scholarly journals Mechanical Stimuli Affect Escherichia coli Heat-Stable Enterotoxin-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Model

2019 ◽  
Vol 88 (3) ◽  
Author(s):  
Laxmi Sunuwar ◽  
Jianyi Yin ◽  
Magdalena Kasendra ◽  
Katia Karalis ◽  
James Kaper ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cell ◽  
Cyclic Gmp ◽  
Cyclic Stretch ◽  
Intestinal Bacterium ◽  
Mechanical Forces ◽  
Mechanical Stimuli ◽  
Content Type ◽  
Heat Stable ◽  
Static Conditions

ABSTRACT Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a virulence factor of a pathogenic intestinal bacterium, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips) exposed to enterotoxigenic Escherichia coli heat-stable enterotoxin A (ST) and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height and apical and basolateral secretion of cyclic GMP (cGMP) under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to the level under static conditions but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to those of static culture conditions under both baseline conditions and with exposure to ST enterotoxin and suggests that further investigations of the application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resemble intact intestine than study under static conditions.

scholarly journals Mechanical Stimuli Affect E. Coli Heat Stable Enterotoxin (ST)-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Diarrhea Model

2019 ◽  
Author(s):  
Laxmi Sunuwar ◽  
Jianyi Yin ◽  
Magdalena Kasendra ◽  
Katia Karalis ◽  
James Kaper ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Animal Studies ◽  
Culture Conditions ◽  
Cyclic Stretch ◽  
Mechanical Forces ◽  
Mechanical Stimuli ◽  
E Coli ◽  
Cell Height ◽  
Heat Stable ◽  
Static Conditions

ABSTRACTModeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a pathogen virulence factor, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips), exposed to enterotoxigenic E. coli heat-stable enterotoxin A (ST), and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height, transcription of the cyclic nucleotide transporting protein MRP4, and apical and basolateral secretion of cGMP under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to static conditions, but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to static culture conditions under both baseline conditions and with exposure to ST enterotoxin, and suggests that further investigations of application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resembles intact intestine than study under static conditions.

scholarly journals Enterotoxigenic Escherichia coli Prevents Host NF-κB Activation by Targeting IκBα Polyubiquitination

2012 ◽  
Vol 80 (12) ◽  
pp. 4417-4425 ◽  
Author(s):  
Xiaogang Wang ◽  
Philip R. Hardwidge
Keyword(s):  
Escherichia Coli ◽  
Immune Responses ◽  
Diarrheal Disease ◽  
Innate Immune ◽  
Host Cells ◽  
Enterotoxigenic Escherichia Coli ◽  
Host Responses ◽  
Innate Immune Responses ◽  
Content Type ◽  
Heat Stable

ABSTRACTThe NF-κB pathway regulates innate immune responses to infection. NF-κB is activated after pathogen-associated molecular patterns are detected, leading to the induction of proinflammatory host responses. As a countermeasure, bacterial pathogens have evolved mechanisms to subvert NF-κB signaling. EnterotoxigenicEscherichia coli(ETEC) causes diarrheal disease and significant morbidity and mortality for humans in developing nations. The extent to which this important pathogen subverts innate immune responses by directly targeting the NF-κB pathway is an understudied topic. Here we report that ETEC secretes a heat-stable, proteinaceous factor that blocks NF-κB signaling normally induced by tumor necrosis factor (TNF), interleukin-1β, and flagellin. Pretreating intestinal epithelial cells with ETEC supernatant significantly blocked the degradation of the NF-κB inhibitor IκBα without affecting IκBα phosphorylation. Data from immunoprecipitation experiments suggest that the ETEC factor functions by preventing IκBα polyubiquitination. Inhibiting clathrin function blocked the activity of the secreted ETEC factor, suggesting that this yet-uncharacterized activity may utilize clathrin-dependent endocytosis to enter host cells. These data suggest that ETEC evades the host innate immune response by directly modulating NF-κB signaling.

scholarly journals Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli

2016 ◽  
Vol 84 (4) ◽  
pp. 1239-1249 ◽  
Author(s):  
Arne M. Taxt ◽  
Yuleima Diaz ◽  
Rein Aasland ◽  
John D. Clements ◽  
James P. Nataro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rational Design ◽  
Diarrheal Disease ◽  
Cross Reactivity ◽  
Single Amino Acid ◽  
Minimal Risk ◽  
Content Type ◽  
Heat Stable ◽  
Low Toxicity ◽  
Mutant Forms

EnterotoxigenicEscherichia coli(ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity.

scholarly journals A Tripartite Fusion, FaeG-FedF-LT192A2:B, of Enterotoxigenic Escherichia coli (ETEC) Elicits Antibodies That Neutralize Cholera Toxin, Inhibit Adherence of K88 (F4) and F18 Fimbriae, and Protect Pigs against K88ac/Heat-Labile Toxin Infection

2011 ◽  
Vol 18 (10) ◽  
pp. 1593-1599 ◽  
Author(s):  
Xiaosai Ruan ◽  
Mei Liu ◽  
Thomas A. Casey ◽  
Weiping Zhang
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
Etec Strain ◽  
Severe Diarrhea ◽  
Heat Stable ◽  
Heat Labile ◽  
Young Pigs ◽  
Gnotobiotic Piglet

ABSTRACTEnterotoxigenicEscherichia coli(ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT192) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT192A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT192A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT192A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrialE. colistrains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea.

scholarly journals Pathogenic Potential, Genetic Diversity, and Population Structure of Escherichia coli Strains Isolated from a Forest-Dominated Watershed (Comox Lake) in British Columbia, Canada

2014 ◽  
Vol 81 (5) ◽  
pp. 1788-1798 ◽  
Author(s):  
Abhirosh Chandran ◽  
Asit Mazumder
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Population Structure ◽  
Virulence Genes ◽  
Repetitive Element ◽  
Fingerprint Analysis ◽  
Pathogenic Potential ◽  
Content Type ◽  
E Coli ◽  
Heat Stable

ABSTRACTEscherichia coliisolates (n= 658) obtained from drinking water intakes of Comox Lake (2011 to 2013) were screened for the following virulence genes (VGs):stx1andstx2(Shiga toxin-producingE. coli[STEC]),eaeand the adherence factor (EAF) gene (enteropathogenicE. coli[EPEC]), heat-stable (ST) enterotoxin (variants STh and STp) and heat-labile enterotoxin (LT) genes (enterotoxigenicE. coli[ETEC]), andipaH(enteroinvasiveE. coli[EIEC]). The only genes detected wereeaeandstx2, which were carried by 37.69% (n= 248) of the isolates. Onlyeaewas harbored by 26.74% (n= 176) of the isolates, representing potential atypical EPEC strains, while onlystx2was detected in 10.33% (n= 68) of the isolates, indicating potential STEC strains. Moreover, four isolates were positive for both thestx2andeaegenes, representing potential EHEC strains. The prevalence of VGs (eaeorstx2) was significantly (P< 0.0001) higher in the fall season, and multiple genes (eaeplusstx2) were detected only in fall. Repetitive element palindromic PCR (rep-PCR) fingerprint analysis of 658E. coliisolates identified 335 unique fingerprints, with an overall Shannon diversity (H′) index of 3.653. Diversity varied among seasons over the years, with relatively higher diversity during fall. Multivariate analysis of variance (MANOVA) revealed that the majority of the fingerprints showed a tendency to cluster according to year, season, and month. Taken together, the results indicated that the diversity and population structure ofE. colifluctuate on a temporal scale, reflecting the presence of diverse host sources and their behavior over time in the watershed. Furthermore, the occurrence of potentially pathogenicE. colistrains in the drinking water intakes highlights the risk to human health associated with direct and indirect consumption of untreated surface water.

scholarly journals Immunizations with Enterotoxigenic Escherichia coli Heat-Stable Toxin Conjugates Engender Toxin-Neutralizing Antibodies in Mice That Also Cross-React with Guanylin and Uroguanylin

2019 ◽  
Vol 87 (7) ◽  
Author(s):  
Yuleima Diaz ◽  
Morten L. Govasli ◽  
Ephrem Debebe Zegeye ◽  
Halvor Sommerfelt ◽  
Hans Steinsland ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Cross Reactivity ◽  
Enterotoxigenic Escherichia Coli ◽  
Middle Income ◽  
Content Type ◽  
Endogenous Peptides ◽  
Heat Stable ◽  
Important Challenge

ABSTRACT Infection with enterotoxigenic Escherichia coli (ETEC) is a common cause of childhood diarrhea in low- and middle-income countries, as well as of diarrhea among travelers to these countries. In children, ETEC strains secreting the heat-stable toxin (ST) are the most pathogenic, and there are ongoing efforts to develop vaccines that target ST. One important challenge for ST vaccine development is to construct immunogens that do not elicit antibodies that cross-react with guanylin and uroguanylin, which are endogenous peptides involved in regulating the activity of the guanylate cyclase-C (GC-C) receptor. We immunized mice with both human ST (STh) and porcine ST (STp) chemically coupled to bovine serum albumin, and the resulting sera neutralized the toxic activities of both STh and STp. This suggests that a vaccine based on either ST variant can confer cross-protection. However, several anti-STh and anti-STp sera cross-reacted with the endogenous peptides, suggesting that the ST sequence must be altered to reduce the risk of unwanted cross-reactivity. Epitope mapping of four monoclonal anti-STh and six anti-STp antibodies, all of which neutralized both STh and STp, revealed that most epitopes appear to have at least one amino acid residue shared with guanylin or uroguanylin. Despite this, only one monoclonal antibody displayed demonstrable cross-reactivity to the endogenous peptides, suggesting that targeted mutations of a limited number of ST residues may be sufficient to obtain a safe ST-based vaccine.

Role of cyclic GMP in the action of heat-stable enterotoxin of Escherichia coli

Nature ◽  
1978 ◽  
Vol 271 (5647) ◽  
pp. 755-756 ◽  
Author(s):  
JAMES M. HUGHES ◽  
FERID MURAD ◽  
BING CHANG ◽  
RICHARD L. GUERRANT
Keyword(s):  
Escherichia Coli ◽  
Cyclic Gmp ◽  
Heat Stable

scholarly journals Evaluating the Occurrence of Escherichia albertii in Chicken Carcass Rinses by PCR, Vitek Analysis, and Sequencing of therpoBGene

2014 ◽  
Vol 81 (5) ◽  
pp. 1727-1734 ◽  
Author(s):  
Rebecca L. Lindsey ◽  
Paula J. Fedorka-Cray ◽  
Melanie Abley ◽  
Jennifer B. Turpin ◽  
Richard J. Meinersmann
Keyword(s):  
Escherichia Coli ◽  
Causative Agent ◽  
Clinical Importance ◽  
Culture Methods ◽  
Hafnia Alvei ◽  
Content Type ◽  
Shiga Toxins ◽  
Heat Stable ◽  
Food Borne ◽  
Chicken Carcass

ABSTRACTEscherichia albertiiis a recently described species that has been associated with gastroenteritis in humans and with healthy and ill birds. Most recently, it has been identified as the causative agent in a food-borne outbreak in Japan. The distribution and clinical importance ofE. albertiiare not well studied because its importance is unclear. Culture methods for clinical isolation frequently missE. albertiior incorrectly identify it asShigellaspp.,Escherichia coli, orHafnia alvei. This study was designed to determine ifE. albertiicould be recovered from chicken carcass rinses collected at slaughter during a 1-year period from November 2009 until October 2010. Colonies were isolated from chicken carcass rinses and tested by PCR for the presence or absence ofclpX, lysP, mdh, intimin (eae), Shiga toxins 1 and 2 (stx1,stx2, andstx2f), heat-stable enterotoxin A (staA), and cytolethal distending toxins 1 and 2 (cdtB) genes. Sixty-five isolates were analyzed by sequencing a section of therpoBgene. Analysis of therpoBgene sequences revealed 14 fixed differences betweenE. albertiiand other, closely related organisms. The fixed differences found in therpoBgene could aid in future discrimination ofE. albertiifrom closely related bacteria.

Sign in / Sign up

Export Citation Format

Share Document