Athanasia Koutsouris

Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function

The mechanisms by which enteropathogenic Escherichia coli (EPEC), an important cause of diarrhea among infants in developing countries, induce symptoms are not defined. EPEC have a type III secretion system required for characteristic attaching and effacing changes that modify the cytoskeleton and apical surface of host cells. Infection of polarized intestinal epithelial cell monolayers by EPEC leads to a loss of transepithelial electrical resistance, which also requires the type III secretion system. We demonstrate here that EspF, a protein that is secreted by EPEC via the type III secretion system, is not required for quantitatively and qualitatively typical attaching and effacing lesion formation in intestinal epithelial cells. However, EspF is required in a dose-dependent fashion for the loss of transepithelial electrical resistance, for increased monolayer permeability, and for redistribution of the tight junction-associated protein occludin. Furthermore, the analysis of EPEC strains expressing EspF-adenylate cyclase fusion proteins indicates that EspF is translocated via the type III secretion system to the cytoplasm of host cells, a result confirmed by immunofluorescence microscopy. These studies suggest a novel role for EspF as an effector protein that disrupts intestinal barrier function without involvement in attaching and effacing lesion formation.

Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions

Enteropathogenic Escherichia coli (EPEC) increases tight junction permeability in part by phosphorylating the 20 kDa myosin light chain (MLC20) 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.

Activation of NF-κB in intestinal epithelial cells by enteropathogenic Escherichia coli

The initial response to infection is recruitment of acute inflammatory cells to the involved site. Interleukin (IL)-8 is the prototypical effector molecule for this process. Transcription of the IL-8 gene is primarily governed by the nuclear transcription factor (NF)-κB. Intestinal epithelial cells produce IL-8 in response to infection by enteric pathogens yet remain quiescent in a milieu where they are literally bathed in normal bacterial flora. We therefore sought to investigate NF-κB activation in response to enteropathogenic Escherichia coli (EPEC), nonpathogenic E. coli, and bacterial lipopolysaccharide in an intestinal epithelial cell (T84) model and to determine whether EPEC-induced activation of NF-κB factor is causally linked to IL-8 production. We report herein that NF-κB is activated by EPEC, yet such a response is not extended to nonpathogenic organisms or purified E. coli lipopolysaccharide. Transcription factor decoys significantly diminished IL-8 production in response to EPEC, demonstrating a causal relationship. Furthermore, deletion of specific EPEC virulence genes abrogates the NF-κB-activating property of this pathogen, suggesting that specific bacterial factors are crucial for inducing this response. These studies show for the first time that infection of intestinal epithelial cells with EPEC activates NF-κB, which in turn initiates IL-8 transcription, and highlight the differential response of these cells to bacterial pathogens vs. nonpathogens.

Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability

BACKGROUND & AIMS Infection of epithelial cells with enteropathogenic Escherichia coli (EPEC) induces phosphorylation of the 20-kilodalton myosin light chain (MLC20). The physiological consequence of this biochemical observation, however, has not been discerned. The aim of this study was to determine if EPEC-induced phosphorylation of MLC20 was involved in the associated perturbation of intestinal epithelial barrier function. METHODS Cultured intestinal epithelial cells, T84, were infected with EPEC. The effects of protein kinase inhibitors on EPEC-induced perturbation of barrier function were assessed using electrophysiological techniques. Alterations in MLC20 phosphorylation were correlated with functional responses. RESULTS Inhibition of myosin light chain kinase, but not protein kinase C or tyrosine kinase, prevented the decrease in resistance caused by EPEC infection and significantly diminished EPEC-induced MLC20 phosphorylation. Epithelial cell monolayers genetically manipulated to constitutively increase MLC20 phosphorylation were relatively resistant to the effects of EPEC on barrier function. CONCLUSIONS For the first time, these data show that a physiological consequence of the long-recognized increase in MLC20 phosphorylation by EPEC is perturbation of intestinal epithelial barrier function, which probably contributes to the diarrhea associated with this infection.

Clostridium difficile toxin B disrupts the barrier function of T84 monolayers

The contribution of toxin B to Clostridium difficile-associated infection is undefined. Toxin B induces dramatic phenotypic alterations (cytotoxic effects) in cultured mesenchymal and nonintestinal epithelial cells, yet its effects on intestinal epithelial cells are not clearly understood. The alterations induced by toxin B in nonintestinal cells appear to be secondary to toxin-induced redistribution of filamentous actin. It has not been determined whether toxin B exerts similar effects on cultured intestinal epithelial cells or whether such phenotypic alterations are of any physiological consequence. To address these questions, we examined the effect of C. difficile toxin B on the phenotype and barrier function of T84 cell monolayers. Our studies show that the cytotoxic effects of toxin B, i.e., cell rounding, do extend to cultured intestinal epithelial cells (T84). In addition, toxin B dramatically reduces the barrier function of T84 monolayers grown on collagen-coated filters. Toxin B-induced redistribution of filamentous actin appears to be responsible for the alterations in both intestinal epithelial cell phenotype and barrier function. Specifically, filamentous actin comprising the perijunctional actomyosin ring, known to be important in regulating tight junction permeability, is condensed into discrete plaques. Flux studies confirm that the permeability defect is at the level of the tight junction. We conclude that toxin-induced changes in actin distribution perturb intercellular junctional contacts and thereby ablate epithelial barrier function. There was no evidence of cell death as determined by lactate dehydrogenase release assays.

Enteropathogenic E. coli disrupts tight junction barrier function and structure in vivo.

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 ∼2 × 108 EPEC organisms or PBS. At 1 or 5 days postinfection, mice were killed and ileal and colonic tissue was mounted in Üssing 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 (ΔespF) had no effect on barrier function at 1 day postinfection. Furthermore, ΔespF 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 ΔespF 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-α 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.

Comparative Analysis of EspF from Enteropathogenic and Enterohemorrhagic Escherichia coli in Alteration of Epithelial Barrier Function

ABSTRACT Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related intestinal pathogens that harbor highly similar pathogenicity islands known as the locus of enterocyte effacement (LEE). Despite their genetic similarity, these two pathogens disrupt epithelial tight junction barrier function with distinct kinetics. EHEC-induced reduction in transepithelial electrical resistance (TER), a measure of barrier function disruption, is significantly slower and more modest in comparison to that induced by EPEC. The variation in bacterial adherence only partially accounted for these differences. The LEE-encoded effector protein EspF has been shown to be critical for EPEC-induced alterations in TER. EspF from both EPEC and EHEC is expressed and secreted upon growth in tissue culture medium. The mutation of EHEC cesF suggested that the optimal expression and secretion of EHEC EspF required its chaperone CesF, as has been shown for EPEC. In contrast to EPEC espF and cesF, mutation of the corresponding EHEC homologs did not dramatically alter the decrease in TER. These differences could possibly be explained by the presence of additional espF-like sequences (designated U- and M-espF, where the letter designations refer to the specific cryptic prophage sequences on the EHEC chromosome closest to the respective genes) in EHEC. Reverse transcription-PCR analyses revealed coordinate regulation of EHEC U-espF and the LEE-encoded espF, with enhanced expression in bacteria grown in Dulbecco-Vogt modified Eagle’s medium compared to bacteria grown in Luria broth. Both EHEC espF and U-espF complemented an EPEC espF deletion strain for barrier function alteration. The overexpression of U-espF, but not espF, in wild-type EHEC potentiated the TER response. These studies reveal further similarities and differences in the pathogenesis of EPEC and EHEC.

Disruption of Cell Polarity by Enteropathogenic Escherichia coli Enables Basolateral Membrane Proteins To Migrate Apically and To Potentiate Physiological Consequences

ABSTRACT Enteropathogenic Escherichia coli (EPEC) disrupts the structure and barrier function of host intestinal epithelial tight junctions (TJs). The impact of EPEC on TJ “fence function,” i.e., maintenance of cell polarity, has not been investigated. In polarized cells, proteins such as β1-integrin and Na+/K+ ATPase are restricted to basolateral (BL) membranes. The outer membrane EPEC protein intimin possesses binding sites for the EPEC translocated intimin receptor (Tir) and β1-integrin. Restriction ofβ 1-integrin to BL domains, however, precludes opportunity for interaction. We hypothesize that EPEC perturbs TJ fence function and frees BL proteins such as β1-integrin to migrate to apical (AP) membranes of host cells, thus allowing interactions with bacterial adhesins such as intimin. The aim of this study was to determine whether EPEC alters the polar distribution of BL proteins, in particular β1-integrin, and if such redistribution contributes to pathogenesis. Human intestinal epithelial T84 cells and EPEC strain E2348/69 were used. Selective biotinylation of AP or BL membrane proteins and confocal microscopy showed the presence of β1-integrin and Na+/K+ ATPase on the AP membrane following infection. β1-Integrin antibody afforded no protection against the initial EPEC-induced decrease in transepithelial electrical resistance (TER) but halted the progressive decrease at later time points. While the effects of EPEC on TJ barrier and fence function were Tir dependent, disruption of cell polarity by calcium chelation allowed a tir mutant to be nearly as effective as wild-type EPEC. In contrast, deletion of espD, which renders the type III secretory system ineffective, had no effect on TER even after calcium chelation, suggesting that the putativeβ 1-integrin-intimin interaction serves to provide intimate contact, like that of Tir and intimin, making translocation of effector molecules more efficient. We conclude that the initial alterations of TJ barrier and fence function by EPEC are Tir dependent but that later disruption of cell polarity and accessibility of EPEC to BL membrane proteins, such asβ 1-integrin, potentiates the physiological perturbations.

The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that adheres to epithelial cells of the small intestine and uses a type III secretion system to inject effector proteins into host cells. EPEC infection leads to disruption of host intestinal tight junctions that are important for maintaining intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Here we show that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. Using the drug Brefeldin A, we demonstrate that the effect of NleA on tight junction integrity is related to its inhibition of host cell protein trafficking through COPII‐dependent pathways. These results suggest that NleAs striking effect on virulence is mediated, at least in part, via its role in disruption of intestinal barrier function.

Enteropathogenic Escherichia coli Activates Ezrin, Which Participates in Disruption of Tight Junction Barrier Function

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is an important human intestinal pathogen, especially in infants. EPEC adherence to intestinal epithelial cells induces the accumulation of a number of cytoskeletal proteins beneath the bacteria, including the membrane-cytoskeleton linker ezrin. Evidence suggests that ezrin can participate in signal transduction. The aim of this study was to determine whether ezrin is activated following EPEC infection and if it is involved in the cross talk with host intestinal epithelial cells. We show here that following EPEC attachment to intestinal epithelial cells there was significant phosphorylation of ezrin, first on threonine and later on tyrosine residues. A significant increase in cytoskeleton-associated ezrin occurred following phosphorylation, suggesting activation of this molecule. Nonpathogenic E. coli and EPEC strains harboring mutations in type III secretion failed to elicit this response. Expression of dominant-negative ezrin significantly decreased the EPEC-elicited association of ezrin with the cytoskeleton and attenuated the disruption of intestinal epithelial tight junctions. These results suggest that ezrin is involved in transducing EPEC-initiated signals that ultimately affect host physiological functions.