THEME
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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ABSTRACT |
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
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INTRODUCTION |
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.
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THE INITIAL APPROACH |
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.
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.
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THE THRILL OF THE MECHANISTIC PURSUIT |
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.
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-
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,
-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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SUMMARY |
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.
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ACKNOWLEDGEMENTS |
I gratefully acknowledge the editorial input and helpful
discussions with Drs. David Acheson, Mark Donowitz, and Gail Hecht.
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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).
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