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Molecular Physiology and Pathophysiology of Tight Junctions V. Assault of the tight junction by enteric pathogens

Cynthia L. Sears

Divisions of Infectious Diseases and Gastroenterology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196


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Studies of the impact of enteric pathogens and their virulence factors on the proteins comprising the tight junction and zonula adherens offer a novel approach to dissection of tight junctional complex regulation. Most studies to date provide only tantalizing clues that select pathogens may indeed assault the tight junctional complex. Information on critical human pathogens such as Campylobacter jejuni and Shigella and Salmonella subspecies is lacking. Mechanistic studies are currently sparse, but available results on pathogenic Escherichia coli and specific virulence factors such as the Rho-modifying and protease bacterial toxins indicate four major mechanisms by which these pathogens may act: 1) direct cleavage of tight junctional structural proteins; 2) modification of the actin cytoskeleton; 3) activation of cellular signal transduction; and 4) triggering transmigration of polymorphonuclear cells across the epithelial cell barrier. New therapeutics may evolve from detailed studies of these pathogens and the cellular processes and proteins they disrupt.

zonula adherens; zonula occludens; intestinal epithelial cell; bacterial toxins; cytoskeleton


    INTRODUCTION
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IN RECENT YEARS, ENTERIC PATHOGENS and their virulence factors have been celebrated for their novel interactions with eukaryotic cells, including polarized epithelial cells, and for their utility in unraveling the regulation of cell structure and function (21). This themes article will focus on evaluating results in which enteric pathogens and/or their virulence factors appear to target the tight junctions (TJs) of epithelial cells. Although the formal TJ is the zonula occludens, this discussion will also consider the impact of these pathogens on the zonula adherens, whose major structural protein is E-cadherin. Nearly all of the relevant experiments have been conducted in vitro using polarized epithelial cells (usually of intestinal origin). Such systems offer the ability to more specifically and conclusively evaluate the action of the pathogen and/or its virulence factor(s) on the TJ complex.


    THE INITIAL APPROACH
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On the basis of the pioneering work of Dharmsathaphorn, Madara, and colleagues in the 1980s, investigators interested in the pathogenesis of enteric infections gradually appreciated both the power of the Ussing chamber to evaluate the electrophysiological response to enteric pathogens and/or their virulence factors and the ability to focus more exactly on epithelial cell effects with the use of polarized monolayers of intestinal epithelial cells. Thus a common (and laudable) initial approach to investigating the impact of an enteric pathogen and/or its virulence factor on intestinal epithelial cell function has been to infect and/or treat monolayers with the enteric pathogen and/or virulence factor of interest. In certain instances, this has yielded results that suggest that the enteric pathogen and/or its virulence factor may directly or indirectly alter the TJ complex structure and/or function (Tables 1 and 2). The result implicating a potential TJ effect has most often been the documentation of a reduction in transepithelial resistance (Rt) in the absence of an increase in a chloride current. The effect of an enteric pathogen or its virulence factor(s) on the TJ is most easily observed in polarized intestinal epithelial cell monolayers in vitro (e.g., T84, Caco-2, HT-29), in which the contribution of any potential feedback loop to intestinal epithelial cell function from lamina propria cells (e.g., immune cells, fibroblasts producing mediators affecting epithelial cell function) and/or the enteric nervous system can be eliminated from consideration.

                              
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Table 1.   Enteric pathogens that modify epithelial barrier function


                              
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Table 2.   Enteric pathogen virulence factors that alter TJ proteins and/or modify epithelial barrier function

A surprisingly short list of enteric pathogens and their virulence factors (Tables 1 and 2) has been reported to impact on Rt or paracellular permeability, most often with an alteration in the arrangement of TJ component proteins. It is of interest that, despite the linkage of the TJ and zonula adherens by actin strands, disruption of TJ proteins occurs not infrequently in the available examples without an apparent structural zonula adherens change [the Bacteroides fragilis toxin (BFT) is one exception (2)]. This suggests perhaps a remarkably specific signaling to the TJ triggered by quite diverse bacteria [e.g., diffusely adherent Escherichia coli (17), enterohemorrhagic E. coli (EHEC) (9), and perhaps Salmonella typhimurium] and even rotavirus (15). Importantly, clear correlations between a change in Rt and structural changes in the TJ are not apparent from these data. Structural TJ changes are not always accompanied by a change in Rt (e.g., diffusely adherent E. coli and rotavirus, although paracellular permeability is increased by both). In contrast, rapid and/or profound decreases in Rt stimulated by a pathogen or a virulence factor may not have an apparent TJ structural correlate [e.g., enteropathogenic E. coli (EPEC) (21), Helicobacter pylori vacuolating toxin A (16), Clostridium difficile toxins A and B (21)]. However, these latter observations are limited by the number of proteins evaluated and the approaches used. For example, transmission electron microscopy alone has been used to evaluate TJ structure in the studies of EPEC and C. difficile toxins A and B, which is too insensitive to detect redistribution of individual proteins such as occludin and ZO-1.

Unfortunately, we have minimal to no information on the TJ effects of common human enteric pathogens such as Shigella flexneri, Campylobacter jejuni and even Salmonella subspecies. In the case of S. flexneri, short-term infection sufficient to stimulate a transepithelial neutrophil response is insufficient to alter Rt (11). Rather surprisingly, the impact of the actin polymerizing activity (necessary for the motoring of the bacteria within the intestinal epithelial cells and to facilitate spread to adjacent cells) of S. flexneri and Listeria monocytogenes (for which the gut is the critical portal of entry) on TJ function has not been studied. Of the two Salmonella species (typhimurium, enteriditis) epidemiologically prominent in human diarrheal disease, only S. typhimurium has been studied in any depth (with the greatest focus on its ability to stimulate polymorphonuclear transmigration), and studies using polarized epithelial cells vary in their results. S. typhimurium induces a rapid decrease in Rt accompanied by rearrangement of TJ proteins in Madin-Darby canine kidney cell (MDCK) II monolayers (7), whereas even heavily infected T84 monolayers (i.e., loaded with 1,500 bacteria/cell) exhibit a delayed decrease in Rt, indicating that the membrane ruffling triggered by contact of S. typhimurium with intestinal epithelial cells is insufficient to modify TJ function (10). These disparate results may represent the impact of different experimental conditions and/or differences in the bacterial strains or cell lines studied. In other cases, incomplete analyses have left a void in our understanding. For example, enteroaggregative E. coli have been shown to dramatically alter the apical structure of T84 monolayers, but electrophysiological measurements are lacking.


    THE THRILL OF THE MECHANISTIC PURSUIT
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Despite the rapid growth of this field of investigation, little attention to date has been directed to detailed studies of mechanisms by which TJ function or structure is altered by infection of epithelial cells with enteric pathogens and/or treatment with purified virulence factors. Table 3 lists potential mechanisms by which enteric pathogens may modify TJ function or structure. Examples exist to validate most of the proposed mechanisms. The most notable indirect mechanism now repeatedly shown to modify TJ function is the biochemical modification of the actin binding protein Rho [i.e., C. difficile toxins A and B (21) and E. coli cytotoxic necrotizing factor 1 (CNF1) (5)]. Intriguingly, despite the fact that monoglucosylation of Rho (C. difficile toxins A and B) depolymerizes F-actin and deamidation of Rho (E. coli CNF1) increases stress fiber formation, all three toxins decrease Rt (5, 21) (Table 2). Additional studies indicate that Rho modulates the apical actin cytoskeleton of polarized intestinal epithelial cells to modify TJ function (13), but, notably, the effect of E. coli CNF1 on precisely this pool of actin has not been examined. Furthermore, although C. difficile toxins A and B modify Rho via an identical mechanism, their effects on Rt differ dramatically (Table 2), illustrating that the pathway from Rho modification to decreased Rt is not direct.

                              
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Table 3.   Potential mechanisms used by enteric pathogens to modify TJ function

Of the organisms demonstrated to decrease Rt (Table 1), only the studies of EPEC have provided substantive mechanistic details to date. These studies provide an excellent example to illustrate the complexity of the analysis required. Three major mechanisms by which EPEC may stimulate a decrease in Rt have been identified: 1) an increase in intracellular calcium; 2) myosin light chain (MLC) phosphorylation (via MLC kinase); and 3) dephosphorylation of occludin (21, 22). Most recently, the EPEC-induced decrease in Rt has been linked to expression by the bacteria of EPEC-secreted protein F (personal communication, G. Hecht). Of the mechanisms implicated in EPEC-induced decrease in Rt, the increased calcium acts, at least in part, by activation of MLC kinase (personal communication, G. Hecht). MLC phosphorylation, in turn, triggers contraction of the perijunctional actomyosin ring beneath the TJ, presumably increasing the tension on the TJ to reduce Rt. A serine/threonine phosphatase (calcium dependence not yet known) may mediate the dephosphorylation of occludin that leads to its dissociation from the TJ, consistent with prior observations. Nonetheless, a clear view of the cellular cascade incited by EPEC infection of the apical intestinal epithelial cell membrane and ending in decreased Rt is not yet available. Of note, blockade of each of these Rt-modifying mechanisms independently (using drug inhibitors) abrogates the EPEC-induced decrease in Rt, suggesting interdependence of these mechanisms of TJ regulation. Neither protein kinase C, tyrosine kinases, nor mitogen-activated protein kinases appear to effect the EPEC-induced decrease in Rt despite their involvement in the normal physiological regulation of TJs. In contrast, EPEC induction of polymorphonuclear transmigration via nuclear factor-kappa B is dependent on mitogen-activated protein kinase and independent of calcium, demonstrating that an organism can trigger signal transduction pathways with independent, but complementary, functional consequences (personal communication, G. Hecht; Ref. 20). Namely, EPEC induction of polymorphonuclear transmigration will also, at least transiently, decrease Rt (Table 3). In contrast, mechanistic studies are far more rudimentary on the genetically related and extremely virulent food-borne pathogen EHEC. In this case, the delayed decrease in Rt observed in EHEC-infected T84 cell monolayers appears dependent on protein kinase C and MLC kinase activation as well as calmodulin but not on the organisms' primary virulence factors, Shiga toxins 1 and 2 (6, 9, 18) (Table 1). These EHEC-dependent epithelial cell effects on TJ function are likely enhanced in vivo by the ability of the organism to stimulate polymorphonuclear transmigration. Polymorphonuclear transmigration, in turn, enhances Shiga toxin translocation across the epithelial cell barrier in vitro, which may be a critical factor in systemic toxin uptake (personal communication, Dr. D. Acheson).

Direct action of a bacterial virulence factor on component proteins of the TJ or the zonula adherens has been proposed in only a few instances (Table 2). In a convincing data set, the internalin protein of L. monocytogenes has been shown to bind to E-cadherin to promote its entry into intestinal epithelial cells (12). A single amino acid (proline 16) of E-cadherin (a residue not involved in cell-cell adhesion) is essential to the interaction of this organism with human, but not mouse, E-cadherin. Nonetheless, there are no data to indicate whether this binding and internalization process alters either Rt or the rich array of signal transduction processes associated with the zonula adherens. Although E-cadherin has been proposed as a requirement for the uptake of S. flexneri, the original observations in transfected fibroblast cell lines have not been extended to polarized intestinal epithelial cells (19). Of specific virulence factors, only BFT and the Clostridium perfringens enterotoxin (CPE) appear to directly cleave structural proteins of the zonula adherens (E-cadherin) and TJ (claudins), respectively (23, 24). In addition, crude culture supernatants of a Vibrio cholerae mutant strain (CVD 110) cleave occludin, an effect attributed to a hemagglutinin/protease (25). Onset of E-cadherin cleavage by BFT occurs within 1 min in unpolarized HT-29/C1 cells, and loss of the extracellular domain precedes loss of the intracellular domain, consistent with direct cleavage of E-cadherin by BFT (24). BFT treatment diminishes Rt by 10 min in polarized T84 monolayers, and by 2 h, morphologically altered or completely dissolved TJs and zonula adherens are observed in BFT-treated T84 monolayers (2). However, neither direct binding of BFT to E-cadherin nor in vitro cleavage of E-cadherin has been documented. Thus this leaves open the question of whether direct cleavage of E-cadherin is indeed the first step in the action of this toxin. In contrast, the activity of CPE is slower, with onset of cleavage of claudins 3 and 4 (but not claudins 1 and 2) by 4 h of incubation in transfected fibroblasts and MDCK I monolayers, respectively (23). This latter result correlates well with the onset of diminished Rt and loss of TJ strands identified in MDCK I monolayers. Direct binding of CPE to claudins 3 and 4 (apparently to an extracellular domain) but not 1 and 2 has been demonstrated, but the sites of cleavage of the claudins and the dependence of cleavage on cellular internalization of CPE have not been defined. Both L. monocytogenes and S. flexneri, which bind to E-cadherin, as well as BFT and CPE act most efficiently from the basolateral membrane, consistent with the site of localization of their target proteins. Of interest, proteases of both Porphyromonas gingivalis (a dental pathogen) and Pseudomonas aeruginosa (which enters by one or more mucosal sites to cause pneumonia, urinary tract infections, and/or systemic disease) also appear to degrade epithelial cell junctional complexes (1, 8). Lastly, the herpes simplex virus glycoprotein complex gE-gI colocalizes with the zonula adherens junction protein, beta -catenin, where it is hypothesized to mediate transfer of herpes simplex virus across cell junctions (3). Together, these results suggest that assault of the TJ and/or entry via the zonula adherens are mechanisms of importance to bacterial and viral pathogenesis at all mucosal sites, an important observation given that mucosal surfaces are, in fact, the leading portal of entry for most infectious pathogens of importance in human disease.


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One aspect of the assault of enteric pathogens on intestinal epithelial cells is the dysregulation and dissolution of the TJ and/or the zonula adherens. To date, the rather bland outcome of reduced Rt has not been accompanied in most cases by studies to address the mechanisms (pathogen and/or host cell) accounting for the decrease in Rt. The lack of synchrony of the TJ proteins evaluated in the available studies makes it difficult at this time to discern whether common or divergent themes of dysregulation of TJ or zonula adherens function are occurring in response to the different enteric pathogens evaluated to date. Conversely, studies of purified virulence factors need to be reconsidered in the context of the intact organism and its effect(s) on the TJ. The challenge is to determine how pathophysiology resulting from enteric infections intertwines with the normal physiological regulation of TJs. Do enteric pathogens merely disrupt the regulatory balance of normal physiology, or are they triggering novel, unrecognized pathways to alter TJ function? New observations on TJs await exploration, such as the impact of enteric pathogens or their virulence factors on the recently recognized clustering of TJ proteins in raftlike membrane microdomains (14). As shown by preliminary studies conducted with the zonula occludens toxin of V. cholerae (4), it is possible that a better understanding of how enteric pathogens and, more critically, specific virulence factors alter TJ function may lead to the development of new therapeutically helpful approaches to modulate gut function. This thought should provide the motivation for investigators to tackle the mountain of serious mechanistic work that lies ahead.


    ACKNOWLEDGEMENTS

I gratefully acknowledge the editorial input and helpful discussions with Drs. David Acheson, Mark Donowitz, and Gail Hecht.


    FOOTNOTES

Because of space limitations, important contributions of many investigators could not be appropriately referenced. Additional references are available from the author on request.

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45496.

Address for reprint requests and other correspondence: C. L. Sears, Johns Hopkins Univ. School of Medicine, 720 Rutland Ave., Ross Bldg. Rm. 1167, Baltimore, MD 21205-2196 (E-mail: csears{at}jhmi.edu).


    REFERENCES
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