scholarly journals Preliminary Characterization of the Transcriptional Response of the Porcine Intestinal Cell Line IPEC-J2 to EnterotoxigenicEscherichia coli,Escherichia coli, andE. coliLipopolysaccharide

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Marisa M. Geens ◽  
Theo A. Niewold
Keyword(s):  
Escherichia Coli ◽  
Cell Line ◽  
Transcriptional Response ◽  
Intestinal Cell ◽  
Adhesion Assay ◽  
Transcriptional Level ◽  
Suitable Model ◽  
E Coli ◽  
Intestinal Cell Line

IPEC-J2, a promisingin vitromodel system, is not well characterized especially on the transcriptional level, in contrast to human counterparts. The aim of this study was to characterize the gene expression in IPEC-J2 cells when coincubated with enterotoxigenicEscherichia coli(ETEC), nonpathogenicE. coli, andE. coliendotoxin. Apical infection of polarized IPEC-J2 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TEER). Microarray analysis showed up-regulation of interleukins when IPEC-J2 were cocultured withE. colistrains this has so far never been measured in this cell line. Highest IL8 expression was found with the ETEC strain possessing the F4 fimbrium, suggesting IPEC-J2 cells to be F4 receptor positive, confirmed in a brush border membrane adhesion assay. It is concluded that the innate immune responses to pathogens and LPS makes the IPEC-J2 cell line a suitable model for research on intestinal host pathogen interaction.

scholarly journals Mannan rich fraction from yeast modulates inflammatory responses in intestinal cells (HT-29) exposed toEscherichia coli

2019 ◽  
Vol 7 ◽  
Author(s):  
Niall Browne ◽  
Aimee Traynor ◽  
Karina A. Horgan
Keyword(s):  
Inflammatory Responses ◽  
Economic Cost ◽  
Intestinal Cell ◽  
Cell Protein ◽  
Intestinal Cells ◽  
E Coli ◽  
Intestinal Cell Line ◽  
Proinflammatory Responses ◽  
Ht 29

AbstractMannan from yeast has been demonstrated to limit infection in animals susceptible to gastrointestinal infection, including pigs, poultry and cows, by blocking the mechanism by which gram-negative bacteria adhere to and invade the intestines. EnterotoxigenicEscherichia coli(ETEC) cause post weaning diarrhoea (PWD) which results in poor weight gain and potential death at great economic cost to the farmer. A mannan rich fraction (MRF) was assessedin vitrofor its impact on ETEC infection of HT-29 intestinal cell line. Gene expression markers for inflammation (TNFαandIL-1β) and TLR4 (TICAM-1andLY96) associated recognition of bacteria were significantly elevated following exposure toE. colialone, but not in combination with MRF compared to the control. HT-29 cells exposed to MRF alone demonstrated significantly reduced expression of immune signalling genesIRAK1,IRF7andJUNwhen compared to the control. HT-29 cell protein abundance for TNFα and TLR4 associated proteins were significantly increased in response toE. coliexposure alone while no significant change was observed for MRF treatment withE. coliinfection.E. coliadhesion to HT-29 cells was significantly decreased with addition of MRF compared toE. coliinfection alone. The action of MRF demonstrated its potential capacity to limit infection on anin vitrolevel through blocking bacterial interaction with the intestines that leads to infection as marked by a reduction in proinflammatory responses. MRF on its own demonstrated potential anti-inflammatory effects on intestinal cells with the reduction of proinflammatory responses observed.

scholarly journals Afa/Dr Diffusely Adhering Escherichia coli Infection in T84 Cell Monolayers Induces Increased Neutrophil Transepithelial Migration, Which in Turn Promotes Cytokine-Dependent Upregulation of Decay-Accelerating Factor (CD55), the Receptor for Afa/Dr Adhesins

2003 ◽  
Vol 71 (4) ◽  
pp. 1774-1783 ◽  
Author(s):  
Frederic Betis ◽  
Patrick Brest ◽  
Véronique Hofman ◽  
Julie Guignot ◽  
Imad Kansau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Laboratory Strain ◽  
Intestinal Cell ◽  
Necrosis Factor Alpha ◽  
Decay Accelerating Factor ◽  
E Coli ◽  
Intestinal Cell Line ◽  
Bowel Diseases ◽  
Escherichia Coli Infection ◽  
Human Intestinal Cell Line

ABSTRACT Ulcerative colitis and Crohn's disease are inflammatory bowel diseases thought to involve strains of Escherichia coli. We report here that two wild-type Afa/Dr diffusely adhering E. coli (DAEC) strains, C1845 and IH11128, which harbor the fimbrial F1845 adhesin and the Dr hemagglutinin, respectively, and the E. coli laboratory strain HB101, transformed with the pSSS1 plasmid to produce Afa/Dr F1845 adhesin, all induced interleukin-8 (IL-8) production and transepithelial migration of polymorphonuclear leukocytes (PMNL) in polarized monolayers of the human intestinal cell line T84 grown on semipermeable filters. We observed that after PMNL migration, expression of decay-accelerating factor (DAF, or CD55), the brush border-associated receptor for Afa/Dr adhesins, was strongly enhanced, increasing the adhesion of Afa/Dr DAEC bacteria. When examining the mechanism by which DAF expression was enhanced, we observed that the PMNL transepithelial migration induced epithelial synthesis of tumor necrosis factor alpha and IL-1β, which in turn promoted the upregulation of DAF.

scholarly journals Piracy of Decay-Accelerating Factor (CD55) Signal Transduction by the Diffusely Adhering Strain Escherichia coli C1845 Promotes Cytoskeletal F-Actin Rearrangements in Cultured Human Intestinal INT407 Cells

1998 ◽  
Vol 66 (9) ◽  
pp. 4036-4042 ◽  
Author(s):  
Isabelle Peiffer ◽  
Alain L. Servin ◽  
Marie-Françoise Bernet-Camard
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Protein Tyrosine Kinase ◽  
Cell Entry ◽  
Intestinal Cell ◽  
Intestinal Epithelial ◽  
Decay Accelerating Factor ◽  
E Coli ◽  
Intestinal Cell Line ◽  
Fimbrial Adhesin

ABSTRACT Diffusely adhering Escherichia coli (DAEC) C1845 (clinical isolate) harboring the fimbrial adhesin F1845 can infect cultured human differentiated intestinal epithelial cells; this process is followed by the disassembly of the actin network in the apical domain. The aim of this study was to examine the mechanism by which DAEC C1845 promotes F-actin rearrangements. For this purpose, we used a human embryonic intestinal cell line (INT407) expressing the membrane-associated glycosylphosphatidylinositol (GPI) protein-anchored decay-accelerating factor (DAF), the receptor of the F1845 adhesin. We show here that infection of INT407 cells by DAEC C1845 can provoke dramatic F-actin rearrangements without cell entry. Clustering of phosphotyrosines was observed, revealing that the DAEC C1845-DAF interaction involves the recruitment of signal transduction molecules. A pharmacological approach with a subset of inhibitors of signal transduction molecules was used to identify the cascade of signal transduction molecules that are coupled to the DAF, that are activated upon infection, and that promote the F-actin rearrangements. DAEC C1845-induced F-actin rearrangements can be blocked dose dependently by protein tyrosine kinase, phospholipase Cγ, phosphatidylinositol 3-kinase, protein kinase C, and Ca2+inhibitors. F-actin rearrangements and blocking by inhibitors were observed after infection of the cells with two E. colirecombinants carrying the plasmids containing the fimbrial adhesin F1845 or the fimbrial hemagglutinin Dr, belonging to the same family of adhesins. These findings show that the DAEC Dr family of pathogens promotes alterations in the intestinal cell cytoskeleton by piracy of the DAF-GPI signal cascade without bacterial cell entry.

scholarly journals PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome

2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Vanessa S. Terra ◽  
Marta Mauri ◽  
Thippeswamy H. Sannasiddappa ◽  
Alexander A. Smith ◽  
Mark P. Stevens ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rapid Identification ◽  
Effect Of Temperature ◽  
Limiting Factor ◽  
Transcriptional Level ◽  
Coding Region ◽  
Temperature Dependent ◽  
Live Attenuated Vaccine ◽  
E Coli

Abstract Background Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. Results We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain. Conclusions We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level.

scholarly journals Gain of Spontaneous clpX Mutations Boosting Motility via Adaption to Environments in Escherichia coli

2021 ◽  
Vol 9 ◽  
Author(s):  
Bingyu Li ◽  
Chaofan Hou ◽  
Xian Ju ◽  
Yong Feng ◽  
Zhi-Qiang Ye ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Promoter Region ◽  
Molecular Mechanisms ◽  
Soft Agar ◽  
Transcriptional Level ◽  
E Coli ◽  
Proteomic Data ◽  
C Terminus ◽  
Enhanced Expression

Motility is finely regulated and is crucial to bacterial processes including colonization and biofilm formation. There is a trade-off between motility and growth in bacteria with molecular mechanisms not fully understood. Hypermotile Escherichia coli could be isolated by evolving non-motile cells on soft agar plates. Most of the isolates carried mutations located upstream of the flhDC promoter region, which upregulate the transcriptional expression of the master regulator of the flagellum biosynthesis, FlhDC. Here, we identified that spontaneous mutations in clpX boosted the motility of E. coli largely, inducing several folds of changes in swimming speed. Among the mutations identified, we further elucidated the molecular mechanism underlying the ClpXV78F mutation on the regulation of E. coli motility. We found that the V78F mutation affected ATP binding to ClpX, resulting in the inability of the mutated ClpXP protease to degrade FlhD as indicated by both structure modeling and in vitro protein degradation assays. Moreover, our proteomic data indicated that the ClpXV78F mutation elevated the stability of known ClpXP targets to various degrees with FlhD as one of the most affected. In addition, the specific tag at the C-terminus of FlhD being recognized for ClpXP degradation was identified. Finally, our transcriptome data characterized that the enhanced expression of the motility genes in the ClpXV78F mutations was intrinsically accompanied by the reduced expression of stress resistance genes relating to the reduced fitness of the hypermotile strains. A similar pattern was observed for previously isolated hypermotile E. coli strains showing high expression of flhDC at the transcriptional level. Hence, clpX appears to be a hot locus comparable to the upstream of the flhDC promoter region evolved to boost bacterial motility, and our finding provides insight into the reduced fitness of the hypermotile bacteria.

scholarly journals Cytotoxicological evaluation of semi-purified extracts of some dye yielding plants of the Kashmir Valley on Normal Intestinal Cell Line (IEC-6) by MTT assay

2018 ◽  
Vol 7 (1) ◽  
pp. 5-9
Author(s):  
Qazi Gazala ◽  
◽  
Shoukat Ara ◽  
KM Ansari ◽  
Imtiyaz Murtaza ◽  
...  
Keyword(s):  
Cell Line ◽  
Plant Extracts ◽  
Mtt Assay ◽  
Cytotoxic Effect ◽  
Intestinal Cell ◽  
Calendula Officinalis ◽  
Rubia Cordifolia ◽  
Intestinal Cell Line ◽  
Calendula Officinalis L

Plant extracts are widely used in many fields and there is a need to evaluate their cytotoxic effect to determine their non-cytotoxic concentration at which they can be used in a safe manner. Keeping this in view, the present study was designed to evaluate the in vitro toxicity of Celosia argentia L. var plumosa (Cockscomb), Calendula officinalis L. (Pot Marigold), Indigofera heterantha Wall. (Himalayan Indigo) and Rubia cordifolia L. (Indian Madder) on Normal Intestinal Cell Line (IEC-6) by MTT assay to test their feasibility for natural edible dye extraction. The experimental material, comprised of inflorescence of Celosia argentia L. var plumose, petals of the two varieties of Calendula officinalis L., leaves of Indigofera heterantha Wall. and leaves and roots of the Rubia cordifolia L. Cell line was exposed to 1, 4, 16, 64 and 256µg/ml concentrations of plant extracts for 24, 48, and 72hr at 37oC. Results revealed that both the varieties of Calendula officinalis L. var. Gitana Orange and Gitana Yellow did not show any cytotoxic effect on IEC-6 cell line while as Celosia argentia L. var plumose, Indigofera heterantha Wall. and Rubia cordifolia L. showed cytotoxicity. From the present study it was concluded that the extracts of the both varieties of Calendula officinalis L. var. Gitana Orange and Gitana Yellow extracts are non-toxic in nature, thus can be utilized for the extraction of natural edible dye while as the extracts of Celosia argentia L. var plumose, Indigofera heterantha Wall. and Rubia cordifolia L. had potent in vitro cytotoxic activity thus they cannot be used for extraction of natural edible food colour. However, to better evaluate the cytotoxic effect of these plant extracts, in vivo experiments on laboratory animal followed by histological analysis should be done

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