue explorar si las vesículas de membrana externa (OMVs) liberadas por EcN fortalecen la barrera epitelial. Este estudio incluye otras cepas de E. coli de origen intestinal humano portadoras del gen tcpC, tales como ECOR63. Los sobrenadantes de cultivos de las cepas tipo salvaje y de los mutantes tcpC derivados se fraccionaron en OMVs y factores secretados. El impacto de estas fracciones extracelulares sobre la barrera epitelial se evaluó midiendo la resistencia transepitelial, la expresión y la redistribución de varias proteínas de TJ en monocapas de células T-84 y Caco-2. Nuestros resultados muestran que la actividad de refuerzo de la barrera por parte de EcN y ECOR63 no depende exclusivamente de TcpC. Tanto las OMVs como factores secretados por ambas cepas promueven la regulación positiva de ZO-1 y claudina-14, y la regulación negativa de claudina-2. Los efectos mediados por OMVs son independientes de TcpC. La proteína TcpC secretada en forma soluble contribuye a la regulación positiva de ZO-1 y claudina-14, pero no tiene ningún efecto sobre claudina-2. Las fracciones secretadas de ambas cepas, también mostraron actividad protectora de la barrera epitelial en un modelo de barrera dañada por infección con E. coli enteropatógena (EPEC). Este patógeno altera la integridad de la barrera epitelial, causando la regulación negativa de ZO-1, ZO-2, ocludina, y claudina-14, y un reordenamiento alterado de ZO-1 a nivel subcelular. Sin embargo, la infección con EPEC en presencia de las OMVs o de los factores solubles secretados por EcN o ECOR63 evitaron la disminución de la expresión de ocludina y claudina-14. A nivel de redistribución subcelular, ambas fracciones de EcN y ECOR63 suprimieron claramente el efecto de EPEC sobre ZO-1, manteniendo su localización asociada a ocludina en la zona de las TJ.[eng] The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses. Gut microbes, and probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions (TJ). Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the TJ proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance (TER) and expression of tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional regulation of claudin-2. Thus, inaddition to OMVs and TcpC, other active factors released by these microbiota strains contribute to there inforcement of the epithelial barrier. Also, we analyze the ability of OMVs and soluble factors to protect EPEC-induced TJ damage. TER and distribution of TJ proteins were monitored in polarized cells infected with EPEC in the absence or presence of both extracellular fractions from EcN and ECOR63. EPEC caused 50% reduction in the TER levels and altered localization of ZO-1, ZO-2 and occludin. However, OMVs and soluble factors secreted avoided TER reduction and promoted redistribution of the altered TJ proteins. Our results indicate the ability of OMVs and factors secreted by EcN and ECOR63 to protect TJ disruption caused by EPEC

Outer membrane vesicles and soluble factors released by probiotic Escherichia coli Nissle 1917 and commensal ECOR63 enhance barrier function by regulating expression of tight junction proteins in intestinal epithelial cells

The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional regulation of claudin-2. Thus, in addition to OMVs and TcpC, other active factors released by these microbiota strains contribute to the reinforcement of the epithelial barrier. Keywords: probiotics, gut microbes, Escherichia coli, phylogenetic group B2, membrane vesicles, tight junctions, intestinal barrier, Tcp

An overview on the modulation of the intestinal barrier and immune response by membrane vesicles secreted by the probiotic Escherichia coli Nissle 1917

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/128014Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut and its efficacy in the inflammatory process undergone in ulcerative colitis has been demonstrated. The probiotic action is mainly through the modulation of intestinal epithelial tight junctions and immune system. Here we review the role of outer membrane vesicles (OMVs) released by this probiotic strain on the modulation of intestinal homeostasis. EcN OMVs enter into host epithelial cells via clathrin-mediated endocytosis and are sorted to lysosomes via endocytic compartments. In cellular models of intestinal barrier, EcN OMVs stimulate the underlying immune system through the intestinal epithelium, triggering immune and defense responses. Thus, the use of probiotic derived OMVs could be a safe probiotic-derived strategy targeting intestinal inflammatory processes

The secreted autotransporter toxin (Sat) does not act as a virulence factor in the probiotic Escherichia coli strain Nissle 1917

BACKGROUND: Escherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells. RESULTS: Here we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections. CONCLUSIONS: Sat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype

The secreted autotransporter toxin (Sat) does not act as a virulence factor in the probiotic Escherichia coli strain Nissle 1917

BACKGROUND: Escherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells. RESULTS: Here we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections. CONCLUSIONS: Sat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype

Fructooligosaccharide supplementation in diets for tropical gar (Atractosteus tropicus) juvenile: Effects on morphophysiology and intestinal barrier function

Supplementary fructooligosaccharides (FOS) were evaluated on growth, survival, gut morphology, digestive enzyme activity and expression of intestinal barrier genes in tropical gar (Atractosteus tropicus). Four treatments were designed to include FOS (5, 10, 15 and 20 g/Kg) and were compared with a control (0 g/Kg) and the current commercial trout diet. Ten fish were stocked (0.25 ± 0.01 g and 4.2 ± 0.4 cm total length) per experimental unit by triplicate in a 45-day experiment. Fish fed with 5 and 10 g/Kg of FOS had the highest average weight and total average length. Fish fed 5 g/Kg FOS showed the best growth indexes (absolute weight gain: 2.16; specific growth rate: 4.96; and feed conversion rate: 1.23) and survival (96.66%) and also had the highest acid protease, chymotrypsin and leucine aminopeptidase activities. Fish fed 5 g/Kg FOS had a high absorption area and accumulation of lipids in the liver. Finally, relative overexpression of occluding (occ), nucleotide-binding oligomerization domain-2 (nod2) and mucin 2 (muc2) was detected in fish fed 10 and 15 g/Kg FOS. The supplementation between 5 and 10 g/Kg FOS in diets for A. tropicus juveniles could be beneficial for growth, digestive enzyme activities, gut morphology and intestinal barrier function.Fil: Sepúlveda Quiroz, Cesar Antonio. Universidad Juárez Autónoma de Tabasco; MéxicoFil: Peña Marín, Emyr Saul. Universidad Juárez Autónoma de Tabasco; MéxicoFil: Pérez Morales, Alfredo. Universidad de Colima; MéxicoFil: Martínez García, Rafael. Universidad Juárez Autónoma de Tabasco; MéxicoFil: Alvarez Villagomez, Carina Shianya. Universidad Juárez Autónoma de Tabasco; MéxicoFil: Maytorena Verdugo, Claudia Ivette. Universidad Juárez Autónoma de Tabasco; MéxicoFil: Camarillo Coop, Susana. Universidad Juárez Autónoma de Tabasco; MéxicoFil: Vissio, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Pérez Sirkin, Daniela Irina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; ArgentinaFil: Tovar Ramírez, Dariel. Centro de Investigaciones Biologicas del Noroeste; MéxicoFil: Galaviz, Mario. Universidad Autónoma de Baja California Sur; MéxicoFil: Álvarez González, Carlos Alfonso. Universidad Juárez Autónoma de Tabasco; Méxic