Novel effects of the prototype translocating Escherichia coli, strain C25 on intestinal epithelial structure and barrier function

The mechanism by which enteropathogenic Escherichia coli (EPEC) causes diarrhea remains elusive. Several alterations within the host cell have been demonstrated to occur following EPEC attachment including increases in intracellular Ca2+ concentration and rearrangement and phosphorylation of several cytoskeletal proteins. The consequences of these intracellular perturbations on host cell function, however, have not been determined. The aim of this study was to examine the effect of EPEC adherence on intestinal epithelial barrier function. T84 cell monolayers were infected with either wild-type EPEC or a nonadherent isogenic derivative. Transepithelial electrical resistance, a measure of barrier function, decreased 33.5 +/- 6.4% after a 6-h incubation with the wild-type strain. Electron microscopy revealed ultrastructurally normal cells, and lactate dehydrogenase release assays failed to demonstrate cytotoxicity. Dual 22Na+ and [3H]mannitol flux studies localized the permeability defect to tight junctions. In addition, cumulative flux of the paracellular marker mannitol was four- to fivefold greater across monolayers infected with wild-type EPEC. Sequestration of intracellular calcium stores by dantrolene completely abrogated the resistance drop associated with EPEC attachment. These data demonstrate that adherence of EPEC to intestinal epithelial cell monolayers disrupts tight junction barrier function via a calcium-requiring event.

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The role of caspase-3 in lipopolysaccharide-mediated disruption of intestinal epithelial tight junctions

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y06-056 ◽

2006 ◽

Vol 84 (10) ◽

pp. 1043-1050 ◽

Cited By ~ 67

Author(s):

Alex C. Chin ◽

Andrew N. Flynn ◽

Jason P. Fedwick ◽

Andre G. Buret

Keyword(s):

Escherichia Coli ◽

Tight Junctions ◽

Intestinal Permeability ◽

Barrier Function ◽

Caspase 3 ◽

Parp Cleavage ◽

Intestinal Epithelial ◽

Epithelial Permeability ◽

Epithelial Monolayers

The mechanisms responsible for microbially induced epithelial apoptosis and increased intestinal permeability remain unclear. This study assessed whether purified bacterial lipopolysaccharide (LPS) increases epithelial apoptosis and permeability and whether these changes are dependent on caspase-3 activation. In nontumorigenic epithelial monolayers, Escherichia coli O26:B6 LPS increased apoptosis, as shown by nuclear breakdown, caspase-3 activation, and PARP cleavage, and induced disruption of tight junctional ZO-1. Apical, but not basolateral, exposure to LPS increased epithelial permeability. Addition of a caspase-3 inhibitor abolished the effects of LPS. The findings describe a novel mechanism whereby apical LPS may disrupt epithelial tight junctional ZO-1 and barrier function in a caspase-3-dependent fashion.

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The Flagella of an Atypical Enteropathogenic Escherichia coli Strain Are Required for Efficient Interaction with and Stimulation of Interleukin-8 Production by Enterocytes In Vitro

Infection and Immunity ◽

10.1128/iai.00177-09 ◽

2009 ◽

Vol 77 (10) ◽

pp. 4406-4413 ◽

Cited By ~ 22

Author(s):

Suely C. F. Sampaio ◽

Tânia A. T. Gomes ◽

Christophe Pichon ◽

Laurence du Merle ◽

Stéphanie Guadagnini ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Coli Strain ◽

Interleukin 8 ◽

Intestinal Epithelial ◽

Protein Subunit ◽

T84 Cells ◽

Transmission Electron

ABSTRACT The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.

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The Oxidoreductase DsbA Plays a Key Role in the Ability of the Crohn's Disease-Associated Adherent-Invasive Escherichia coli Strain LF82 To Resist Macrophage Killing

Journal of Bacteriology ◽

10.1128/jb.00233-07 ◽

2007 ◽

Vol 189 (13) ◽

pp. 4860-4871 ◽

Cited By ~ 45

Author(s):

Marie-Agnès Bringer ◽

Nathalie Rolhion ◽

Anne-Lise Glasser ◽

Arlette Darfeuille-Michaud

Keyword(s):

Escherichia Coli ◽

Crohn’S Disease ◽

Crohn's Disease ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Coli Strain ◽

Intestinal Epithelial ◽

Type 1 Pili ◽

K 12

ABSTRACT Adherent-invasive Escherichia coli (AIEC) isolated from Crohn's disease patients is able to adhere to and invade intestinal epithelial cells and to replicate in mature phagolysosomes within macrophages. Here, we show that the dsbA gene, encoding a periplasmic oxidoreductase, was required for AIEC strain LF82 to adhere to intestinal epithelial cells and to survive within macrophages. The LF82-ΔdsbA mutant did not express flagella and, probably as a consequence of this, did not express type 1 pili. The role of DsbA in adhesion is restricted to the loss of flagella and type 1 pili, as forced contact between bacteria and cells and induced expression of type 1 pili restored the wild-type phenotype. In contrast, the dsbA gene is essential for AIEC LF82 bacteria to survive within macrophages, irrespective of the loss of flagella and type 1 pilus expression, and the survival ability of LF82-ΔdsbA was as low as that of the nonpathogenic E. coli K-12, which was efficiently killed by macrophages. We also provide evidence that the dsbA gene is needed for LF82 bacteria to grow and survive in an acidic and nutrient-poor medium that partly mimics the harsh environment of the phagocytic vacuole. In addition, under such stress conditions dsbA transcription is highly up-regulated. Finally, the CpxRA signaling pathway does not play a role in regulation of dsbA expression in AIEC LF82 bacteria under conditions similar to those of mature phagolysosomes.

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Infection of T84 cells with enteropathogenic Escherichia coli alters barrier and transport functions

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.270.4.g634 ◽

1996 ◽

Vol 270 (4) ◽

pp. G634-G645 ◽

Cited By ~ 29

Author(s):

D. J. Philpott ◽

D. M. McKay ◽

P. M. Sherman ◽

M. H. Perdue

Keyword(s):

Escherichia Coli ◽

Tight Junction ◽

Diarrheal Disease ◽

Chloride Secretion ◽

Coli Strain ◽

Intestinal Epithelial ◽

Enteropathogenic Escherichia Coli ◽

T84 Cells ◽

Junction Protein ◽

Barrier Defect

The effect of enteropathogenic Escherichia coli (EPEC) infection on electrophysiology of T84 cell monolayers was examined. After 18 h of infection with EPEC (E2348), transepithelial electrical resistance was decreased (30 +/- 5% of uninfected values) compared with monolayers infected with a nonpathogenic E. coli strain (104 +/- 13%). Resistance of monolayers infected with EPEC mutant strain CVD206, deficient in attaching and effacing lesion formation, was partially reduced (66 +/- 10%). In addition, permeability of EPEC-infected T84 monolayers increased compared with uninfected cells. Associated with these changes was an altered distribution of the tight junction protein, ZO-1. Taken together, these findings suggest that the barrier defect induced by EPEC was at the level of the tight junction. Adenosine 3'5'-cyclic monophosphate-stimulated chloride secretion was also diminished in EPEC-infected cells, whereas Ca2+ -dependent chloride secretion was not different from uninfected cells. These findings indicate that EPEC infection alters intestinal epithelial barrier and transport functions. Furthermore, these results provide a possible mechanism for EPEC-induced diarrheal disease.

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