Lactobacillus reuteri glyceraldehyde-3-phosphate dehydrogenase functions in adhesion to intestinal epithelial cells

2015 ◽  
Vol 61 (5) ◽  
pp. 373-380 ◽  
Author(s):  
Wen-Ming Zhang ◽  
Hai-Feng Wang ◽  
Kan Gao ◽  
Cong Wang ◽  
Li Liu ◽  
...  
Keyword(s):  
Surface Layer ◽  
Epithelial Cells ◽  
Lactobacillus Reuteri ◽  
Intestinal Epithelial Cells ◽  
Lactobacillus Rhamnosus ◽  
Protein Band ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Intestinal Epithelial ◽  
Spectrometry Analysis

This study was aimed to identify key surface proteins mediating the adhesion of lactobacilli to intestinal epithelial cells. By using Caco-2 and IPEC-J2 cells labeled with sulfo-NHS-biotin in the western blotting, a protein band of an approximately 37 kDa was detected on the surface layer of Lactobacillus reuteri strains ZJ616, ZJ617, ZJ621, and ZJ623 and Lactobacillus rhamnosus GG. Mass spectrometry analysis using the adhesion-related protein from L. reuteri ZJ617 showed that it was 100% homologous to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. reuteri JCM 1112 (GenBank: YP_001841377). The ability of L. reuteri ZJ617 to adhere to epithelial cells decreased significantly by treatment with LiCl or by blocking with an anti-GAPDH antibody, in comparison with the untreated strain (p < 0.05). Immunoelectron microscopic and immunofluorescence analyses confirmed that GAPDH is located on the surface layer of L. reuteri ZJ617. The results indicated that the GAPDH protein of L. reuteri ZJ617 acts as an adhesion component that plays an important role in binding to the intestinal epithelial cells.

Mass Spectrometry Analysis of Differential Protein Spots from Intestinal Side Population Cells and Epithelial Cells in Newborn Mice

2013 ◽  
Vol 749 ◽  
pp. 232-235
Author(s):  
Yong Feng Zhang ◽  
Jin Yang ◽  
Yong Gang Tan ◽  
Man Yin Chen ◽  
Min Yang
Keyword(s):  
Mass Spectrometry ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Side Population ◽  
Mass Spectrometry Analysis ◽  
Intestinal Epithelial ◽  
Side Population Cells ◽  
Differential Protein ◽  
Spectrometry Analysis ◽  
Database Retrieval

objective:To screen differential proteins or protein groups with stem cell characteristics.methods:Two-dimensional gel electrophoresis was used to separate total proteins from intestinal side population cells and intestinal epithelial cells of neonatal mice. Proteins which were differentially expressed more than 1.5 times were selected for mass spectrometry analysis as well as database retrieval and identification.results:The two-dimensional electrophoresis patterns of intestinal side population cells and intestinal epithelial cells were successfully established to screen 39 differential protein spots. Thirty-two kinds of proteins were identified using the mass spectrometry analysis and database retrieval.conclusion:There were differences between the protein expression profiles of mouse intestinal epithelial cells and stem cells. The identified 32 kinds of proteins may become potential and valuable molecular markers for identification of intestinal stem cells.

7 LACTOBACILLUS REUTERI SUPPRESSES PRO-INFLAMMATORY DRIVEN REACTIVE OXYGEN SPECIES IN VITRO IN HUMAN INTESTINAL EPITHELIAL CELLS AND IN VIVO IN A TNBS COLITIS MOUSE MODEL

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S41-S41 ◽  
Author(s):  
Wenly Ruan ◽  
Melinda Engevik ◽  
Alexandra Chang-Graham ◽  
Joseph Hyser ◽  
James Versalovic
Keyword(s):  
Reactive Oxygen Species ◽  
Epithelial Cells ◽  
Lactobacillus Reuteri ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Oxygen Species ◽  
Intestinal Epithelial ◽  
Colitis Model

Abstract Background Reactive oxygen species (ROS) play a role in maintaining intestinal epithelial homeostasis and are normally kept at low levels via antioxidant compounds. Dysregulation of ROS can lead to intestinal inflammation and contribute to inflammatory bowel disease (IBD). Select gut microbes possess the enzymatic machinery to produce antioxidants whereas others can dysregulate levels of ROS. Our model microbe, Lactobacillus reuteri (ATCC PTA 6475), has been demonstrated to reduce intestinal inflammation in mice models. It contains the genes encoding two distinct GshA-like glutamylcysteine ligases. We hypothesize that L. reuteri can secrete γ-glutamylcysteine to suppress ROS, minimize NFκB activation and regulate secretion of e pithelial cytokines. Methods & Results Conditioned media from L. reuteri was analyzed via mass spectrometry to confirm the presence of γ-glutamylcysteine. All cysteine containing products including γ-glutamylcysteine were fluorescently tagged in the conditioned media and then incubated with HT29 cell monolayers as well as human jejunal enteroid (HJE) monolayers. γ-glutamylcysteine was demonstrated to enter intestinal epithelial cells based on microscopy. Next, a Thioltracker assay was used to show increased intracellular glutathione levels by L. reuteri secreted γ-glutamylcysteine. HT29 cells and HJEs were then treated with IL-1β or hydrogen peroxide, and L. reuteri metabolites as well as γ-glutamylcysteine significantly suppressed pro-inflammatory cytokine driven ROS and IL-8 production. L. reuteri secreted products also reduced activity of NFκB as determined by a luciferase reporter assay. γ-glutamylcysteine deficient mutants were generated by targeted mutagenesis of GshA genes, and these mutant L. reuteri strains had a diminished ability to suppress IL-8 production and ROS. To further test the role of L. reuteri secreted γ-glutamylcysteine in vivo, a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)- induced mouse colitis model was used. Adolescent mice were orogavaged with PBS, L. reuteri, L. reuteri GshA2 mutant, or γ-glutamylcysteine for a week after which TNBS was rectally administered to induce colitis. We demonstrate that L. reuteri and γ-glutamylcysteine can suppress histologic inflammation compared to PBS control and L. reuteri GshA2 mutant groups. Conclusions Together these data indicate that L. reuteri secretes γ-glutamylcysteine which can enter the intestinal epithelial cells and modulate epithelial cytokine production. It acts via suppression of ROS and NFκB which then decreases IL-8 production. We are able to demonstrate this in vitro in both HT 29 cells and HJEs. We now also demonstrate this in vivo in a mouse colitis model. These experiments highlight a prominent role for ROS intermediates in microbiome-mammalian cell signaling processes involved in immune responses and intestinal inflammation.

scholarly journals Amelioration of Congenital Tufting Enteropathy in EpCAM (TROP1)-Deficient Mice via Heterotopic Expression of TROP2 in Intestinal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1847
Author(s):  
Gaku Nakato ◽  
Sohshi Morimura ◽  
Michael Lu ◽  
Xu Feng ◽  
Chuanjin Wu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Epithelial Cells ◽  
Cell Function ◽  
Intestinal Epithelial Cells ◽  
Surface Proteins ◽  
Stem Cell Markers ◽  
Intestinal Epithelial ◽  
Mesenteric Lymph Nodes ◽  
Function Expression ◽  
Deficient Mice

TROP1 (EpCAM) and TROP2 are homologous cell surface proteins that are widely expressed, and often co-expressed, in developing and adult epithelia. Various functions have been ascribed to EpCAM and TROP2, but responsible mechanisms are incompletely characterized and functional equivalence has not been examined. Adult intestinal epithelial cells (IEC) express high levels of EpCAM, while TROP2 is not expressed. EpCAM deficiency causes congenital tufting enteropathy (CTE) in humans and a corresponding lethal condition in mice. We expressed TROP2 and EpCAM in the IEC of EpCAM-deficient mice utilizing a villin promoter to assess EpCAM and TROP2 function. Expression of EpCAM or TROP2 in the IEC of EpCAM knockout mice prevented CTE. TROP2 rescue (T2R) mice were smaller than controls, while EpCAM rescue (EpR) mice were not. Abnormalities were observed in the diameters and histology of T2R small intestine, and Paneth and stem cell markers were decreased. T2R mice also exhibited enlarged mesenteric lymph nodes, enhanced permeability to 4 kDa FITC-dextran and increased sensitivity to detergent-induced colitis, consistent with compromised barrier function. Studies of IEC organoids and spheroids revealed that stem cell function was also compromised in T2R mice. We conclude that EpCAM and TROP2 exhibit functional redundancy, but they are not equivalent.

scholarly journals Surface proteins involved in the adhesion of Streptococcus salivarius to human intestinal epithelial cells

2018 ◽  
Vol 102 (6) ◽  
pp. 2851-2865 ◽  
Author(s):  
Fanny Chaffanel ◽  
Florence Charron-Bourgoin ◽  
Claire Soligot ◽  
Mounira Kebouchi ◽  
Stéphane Bertin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Surface Proteins ◽  
Intestinal Epithelial ◽  
Streptococcus Salivarius ◽  
Human Intestinal Epithelial Cells

scholarly journals Probiotics inhibit enteropathogenicE. coliadherence in vitro by inducing intestinal mucin gene expression

1999 ◽  
Vol 276 (4) ◽  
pp. G941-G950 ◽  
Author(s):  
David R. Mack ◽  
Sonia Michail ◽  
Shu Wei ◽  
Laura McDougall ◽  
Michael A. Hollingsworth
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Lactobacillus Rhamnosus ◽  
Intestinal Epithelial ◽  
Binding Assays ◽  
Beneficial Effects ◽  
Treatment And Prevention ◽  
Intestinal Mucin ◽  
Ht 29

Probiotic agents, live microorganisms with beneficial effects for the host, may offer an alternative to conventional antimicrobials in the treatment and prevention of enteric infections. The probiotic agents Lactobacillus plantarum 299v and Lactobacillus rhamnosus GG quantitatively inhibited the adherence of an attaching and effacing pathogenic Escherichia coli to HT-29 intestinal epithelial cells but did not inhibit adherence to nonintestinal HEp-2 cells. HT-29 cells were grown under conditions that induced high levels of either MUC2 or MUC3 mRNA, but HEp-2 cells expressed only minimal levels of MUC2 and no MUC3 mRNA. Media enriched for MUC2 and MUC3 mucin were added exogenously to binding assays and were shown to be capable of inhibiting enteropathogen adherence to HEp-2 cells. Incubation of L. plantarum 299v with HT-29 cells increased MUC2 and MUC3 mRNA expression levels. From these in vitro studies, we propose the hypothesis that the ability of probiotic agents to inhibit adherence of attaching and effacing organisms to intestinal epithelial cells is mediated through their ability to increase expression of MUC2 and MUC3 intestinal mucins.

scholarly journals Cell Surface-Associated Elongation Factor Tu Mediates the Attachment of Lactobacillus johnsonii NCC533 (La1) to Human Intestinal Cells and Mucins

2004 ◽  
Vol 72 (4) ◽  
pp. 2160-2169 ◽  
Author(s):  
Dominique Granato ◽  
Gabriela E. Bergonzelli ◽  
Raymond David Pridmore ◽  
Laure Marvin ◽  
Martine Rouvet ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Binding Capacity ◽  
Elongation Factor ◽  
Mass Spectrometry Analysis ◽  
Intestinal Cells ◽  
Elongation Factor Tu ◽  
Intestinal Epithelial ◽  
Lactobacillus Johnsonii ◽  
Proinflammatory Response

ABSTRACT The aim of this work was to identify Lactobacillus johnsonii NCC533 (La1) surface molecules mediating attachment to intestinal epithelial cells and mucins. Incubation of Caco-2 intestinal epithelial cells with an L. johnsonii La1 cell wall extract led to the recognition of elongation factor Tu (EF-Tu) as a novel La1 adhesin-like factor. The presence of EF-Tu at the surface of La1 was confirmed by analysis of purified outer surface protein extract by immunoblotting experiments, by electron microscopy, and by enzyme-linked immunosorbent assays of live bacteria. Furthermore, tandem mass spectrometry analysis proved that EF-TU was expressed at the La1 surface as an intact molecule. Using recombinant La1 EF-Tu protein, we were able to determine that its binding to intestinal cells and to mucins is pH dependent. Competition experiments suggested that EF-Tu has an important role in La1 mucin binding capacity. In addition, immunomodulation studies performed on HT29 cells showed that EF-Tu recombinant protein can induce a proinflammatory response in the presence of soluble CD14. Our in vitro results indicate that EF-Tu, through its binding to the intestinal mucosa, might participate in gut homeostasis.

Sign in / Sign up

Export Citation Format

Share Document