Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner (original) (raw)
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Enteropathogenic E. coli disrupts tight junction barrier function and structure in vivo
Laboratory Investigation, 2005
Enteropathogenic Escherichia coli (EPEC) infection disrupts tight junctions (TJs) and perturbs intestinal barrier function in vitro. E. coli secreted protein F (EspF) is, in large part, responsible for these physiological and morphological alterations. We recently reported that the C57BL/6J mouse is a valid in vivo model of EPEC infection as EPEC colonizes the intestinal epithelium and effaces microvilli. Our current aim was to examine the effects of EPEC on TJ structure and barrier function of the mouse intestine and to determine the role of EspF in vivo. C57BL/6J mice were gavaged with B2 Â 10 8 EPEC organisms or PBS. At 1 or 5 days postinfection, mice were killed and ileal and colonic tissue was mounted in Ü ssing chambers to determine barrier function (measured as transepithelial resistance) and short circuit current. TJ structure was analyzed by immunofluorescence microscopy. Wild-type (WT) EPEC significantly diminished the barrier function of ileal and colonic mucosa at 1 and 5 days postinfection. Deficits in barrier function correlated with redistribution of occludin in both tissues. Infection with an EPEC strain deficient of EspF (DespF) had no effect on barrier function at 1 day postinfection. Furthermore, DespF had no effect on ileal TJ morphology and minor alterations of colonic TJ morphology at 1 day postinfection. In contrast, at 5 days postinfection, WT EPEC and DespF had similar effects on barrier function and occludin localization. In both cases this was associated with immune activation, as demonstrated by increased mucosal tumor necrosis factor-a levels 5 days postinfection. In conclusion, these data demonstrate that WT EPEC infection of 6-8-week-old C57BL/6J mice (1) significantly decreases barrier function in the ileum and colon (2) redistributes occludin in the ileum and colon and (3) is dependent upon EspF to induce TJ barrier defects at early, but not late, times postinfection.
Enteroaggregative Escherichia coli Disrupts Epithelial Cell Tight Junctions
Infection and Immunity, 2010
Enteroaggregative Escherichia coli (EAEC) is responsible for inflammatory diarrhea in diverse populations, but its mechanisms of pathogenesis have not been fully elucidated. We have used a previously characterized polarized intestinal T84 cell model to investigate the effects of infection with EAEC strain 042 on tight junction integrity. We find that infection with strain 042 induces a decrease in transepithelial electrical resistance (TER) compared to uninfected controls and to cells infected with commensal E. coli strain HS. When the infection was limited after 3 h by washing and application of gentamicin, we observed that the TER of EAEC-infected monolayers continued to decline, and they remained low even as long as 48 h after the infection. Cells infected with the afimbrial mutant strain 042 aafA exhibited TER measurements similar to those seen in uninfected monolayers, implicating the aggregative adherence fimbriae II (AAF/II) as necessary for barrier dysfunction. Infection wit...
Cellular Microbiology, 2000
Enteropathogenic Escherichia coli (EPEC) increases tight junction permeability in part by phosphorylating the 20 kDa myosin light chain (MLC 20 ) that induces cytoskeletal contraction. The impact of this enteric pathogen on specific tight junction (TJ) proteins has not been investigated. We examined the effect of EPEC infection on occludin localization and phosphorylation in intestinal epithelial cells. After infection by EPEC, a progressive shift of occludin from a primarily TJ-associated domain to an intracellular compartment occurred, as demonstrated by immunofluorescent staining. A reverse in the ratio of phosphorylated to dephosphorylated occludin accompanied this morphological change. Eradication of EPEC with gentamicin resulted in the normalization of occludin localization and phosphorylation. The serine/threonine phosphatase inhibitor, calyculin A, prevented these events. The EPEC-associated decrease in transepithelial electrical resistance, a measure of TJ barrier function, returned to baseline after gentamicin treatment. Non-pathogenic E. coli, K-12, did not induce these changes. Transformation of K-12 with the pathogenicity island of EPEC, however, conferred the phenotype of wild-type EPEC. Deletion of specific EPEC genes encoding proteins involved in EPEC type III secretion markedly attenuated these effects. These findings suggest that EPEC-induced alterations in occludin contribute to the pathophysiology associated with this infection.
Infection and Immunity, 1998
Enterohemorrhagic Escherichia coli (EHEC) infection is associated with watery diarrhea and can lead to complications, including hemorrhagic colitis and the hemolytic-uremic syndrome. The mechanisms by which these organisms produce diarrheal disease remain to be elucidated. Changes in T84 epithelial cell electrophysiology were examined following EHEC infection. T84 cell monolayers infected with EHEC O157:H7 displayed a time-dependent decrease in transepithelial resistance. Increases in the transepithelial flux of both [ 3 H]mannitol and 51 Cr-EDTA accompanied the EHEC-induced decreases in T84 resistance. Altered barrier function induced by EHEC occurred at the level of the tight junction since immunofluorescent staining of the tight-junction-associated protein ZO-1 was disrupted when examined by confocal microscopy. Decreased resistance induced by EHEC involved a protein kinase C (PKC)-dependent pathway as the highly specific PKC inhibitor, CGP41251, abrogated the EHEC-induced drop i...
BMC Microbiology
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. Conclusion: 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.
The role of epithelial malfunction in the pathogenesis of enteropathogenic E. coli-induced diarrhea
Laboratory Investigation, 2009
The homeostatic balance of the gastrointestinal tract relies on a single layer of epithelial cells, which assumes both digestive and protective functions. Enteric pathogens, including enteropathogenic Escherichia coli (EPEC), have evolved numerous mechanisms to disrupt basic intestinal epithelial functions, promoting the development of gastrointestinal disorders. Despite its non-invasive nature, EPEC inflicts severe damage to the intestinal mucosa, including the dysregulation of water and solute transport and the disruption of epithelial barrier structure and function. Despite the high prevalence and morbidity of disease caused by EPEC infections, the etiology of its pathogenesis remains incompletely understood. This review integrates the newest findings on EPEC-epithelial interactions with established mechanisms of disease in an attempt to give a comprehensive understanding of the cellular processes whereby this common pathogen may cause diarrheal illness.
Frontiers in Cellular and Infection Microbiology, 2016
The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions, and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption.
Interaction of enterohemorrhagic Escherichia coli O157:H7 with mouse intestinal mucosa
FEMS Microbiology Letters, 2008
In this study, we used mouse ileal loops to investigate the interaction of enterohemorrhagic Escherichia coli (EHEC) O157:H7 with the mouse intestinal mucosa. With a dose of 10 9 and 3 h incubation, EHEC O157 was detected in the lumen and to a lesser extent associated with the epithelium. Typical attaching and effacing (A/E) lesions were seen, albeit infrequently. While the effector protein Tir was essential for A/E lesion formation, the bacterial type III secretion system adaptor protein TccP was dispensable. These results suggest that A/E lesions on mouse intestinal mucosa can be formed independently of robust actin polymerization.
Journal of Clinical Investigation, 1988
Toxin A of Clostridium difficile causes severe inflammatory enterocolitis in man and animals that appears to be mediated in part by acute inflammatory cells that migrate into the toxin A-exposed mucosa. To determine the direct effects of toxin A on intestinal epithelial permeability and structure in the absence of other modulating factors, we used cultured monolayers of a human intestinal epithelial cell line (T84). A toxin A concentration of 7 X 10-1 ,ug/ml (3 X i0-' M) nearly abolished monolayer transepithelial resistance within 6-8 h. This marked permeability defect occurred while the monolayers were still confluent. Dual sodium-mannitol flux studies localized the permeability defect to the intercellular tight junction. Cytotoxicity assays and morphological evaluation using Nomarski optics and electron microscopy failed to demonstrate any evidence of cell damage at the time the maximum resistance response was observed. Fluorescent staining for F actin, however, revealed a marked decrease in fluorescent intensity in toxin-treated monolayers versus controls. These data show that toxin A can directly affect the barrier function of this model intestinal epithelium and initially does so by selectively enhancing tight junction permeability. Furthermore, cytoskeletal structure is markedly altered over the same time course, although the integrity of individual cells is maintained. Because the cytoskeleton of intestinal epithelial cells is known to be capable of regulating tight junction permeability, we speculate that the above effects of toxin A on epithelial barrier function result from alterations of the cytoskeleton.