Departments of 1 Medicine and 2 Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, New York 14214
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ABSTRACT |
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Enteropathogenic Escherichia coli (EPEC) causes severe, watery diarrhea in children. We investigated ATP release during EPEC-mediated killing of human cell lines and whether released adenine nucleotides function as secretory mediators. EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7, and T84 (colon cells) as measured using a luciferase kit. Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5'-ectonucleotidase was included and was further enhanced if an ATP-regenerating system was added. In the presence of the inhibitor/regenerator, ATP concentrations in the supernatant medium reached 1.5-2 µM 4 h after infection with wild-type EPEC strains. In the absence of the inhibitor/regenerator system, extracellular ATP was rapidly broken down to ADP, AMP, and adenosine. Conditioned medium from EPEC-infected cells triggered a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber (rabbit distal colon and T84 cell monolayers), whereas conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria did not. The short-circuit current response to EPEC-conditioned medium was completely reversed by adenosine receptor blockers, such as 8-(p-sulfophenyl)-theophylline and MRS1754. EPEC killing of host cells releases ATP, which is broken down to adenosine, which in turn stimulates secretion via apical adenosine A2b receptors. These findings provide new insight into how EPEC causes watery diarrhea.
extracellular nucleotides; adenosine receptors; ATP efflux; purinergic receptors; apoptosis
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INTRODUCTION |
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ENTEROPATHOGENIC Escherichia coli (EPEC) is a common cause of diarrhea in children in developing countries. Unlike other types of diarrheagenic E. coli, EPEC produces no toxins, and the way it produces watery diarrhea is unknown. EPEC kills host intestinal cells in vitro, and this cell death has mixed features of apoptosis and necrosis (2, 12, 14). Intestinal cell death also occurs in vivo in rabbits infected with rabbit EPEC (REPEC) and rabbit diarrheagenic E. coli (RDEC-1; E. Boedeker and J. Crane, unpublished data).
Many EPEC virulence factors are encoded on a pathogenicity island known as the locus of enterocyte effacement (LEE), including the type III secretion machinery and the EPEC secreted proteins. Type III secretion is a property of certain gram-negative bacteria, including Salmonella, Shigella, Yersinia, and EPEC, that allows secretion of bacterial proteins into the host cell cytosol or plasma membrane (26). One LEE-encoded protein, EPEC-secreted protein F (EspF), is necessary for host cell killing (11). EspF is one of a growing list of effector proteins secreted into the host cell during the process of infection, in EspF's case, into the cytosol (33). EspF is also required for the EPEC-mediated drop in transepithelial electrical resistance (TER) seen in response to EPEC infection (34).
While investigating the potential role of ectoprotein kinases in the pathophysiology of EPEC infection, we examined whether EPEC would trigger a release of ATP from the host cell. In this study, we report the rate and extent of ATP release from the host cell in response to EPEC infection, demonstrate the rapid breakdown of released ATP to less phosphorylated adenine nucleotides and adenosine, and show that these extracellular nucleotides and nucleosides trigger a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber. EPEC-infected host cells release nucleotide mediators capable of triggering a secretory response from neighboring, healthy cells. These findings confirm and extend the importance of host cell killing by EPEC and suggest a mechanism for the watery diarrhea produced by this pathogen.
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MATERIALS AND METHODS |
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Materials.
The following reagents were obtained from Sigma-Aldrich:
,
-methylene-ADP, creatine kinase, phosphocreatine, forskolin,
adenosine, AMP, ADP, Tris · acetate, tetrabutylammonium
dihydrogen phosphate, and type III collagen.
8-(p-sulfophenyl)-theophylline was from Research
Biochemicals International (Natick, MA).
Bacterial culture, tissue culture, and animal tissue.
Bacterial strains used in this study are described in Table
1. Bacteria were grown overnight
in LB broth supplemented with 1% mannose at 37°C with 300 rpm
shaking. The next morning, the strains were subcultured 1:8 into EPEC
adherence medium consisting of DMEM/F-12 (GIBCO-BRL, Grand Island, NY)
supplemented with 40 mM HEPES, pH 7.4, 18 mM NaHCO3, 2%
heat-inactivated newborn calf serum, and 1% mannose for 2 h with
the same temperature and shaking conditions. EPEC adherence medium was
warmed overnight at 37°C. T84 cells were infected at a multiplicity
of infection of 100:1 and HeLa cells at 200:1 unless otherwise stated.
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Preparation of colon tissue for Ussing chamber experiments. Male New Zealand White rabbits were killed by exposure to 100% CO2. The descending colon was removed, opened into a flat sheet, and washed in standard tissue bathing solution (see below). The epithelium was stripped from its underlying musculature with a glass microscope slide.
ATP assay.
ATP concentrations in conditioned medium from infected and uninfected
cells were measured using an ATP bioluminescence assay kit (model HS
II; Roche Molecular Biochemicals, Indianapolis, IN). Cells in 24-well
plates were infected for 1 h, then the medium was changed to
serum-free adherence medium to remove most of the unbound bacteria. At
various times after the medium change, the plates were swirled to mix,
and 100-µl aliquots were removed from wells and placed into the upper
chamber of Spin-X tubes (Corning Costar, Corning, NY). Spin-X tubes are
microcentrifuge tubes containing a 0.45-µm filter insert and were
used to remove bacteria and any detached human cells. Before adding the
experimental sample, the lower portion of the Spin-X tube was loaded
with 100-µl of preservative/lysis buffer supplied with the ATP assay
kit. As soon as possible after the collection of the experimental
sample, the Spin-X tubes were centrifuged for 1 min at 14,000 g in an Eppendorf centrifuge, then the filter insert was
removed and discarded. ATP samples so collected were then stored frozen
at 70°C until the day of the ATP assay, when they were diluted an
additional fivefold in 100 mM Tris · acetate-2 mM EDTA.
One hundred microliters of sample was mixed together with 100 µl of
luciferase reagent and immediately read on a Bio-Orbit 1250 luminometer
(LKB-Wallac, Turku, Finland). ATP standards used to prepare the
standard curve were 0, 0.01, 0.03, 0.05, and 0.1 µM. To exclude the
possibility of an artifactual inhibition of ATP measurement by
antibiotics, as in Fig. 4, the luminescence reading observed from 100 nM ATP was compared in the presence and absence of antibiotics. The
addition of 75 µg/ml ciprofloxacin and 80 µg/ml polymixin B (final
concentrations 50 times higher than achieved in Fig. 4 after dilution)
gave readings 94 and 95% of the no-antibiotic standard, respectively.
Induction of apoptosis by UV irradiation, and LDH release assay. Ultraviolet (UV) light was used to induce apoptosis in HeLa cells using 2 min of irradiation on a UV transilluminator box as previously described (11). Lactate dehydrogenase (LDH) release was measured as described previously (12).
Transepithelial electrophysiological measurements.
Monolayers of T84 cells were inserted into a modified Ussing chamber
(model DCH; NaviCyte, San Diego, CA) at 37°C and continuously short
circuited by a four-electrode, automatic voltage clamp apparatus (model
616C; Department of Bioengineering, University of Iowa, Iowa City, IA),
which measured short-circuit current (Isc) and TER; chamber fluid resistance was automatically subtracted.
Isc was measured by passing sufficient current
through the tissues via Ag/AgCl electrodes to reduce the spontaneous
transepithelial potential to zero. Transepithelial resistance was
determined by passing 2-s, 20-mV current pulses through the tissues.
The composition of the tissue bathing solution was as follows (in mM):
140 Na+, 124 Cl, 21 HCO
HPLC analysis. Nucleotides were separated by HPLC on a 25-cm Supelco C18 reversed-phase column. The aqueous phase was 0.1 M KH2PO4, pH 7.5, with 4 mM tetrabutylammonium dihydrogen phosphate, an ion-pairing reagent. After injection of a 0.5-ml sample, a linear gradient of methanol from 0 to 27% was developed over 17 min, followed by a shallower gradient of 27 to 33% methanol from 17 to 24 min, followed by a 80% methanol wash and returned to initial conditions. In this system, the order of elution and the retention times were: AMP, 10.1 min; ADP, 15.3 min; adenosine, 15.8 min; and ATP, 18.6 min. Detection was by UV absorbance at 254 nm, and data were collected using Rainin Dynamax HPLC software that calculates the size of each peak (in µV/s and as a percentage of the total).
To analyze breakdown of ATP, exogenous ATP was added to a final concentration of 0.2 mM to a 75 cm2 flask of uninfected T84 cells in 20 ml of serum-free medium and 3-ml aliquots were withdrawn at 15-min intervals. The aliquots were frozen atData analysis and presentation. ATP values in unknown samples were calculated from luminescence data from the ATP standard curve using the curve-fitting function of GraphPad Prism software for the Macintosh (GraphPad software, San Diego, CA). Graphs shown were generated with the same computer program. ATP assays shown were performed in triplicate, and each experiment shown was performed at least three times. Ussing chamber traces are representative of a least three experiments. Statistical testing was by ANOVA. Error bars shown in graphs, figures, or the text are SD.
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RESULTS |
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Initial experiments to detect ATP release during EPEC infection
were performed in ordinary, serum-free medium without any special
additives, in HeLa and T84 cells (Fig.
1A and 1C). ATP release in these conditions was readily detected in HeLa cells (Fig.
1A), but not in T84 cells (Fig. 1C), which was
surprising, because the extent of EPEC-induced cell death is about the
same in these two cell lines (12). Because the literature
indicated that T84 cells, like other intestinal cells, have abundant
amounts of several ectonucleotidases (46, 51), we felt
that the failure to detect ATP in EPEC-infected T84 cell supernatants
might be due to its rapid destruction, rather than due to actual lack
of release. The addition of an inhibitor of 5'-ectonucleotidase, ,
-methylene-ADP, by itself did not increase the amount of
detectable ATP a great deal, nor did addition by itself of an
ATP-regenerating system consisting of creatine kinase and
phosphocreatine (Table 2). The combination of
,
-methylene-ADP and
the ATP regenerating system, however, resulted in significantly higher
ATP concentrations in the supernatant medium of EPEC-infected cells
(Table 2 and Fig. 1, B and
D; note the difference in scales on the ordinate). Thus when
ATP was "trapped" by the inhibitor/regenerator system the ATP
accumulated to concentrations exceeding 1.5 µM in both cell lines.
ATP release was markedly less in response to infection with the
cell-death deficient espF mutant (strain UMD874), the plasmid-cured strain JPN15, or nonpathogenic E. coli strain
H.S. ATP concentrations in the medium above uninfected cells remained neglible even in the presence of the inhibitor/regenerator system. Other wild-type EPEC strains tested, including E851/71, B171-8, and JCP88 triggered a release of ATP similar in magnitude to that of
strain E2348/69 (Table 2).
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Performance of the espF mutant UMD874 relative to wild-type strains depended on the host cell line used. In HeLa cells, ATP release by strain UMD874 was 32.8 ± 13% of that released by the wild-type strains (mean of 3 separate experiments each done in triplicate; see also Fig. 1B), whereas in T84 cells, ATP release by UMD874 was 68.9 ± 10% of the wild-type strain (mean of 5 separate experiments; P = 0.005 compared with HeLa; see also Fig. 1D). In COS-7 cells, the performance of UMD874 relative to wild-type strains was similar to that in HeLa cells (data not shown).
To determine whether ATP breakdown was indeed rapid enough in T84 cells
to account for the lack of detectable ATP accumulation seen in Fig.
1C, ATP levels were measured after treatment with exogenous
ATP (Fig. 2). Because EPEC and other
diarrheagenic E. coli infections can alter intestinal cell
surface enzymes, the fate of ATP was compared in uninfected cells,
cells infected for 1 h with EPEC, and for 1 h with
enteroaggregative E. coli strain 042. Figure 2A
shows that ATP was rapidly destroyed by T84 cells and the rate of
disappearance was the same in infected as in uninfected monolayers.
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Figure 2B shows that the addition of the inhibitor/regenerator system prevented the destruction of exogenous ATP over the short term. Other experiments (not shown) demonstrated that the regenerator system was eventually exhausted 4-6 h after infection but could be replenished by readdition of the creatine kinase and phosphocreatine.
Although the luminescence method used in Fig. 2, A and B documented the rapid breakdown of ATP in T84 cells, that method could not determine the products of ATP hydrolysis. To accomplish this, HPLC analysis was used (Fig. 2C). In these experiments, a higher concentration of exogenous ATP was added (0.2 mM) to unequivocally identify products generated. The HPLC tracings confirmed the rapid breakdown of ATP and showed, in agreement with the work of others (16), that first ADP, and then AMP and adenosine appear in the cell supernatants as a result of ATP breakdown. The loss of ATP from the culture medium (Fig. 2, B and C) was well fit by a single-component exponential decay curve with an ATP half-life of 22-27 min. To extend the analysis further, exogenous AMP was also added to T84 monolayers, and its fate was determined by HPLC. Figure 2D shows that AMP is rapidly broken down to adenosine; at later times, another peak presumptively identified as inosine monophosphate is also detected.
Figure 3A shows that, in
contrast with EPEC infection, ATP was not released from HeLa cells by a
purely apoptotic stimulus, i.e., irradiation with UV light.
Similarly the cytosolic enzyme LDH was not released in response to UV
irradiation (Fig. 3B), although this UV treatment did
trigger widespread apoptosis by morphologic criteria (Fig.
3C). These results are in accord with a large body of data
showing that cytoplasmic contents are not released in apoptosis
(7). Therefore, the release of ATP by EPEC illustrates a
nonapoptotic feature of EPEC-mediated killing.
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We considered the question of the origin of the ATP released during
EPEC infection and whether the bacteria themselves could release ATP.
The first and most persuasive evidence on this question is
quantitative. EPEC, like all bacteria, contains ATP, and this ATP
content is readily measurable. The amount of ATP detectable in T84 or
HeLa supernatants, as shown in Fig. 1, is far greater than can be
accounted for by the bacterial ATP content. For example, in our typical
assay well of 0.5 ml, if 100% of the bacterial inoculum adhered (which
does not occur) and if 100% of the bacteria spontaneously lysed
[which does not occur (12)], the amount of ATP released
from the bacteria would only yield a concentration of ~70 nM. Thus
the amount of ATP actually released is >20 times that which could
possibly be contributed by the bacteria, even in the most unrealistic,
worst-case scenario. The addition of antibiotics after infection also
indicates that the source of the released ATP is of host cell origin
(Fig. 4). Antibiotics with diverse
mechanisms of action all inhibit the ATP release observed in EPEC
infection. This inhibition is observed even with polymixin B, which
triggers bacterial lysis by binding to the bacterial outer membrane.
Other antibiotics not shown in Fig. 4, including ampicillin and
tetracycline, also inhibited ATP release by 50-75%. This is
consistent with our previous report that EPEC-induced cell death
required prolonged contact of live bacteria with the host cell
(12). If, in contrast, the ATP were coming from the bacteria, the antibiotic treatment should increase the amount liberated
via bacterial cell lysis. Also, there are the results with the
espF mutant, deficient in host cell killing. The
espF mutant UMD874 adheres like the wild-type
strain, but ATP release is markedly less (Fig. 1, A,
B, and D). Again, these results are only
consistent with ATP release from the host cell. Finally, we have
measured the ATP content of the monolayers after infection with
wild-type EPEC. In T84 cells, the ATP content of the monolayer decreased from 3.7 ± 0.1 nmol/well in control cells to 3.0 ± 0.3 nmol/well 4 h after infection. The decline in the monolayer
ATP accounted for the concentration of ATP measured in the supernatant medium for wells containing 0.5 ml of medium.
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In experiments with cells grown in 6.5 mm Transwell inserts, EPEC infection triggered a release of ATP only into the upper, apical compartment. No ATP was detected in the basolateral chamber in polarized cells, even when the inhibitor/regenerator was added on both sides (ATP in the lower chamber 1.2 ± 0.8 vs. 310 ± 54 nM in the upper chamber after EPEC infection). This indicated that ATP release was directed to the same (apical) side of the cell exposed to the bacteria.
The experiment shown in Fig.
5A compared the ATP-releasing
ability of EPEC with that of other known enteric pathogens and the
nonadherent laboratory strain HB101. Of the strains tested, only three
triggered a release of ATP greater than that of HB101: EPEC E2348/69,
E. coli O157:H7, and Salmonella enterica. The
failure of Shigella sonnei to trigger any detectable ATP
release is well explained by the inability of Shigella
species to kill epithelial cells, as opposed to macrophages and
lymphocytes, which Shigellae kill quickly (35).
More surprising was the lack of detectable ATP release by
enteroaggregative E. coli strain 042. Enteroaggregative E. coli are known to damage host cell enterocytes in vitro
and in vivo (38, 40, 41), but the kind of damage inflicted
by strain 042 must differ qualitatively in such a way as to not release ATP. The low amount of ATP released by E. coli O157:H7 is
consistent with the much lower adherence observed with this pathogen,
which lacks the bundle-forming pilus. The ATP release seen with
E. coli O157:H7 reflects a poorly studied, contact-mediated
damage, not the effects of the Shiga-like toxins (SLTs) in the T84
cells (2). T84 cells lack the Gb3 glycolipid
receptor for SLT-I and SLT-II, and furthermore we showed above that a
purely apoptotic stimulus does not trigger ATP release (Fig. 3).
Finally, it is worth noting that ATP release by EPEC was 66% of that
released by S. enterica. This illustrates a recurring theme
in EPEC-mediated host cell killing: that although EPEC is relatively
slow and inefficient in assays of cell death that measure the final
completion of cell death (such as nuclear fragmentation and DNA
cleavage), EPEC performs quite well in assays that reflect earlier,
membrane changes in the host, such as externalization of
phosphatidylserine and phosphatidylethanolamine, membrane permeability
to dyes, and ATP release.
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Figure 5B illustrates the ATP-releasing abilities of several mutants derived from EPEC strain E2348/69. The reduced ATP release of the plasmid-cured derivative, JPN15, and the eae (intimin-deletion) mutant, CVD206, are consistent with their previously described defects in host cell killing and adherence (11, 12). Strains with mutations in the LEE-encoded regulator (ler) and sepZ (a part of the type III secretion machinery) were also markedly reduced in ability to trigger ATP release from the host cell. Results with these mutants demonstrate the importance of genes encoded in the EPEC LEE in the EPEC-mediated ATP release just as they are in EPEC-induced cell death and other types of EPEC-mediated damage (34). The espF mutant is particularly instructive, because this mutant is equivalent to wild-type EPEC in total adherence, intimate adherence, actin condensation, tyrosine phosphorylation, and secretion of the other EPEC-secreted proteins yet is deficient in its ability to induce host cell death (11).
ATP release from EPEC-infected cells could occur by the formation of
large pores in the host plasma membrane, as occurs with other pathogens
possessing the type III secretion system (5, 24), or by
some other mechanism. Several laboratories have reported that the
cystic fibrosis transmembrane regulator (CFTR) may allow efflux of ATP
when it is activated by cell swelling or by cAMP (25, 45,
54). Other members of the ATP-binding cassette family, such as
the P-glycoprotein, which mediates the miltidrug resistance phenotype,
also may allow ATP efflux, albeit in a cAMP-insensitive manner
(1). To determine whether cAMP elevation affected
EPEC-induced ATP release, we compared the effects of forskolin, a
direct stimulator of adenylyl cyclase, on ATP release in cells with and
without CFTR. Figure 6 shows that
although forskolin alone did not trigger detectable ATP release in
either cell line, in T84 cells, which express CFTR, forskolin
dramatically increased the amount of ATP effluxed in response to EPEC
infection (Fig. 6A). In HeLa cells, which do not express
CFTR, forskolin had no effect on EPEC-induced ATP release (Fig.
6B). As with forskolin, cholera toxin treatment of T84 cells
increased ATP efflux in response to EPEC infection (Fig.
6C). These findings show that, in some cell lines, cAMP elevations significantly potentiate EPEC-induced ATP release. The
enhanced ATP release is not due to increased cytotoxicity, i.e.,
forskolin did not increase EPEC-induced cell death as measured by the
propidium iodide uptake method (data not shown). The CFTR is one
plausible target of cAMP that could be involved in the increased ATP
release. If EPEC is shown to interact with the CFTR, it would join
Salmonella as a bacterial pathogen that exploits the
activity of the CFTR during intestinal infection (43).
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Results showed that EPEC infection triggered ATP release from the host cell (Figs. 1 and 4-6). Over the past decade, investigators from many laboratories have shown that extracellular nucleotides may trigger anion secretion in respiratory and gastrointestinal tissues and cell lines (3, 31, 46, 53). Therefore, we sought to determine whether the ATP released in response to EPEC infection was sufficient to cause chloride secretion in intestinal tissues, because such a response could have relevance to the secretory diarrhea seen in EPEC infection. We studied intestinal tissues in the Ussing chamber using the voltage-clamp technique to measure Isc. Initial Ussing chamber experiments were carried out using stripped distal colon from the normal rabbit. Rabbit colon was chosen because agonist-stimulated Isc in this tissue is predominantly due to chloride secretion and because it has been studied in the Ussing chamber with regard to adenosine-induced secretion (21). In these experiments, 5 ml of conditioned medium from EPEC-infected T84 cells collected in the absence and presence of the inhibitor/regenerator triggered Isc responses of 13.8 ± 5.4 and 29.5 ± 3.5 µA/cm2, respectively, when added to the mucosal side of the tissue (data not shown, results from 7-8 colon tissues from 4 separate rabbits). Conditioned medium from uninfected HeLa and T84 cells did not trigger Isc (<1.5 µA/cm2), nor did sterile filtrates of EPEC bacteria (n = 2 separate experiments).
On the basis of the results of the Ussing chamber experiments with rabbit colon, we extended those observations with similar experiments using T84 cell monolayers grown on Snap-Well inserts. This model was chosen because it has been well characterized and offers the potential of less tissue-to-tissue variability than with native colon, and the Isc in T84 cells reflects chloride secretion and not cation absorption. (15, 30). Using T84 cell monolayers as the test tissue in the Ussing chamber, we again tested conditioned media from EPEC-infected HeLa and EPEC-infected T84 cells for their ability to stimulate chloride secretion.
Figure 7 shows the results obtained with
conditioned medium from HeLa cells. Figure 7A shows that
conditioned medium from uninfected HeLa cells failed to trigger an
Isc response, whether collected in the absence
(Fig. 7A, 1) or presence of the
inhibitor/regenerator (Fig. 7A, 2). Similarly, a
filtrate of EPEC bacteria did not trigger chloride secretion (Fig.
7B, 1). In contrast, conditioned medium from
EPEC-infected HeLa cells, without any inhibitor/regenerator, triggered
a brisk and sustained Isc (Fig. 7C).
Figure 7C demonstrates the response observed when the test
sample was diluted 10-fold (0.6 ml of sample into a hemichamber
of 6 ml volume). In four separate experiments, the
Isc generated by the application of 1 ml of
EPEC-HeLa-conditioned medium was 5.3 ± 1.8 µA/cm2
in the absence of the inhibitor/regenerator and 9.3 ± 2.3 µA/cm2 in its presence (P = 0.02). For
EPEC-T84-conditioned media, the Isc values
observed were 3.0 ± 1.9 and 8.4 ± 3.4 µA/cm2
for media collected in the absence and presence of the
inhibitor/regenerator, respectively. In general, the amplitude of the
Isc response was proportional to the amount of
test medium applied over the range of 0.5 to 1.8 ml. As a basis for
comparison, maximal doses of adenosine (10-20 µM) in this system
produce Isc responses of 23-33 µA/cm2. Isc triggered by
EPEC-HeLa- and EPEC-T84-conditioned media were quickly and completely
reversed by 8-(p-sulfophenyl)-theophylline, a
non-cell-permeable adenosine receptor antagonist (Figs. 7C
and 8F). Complete reversal of the secretory response by
8-(p-sulfophenyl)-theophylline indicates that it is
adenosine, not AMP, ADP, or ATP, that is the final agonist acting on
the T84 cells to trigger chloride secretion. This finding is consistent
with the reports of several other laboratories indicating that apically
applied adenine nucleotides in T84 cells trigger secretion via
adenosine receptors, specifically the adenosine A2b
adenosine receptor subtype (3, 31, 52, 53).
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Fig. 8 shows the results obtained with conditioned medium from EPEC-infected T84 cells. Once again, conditioned medium from uninfected T84 cells triggered no secretion whether the inhibitor/regenerator was present or not (Fig. 8A, 1 and 2). Conditioned medium from T84 cells infected with wild-type EPEC E2348/69 did trigger a secretory response, and it was larger with medium collected in the presence of the inhibitor/regenerator (Fig. 8C) than in its absence (Fig. 8B). Conditioned medium from cells infected with the espF mutant UMD874 triggered an Isc less than that from E2348-infected cells (Fig. 8, D and E, without and with inhibitor/regenerator, respectively). In four separate experiments similar to Fig. 8, C and E, the ratio of the Isc triggered by UMD874-conditioned medium was 66.4 ± 22% of that triggered by wild-type EPEC-conditioned medium (UMD874 significantly less than wild EPEC by paired t-test, P = 0.02). The decreased Isc observed after infection with the espF mutant UMD874 is quantitatively similar to the reduced ATP release induced by this mutant (Figs. 1 and 5B). Results shown in Figs. 7 and 8 also demonstrated that the amount of ATP released during EPEC infection was sufficient to trigger chloride secretion, not only when added at full strength, but even after dilution 6- to 10-fold.
Additional experiments were performed to determine whether the secretagogue activity in conditioned medium from EPEC-infected cells was accounted for by its content of adenine nucleotides or adenosine, as opposed to some other bioactive molecule. The secretagogue in the conditioned medium retained its activity after filtration through a 10,000 mol weight cut-off membrane, consistent with the low molecular size of a nucleotide or nucleoside. After boiling, conditioned media from EPEC-infected cells retained 75 ± 3% of their original activity (4 separate experiments), which is consistent with the properties of AMP or adenosine (37).
The presence of the inhibitor/regenerator had more prominent effects in
EPEC-T84-conditioned medium than in EPEC-HeLa-conditioned medium
(compare Fig. 7C with Fig. 8, B and
C). Initially, the enhancing effect of the
inhibitor/regenerator led us to believe that phosphorylated adenine
nucleotides, such as ATP or ADP, might be triggering a secretory
response in the Ussing chamber. Instead, however, the discrepancy is
most likely explained by more avid reuptake of adenosine in T84 cells.
Mun et al. (36) found that T84 cells took up adenosine via
both the apical and basolateral surfaces of the cell, although the
capacity of the latter was much larger. Because EPEC infection markedly
increases transmembrane permeability in the 5-h duration of infection
we used (6, 34, 42), adenosine could reach the basolateral
space and be susceptible to reuptake via the basolateral transport
system. This system is of sufficient affinity and capacity to reduce
extracellular adenosine levels below the threshold for activation of
adenosine receptors (36). Thus the avid adenosine reuptake
ability of T84 cells limits the accumulation of extracellular adenosine
in media collected in the absence of the inhibitor/regenerator. In contrast, ATP, ADP, and AMP are taken up poorly or not at all by either
mammalian or bacterial cells. The inhibitor/regenerator temporarily
traps nucleotides in their phosphorylated forms, in which they are not
susceptible to reuptake, but the phosphocreatine in the regenerating
system becomes exhausted toward the end of the 4-h collection period.
In addition, injection of the sample into the Ussing chamber results in
a 6- to 10-fold dilution, such that the concentration of the
,
-methylene ADP inhibitor drops well below its IC50
for 5'-nucleotidase. Evidently, nucleotidases in the test tissue
are able to convert some of the nucleotides to adenosine and generate a
secretory response. In further support of the hypothesis that adenosine
is the mediator of secretion, apyrase treatment of EPEC-T84-conditioned
medium (5 U/ml apyrase for 5 min, 37°C) did not abolish the
Isc response in the Ussing chamber, but instead
actually increased the Isc response. In four experiments, the ratio of the Isc observed after
apyrase treatment relative to sham-treated control was 154 ± 42%
(significantly increased; P = 0.018 by paired
t-test). Furthermore, the Isc
triggered by EPEC-T84-conditioned medium in the presence of the
inhibitor/regenerator was completely reversed by
8-(p-sulfophenyl)-theophylline (Fig. 8F, 2) an adenosine receptor antagonist. In
addition, the secretory response of EPEC-T84-conditioned medium was
reversed by 200 nM MRS1754, a highly selective adenosine
A2b receptor blocker (29). Figure
9 shows the dose-response relationship
for reversal of the Isc by MRS1754. The apparent
IC50 of MRS1754 in Fig. 9 (~50 nM) is somewhat higher
than values reported in the literature (2-20 nM)
(29). MRS1754, a hydrophobic compound, binds strongly to
plastic, so the modestly decreased potency observed in Fig. 9 may be
due to absorption to the Ussing chamber itself, which has a large
internal surface area and is composed of acrylic plastic. In contrast,
the Isc triggered by EPEC-conditioned medium was not blocked by 10 µM MRS2179, a potent antagonist of P2Y1
receptors and a weaker antagonist of P2X receptors (data not shown).
These findings indicate again that it is adenosine, and not a
phosphorylated adenine nucleotide, that triggers chloride secretion
when applied to the apical side of T84 cells (3, 31, 53),
and this adenosine effect is mediated via adenosine A2b
receptors (52).
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DISCUSSION |
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The results presented here show that EPEC infection of cells triggers a release of ATP from host cells that is large in magnitude and, in polarized cells, is solely into the apical culture medium. Extracellular ATP is quickly broken down to less phosphorylated nucleotides and adenosine. These products accumulate in sufficient quantity that they are capable of triggering a chloride secretory response in intestinal tissues in the Ussing chamber (Figs. 7C and 8B). An inhibitor/regenerator system was used as a nonphysiological way of trapping ATP to allow its convenient and accurate measurement. On the basis of the data shown, concentrations of ATP measured in the presence of the inhibitor/regenerator appear to provide a reasonable estimate of the total adenine nucleotide pool (ATP, ADP, AMP, and adenosine), which accumulates in the absence of the inhibitor/regenerator.
Because the regenerating system can rephosphorylate ADP to ATP, it is formally possible that release of ADP contributes to the extracellular adenine nucleotide accumulation. This seems unlikely, however, for two reasons. First, ATP release is observed in HeLa cells in the absence of the inhibitor/regenerator (Fig. 1A). Second, the ratio of ATP to ADP in healthy cells is >5:1, a situation that applies early in the course of infection. Even after 4 h of EPEC infection, the ATP content of the monolayer is still ~80% of the uninfected control, so a severe alteration in the "energy charge," or ATP:ADP ratio, of the cell is unlikely. However, our data do not rule out the possibility of ADP efflux from the infected host cell, especially late in the course of EPEC infection.
The mechanism of ATP release in response to EPEC infection clearly involves the type III secretion system of this pathogen but may be regulated in more subtle ways as well. The reduced ATP release observed with the EPEC sepZ, ler, and espF mutants clearly implicate the type III secretion system in general and the EspF in particular in the ATP release process.
Detailed studies (5) of the mechanism of type III secretion by Shigella show that assembly of the Shigella "secretion" or "injectosome" in the red blood cell membrane creates a pore of ~25 Å. This pore is sufficiently large to allow permeability of the trisaccharide raffinose (504 mol weight), which is very close in size to ATP (507 mol weight for the free acid). The concentration of ATP inside a mammalian cell is as high as 3-5 mM, so ATP could exit by passive diffusion through this pore. We (12) have already shown that EPEC-infected cells become permeable to dyes such as trypan blue and propidium iodide. Ide et al. (24) recently showed that the pore created in the host cell membrane by EPEC resembles that of Shigella, with a estimated pore size of 30-50 Å.
The finding that forskolin and cholera toxin markedly increase the ATP efflux from EPEC-infected T84 cells (Fig. 6A) suggests that, in addition to the bacteria-produced pore, other cellular mechanisms may influence or regulate the rate of ATP release. Research from other laboratories shows that the CFTR itself, or a molecule closely regulated by the CFTR, permits ATP efflux in response to nonlethal stimuli such as cell swelling, stretching, or cAMP (25, 45, 54). Even in the absence of exogenous stimulators of cAMP, this CFTR-dependent pathway could be activated in response to EPEC infection. For example, EPEC infection could activate this pathway by cell swelling triggered by type III secretion, because both Shigella and EPEC have been shown to induce a contact-dependent osmotic lysis of red blood cells (5, 48, 58). Interestingly, two laboratories have shown that the espF mutant was fully competent in this osmotic hemolysis (48, 58). If EPEC-induced osmotic swelling allows a second, CFTR-dependent pathway of ATP release, this could explain why the performance of the espF mutant was so different in cell lines with and without CFTR. According to this hypothesis, in T84 cells, the CFTR-dependent pathway of ATP release is still activated by the espF mutant, so the mutant appears less attenuated than in cells lacking CFTR.
Although EPEC strains do not produce cAMP-elevating toxins, enterotoxigenic (ETEC) strains do produce the labile toxin. Dual infections with EPEC and ETEC are common in developing countries (32); our results suggest a possible interaction between two distinct types of diarrheagenic E. coli, which could result in more severe disease with dual infection.
The findings presented here may have relevance to the pathogenesis of diarrhea by EPEC, which has remained a puzzle despite decades of research. Malabsorption was one of the earliest explanations proposed for EPEC diarrhea. Indeed, malabsorption has been documented in human cases of EPEC infection (56, 57) as well as in rabbits infected with RDEC-1 (55). However, a purely malabsorptive diarrhea would not explain the rapid onset of diarrhea observed in fasting humans in volunteer challenge studies nor the persistence of diarrhea for weeks in children maintained on total parenteral nutrition without oral intake (47). Therefore, there must be a secretory component to EPEC diarrhea, but how this is triggered is not known.
The idea that adenine nucleotides released from EPEC-infected cells could trigger a fluid secretory response has several attractive features. Biopsy and autopsy specimens from cases of human EPEC infection and from rabbits infected with REPEC or RDEC show that EPEC infection is patchy, with many spared, apparently normal areas (56, 57). Furthermore, EPEC seem to preferentially adhere to villi, whereas it is the crypt cells that possess the secretory capacity to generate a watery diarrhea. Furthermore, several laboratories have shown that after a few hours of EPEC infection, the transmembrane electrical potential of intestinal cells collapses (50) and cells show decreased secretory responses to agonists such as carbachol and forskolin (22, 42). This makes it unlikely that the intestinal cells actually adhered to by EPEC are capable of generating the sustained fluid secretion necessary to explain an ongoing diarrhea. In contrast, a new paradigm for EPEC-induced diarrhea is suggested by the current report. According to this hypothesis, adenine nucleotides released from EPEC-infected cells act on nearby, uninfected cells, including crypt cells, to generate a secretory response in the intestine.
Invasive pathogens, such as Salmonella and Shigella, trigger an influx of polymorphonuclear leukocytes (PMNs) into the gut epithelium, and these transmigrating PMNs release 5'-AMP, the neutrophil-derived secretagogue (30, 31). EPEC, in contrast, behaves clinically as a noninvasive organism and does not trigger an influx of fecal leukocytes (27). Our finding that large concentrations of adenine nucleotides can be released directly from intestinal cells, without the need for PMNs, shows that these nucleotides may play a pathophysiological role in diarrhea even for noninflammatory pathogens such as EPEC.
Theoretically, adenine nucleotides could trigger chloride secretion from epithelial cells by interacting with several types of purinergic receptors on both the apical and basolateral surfaces of the cell. Adenosine A2 receptors coupled to chloride secretion are associated with a stimulation of adenylyl cyclase. Purinergic P2Y receptors, which respond to ADP, ATP, and UTP, are also found in the intestine and are coupled to chloride secretion via various intracellular signaling pathways (46). P2Y receptors capable of responding to adenine nucleotides are apparently not found on the apical surface of T84 cells (3, 16, 53), although one recent report did describe an Isc response to UTP applied apically to T84 cells (49). P2X receptors are found on sensory neurons in intestine and other hollow organs and may mediate the sensation of pain as well as alterations in peristalsis (9, 10) but are apparently not linked to ion secretion. Our results indicate that adenosine A2b receptors are responsible for the chloride secretory response to EPEC-conditioned media applied apically to T84 cells in agreement with others (53) who investigated the effects of ATP on this cell line.
Although the focus in this study has been on the effects of released adenine nucleotides on the host, it has not escaped our attention that the release of nucleotides may provide benefits to the pathogen. E. coli, including EPEC, is capable of scavenging adenosine from the environment with an even higher avidity than mammalian cells (to concentrations <0.1 µM) and utilizing the adenosine in lieu of de novo purine biosynthesis (59), which is energetically expensive. This is particularly relevant in light of increasing appreciation that the intestinal mucosa is a highly purine-limited environment, not just for invasive bacteria (23) but also for noninvasive pathogens such as Vibrio cholerae (8).
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ACKNOWLEDGEMENTS |
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We thank Drs. James B. Kaper and Simon Elliott, University of Maryland, for sharing EPEC LEE mutants; Dr. Tom Cleary, University of Texas Medical School at Houston, for providing E. coli O157:H7 strain 987; Dr. Terry Connell, University at Buffalo, for providing cholera toxin; and Dr. Joel Linden, University of Virginia, for MRS1754 and helpful discussions.
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FOOTNOTES |
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This work was supported by a grant from Research for Health in Erie County (to J. K. Crane) and bridge funds from the Department of Medicine, University at Buffalo.
Address for reprint requests and other correspondence: J. K. Crane, Infectious Diseases Division, Bimedical Research Bldg., 3435 Main St., Buffalo, NY 14214 (E-mail: jcrane{at}acsu.buffalo.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 13, 2002;10.1152/ajpgi.00484.2001
Received 15 November 2001; accepted in final form 25 February 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abraham, E,
Prat A,
Gerweck L,
Seneveratne T,
Arceci R,
Kramer R,
Guidotti G,
and
Cantiello H.
The multidrug resistance (mdr1) gene product functions as an ATP channel.
Proc Natl Acad Sci USA
90:
312-316,
1993[Abstract].
2.
Barnett-Foster, D,
Abul-Milh M,
Huesca M,
and
Lingwood C.
Enterohemorrhagic Escherichia coli induces apoptosis which augments bacterial binding and phosphatidylethanolamine exposure on the plasma membrane outer leaflet.
Infect Immun
68:
3108-3115,
2000
3.
Barrett, K,
Huott P,
Shah S,
Dharmsathophorn K,
and
Wasserman S.
Differing effects of apical and basolateral adenosine on colonic epithelial cell line T84.
Am J Physiol Cell Physiol
256:
C197-C203,
1989
4.
Bieber, D,
Ramer S,
Wu CY,
Murray W,
Tobe T,
Fernandez R,
and
Schoolnik G.
Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli.
Science
280:
2114-2118,
1998
5.
Blocker, A,
Gounon P,
Larquet E,
Niebuhr K,
Cabiaux V,
Parsot C,
and
Sansonetti P.
The tripartite type III secretion system of Shigella flexneri inserts IpaB and IpaC into host membranes.
J Cell Biol
147:
683-693,
1999
6.
Canil, C,
Rosenshine I,
Ruschkowski S,
Donnenberg M,
Kaper J,
and
Finlay B.
Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers.
Infect Immun
61:
2755-2762,
1993[Abstract].
7.
Chang, H,
and
Yang X.
Proteases for cell suicide: functions and regulation of caspases.
Microbiol Molec Biol Rev
64:
821-846,
2000
8.
Chiang, S,
and
Mekalanos J.
Use of signature-tagged mutagenesis to identify Vibrio cholerae genes critical for colonization.
Mol Microbiol
27:
797-805,
1998[ISI][Medline].
9.
Cockayne, D,
Hamilton S,
Zhu QM,
Dunn P,
Zhong Y,
Novakovic S,
and
Malmberg A.
Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice.
Nature
407:
1011-1015,
2000[ISI][Medline].
10.
Cook, S,
and
McCleskey E.
ATP, pain, and a full bladder.
Nature
407:
951-952,
2000[ISI][Medline].
11.
Crane, J,
McNamara B,
and
Donnenberg M.
Role of EspF in host cell death due to enteropathogenic Escherichia coli.
Cell Microbiol
3:
197-211,
2001[ISI][Medline].
12.
Crane, JK,
Majumdar S,
and
Pickhardt DP.
Host cell death due to enteropathogenic Escherichia coli has features of apoptosis.
Infect Immun
67:
2575-2584,
1999
13.
Crane, JK,
and
Oh JS.
Activation of host cell protein kinase C by enteropathogenic Escherichia coli.
Infect Immun
65:
3277-3285,
1997[Abstract].
14.
Czerucka, D,
Dahan S,
Mograbi B,
Rossi B,
and
Rampal P.
Saccharomyces boulardii preserves the barrier function and modulates the signal transduction pathway induced in enteropathogenic Eschericia coli-infected T84 cells.
Infect Immun
68:
5998-6004,
2000
15.
Dharmsathaphorn, K,
and
Pandol SJ.
Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line.
J Clin Invest
77:
348-354,
1986[ISI][Medline].
16.
Dho, S,
Stewart K,
and
Foskett JK.
Purinergic receptor activation of Cl secretion in T84 cells.
Am J Physiol Cell Physiol
262:
C67-C74,
1992
17.
Donnenberg, M,
and
Kaper J.
Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector.
Infect Immun
59:
4310-4317,
1991[ISI][Medline].
18.
Donnenberg, MS,
Lai LC,
and
Taylor KA.
The locus of enterocyte effacement pathogenicity island of enteropathogenic Escherichia coli encodes secretion functions and remnants of transposons at its extreme right end.
Gene
184:
107-114,
1997[ISI][Medline].
19.
Elliott, S,
Sperandio V,
Giron J,
Shin S,
Mellies J,
Wainwright L,
Jutcheson S,
McDaniel T,
and
Kaper J.
The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli.
Infect Immun
68:
6115-6126,
2000
20.
Elliott, SJ,
Wainwright LA,
McDaniel TK,
Jarvis KG,
Deng YK,
Lai LC,
McNamara BP,
Donnenberg MS,
and
Kaper JB.
The complete sequence of the locus for enterocyte effacement (LEE) from enteropathogenic E. coli E2348/69.
Mol Microbiol
28:
1-4,
1998[ISI][Medline].
21.
Grasl, M,
and
Turnheim K.
Stimulation of electrolyte secretion in rabbit colon by adenosine.
J Physiol (Lond)
346:
93-110,
1984[Abstract].
22.
Hecht, G,
and
Koutsouris A.
Enteropathogenic E. coli attenuates secretagogue-induced net intestinal ion transport but not Cl secretion.
Am J Physiol Gastrointest Liver Physiol
276:
G781-G788,
1999
23.
Heithoff, D,
Sinsheimer R,
Low D,
and
Maham M.
In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials.
Philos Trans R Soc Lond B Biol Sci
355:
633-642,
2000[ISI][Medline].
24.
Ide, T,
Laarmann S,
Greune L,
Schillers H,
Oberleithner H,
and
Schmidt M.
Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli.
Cell Microbiol
3:
669-679,
2001[ISI][Medline].
25.
Jiang, Q,
Mak D,
Dividas S,
Schweibert E,
Bragin A,
Zhang Y,
Skach W,
Guggino W,
Foskett J,
and
Engelhardt J.
Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor.
J Cell Biol
143:
645-657,
1998
26.
Lee, C.
Type III secretion systems: machines to deliver bacterial proteins into eukaryotic cells?
Trends Microbiol
5:
148-155,
1997[ISI][Medline].
27.
Levine, M,
Nalin D,
Hornick R,
Bergquist E,
Waterman D,
Young C,
Sotman S,
and
Rowe B.
Escherichia coli strains that cause diarrhea but do not produce heat-labile or heat-stable enterotoxins and are not invasive.
Lancet
1:
1119-1122,
1978[ISI][Medline].
28.
Levine, M,
Nataro J,
Karch H,
Baldini M,
Kaper J,
Black R,
Clements M,
and
O'Brien A.
The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor.
J Infect Dis
152:
550-559,
1985[ISI][Medline].
29.
Linden, J.
Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection.
Annu Rev Pharmacol Toxicol
41:
775-787,
2001[ISI][Medline].
30.
Madara, J,
Parkos C,
Colgan S,
MacLeod R,
Nash S,
Matthews J,
Delp C,
and
Lencer W.
Cl secretion in a model intestinal epithelium induced by a neutrophil-derived secretagogue.
J Clin Invest
89:
1938-1944,
1992[ISI][Medline].
31.
Madara, J,
Patapoff T,
Gillece-Castro B,
Colgan S,
Parkos C,
Delp C,
and
Mrsny R.
5'-adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial monolayers.
J Clin Invest
91:
2320-2325,
1993[ISI][Medline].
32.
Mathewson, J,
Oberhelman R,
DuPont H,
de la Cabada F,
and
Vasquez-Garibay E.
Enteroadherent Escherichia coli as a cause of diarrhea among children in Mexico.
J Clin Microbiol
25:
1917-1919,
1987[ISI][Medline].
33.
McNamara, B,
and
Donnenberg M.
A novel proline-rich protein, EspF, is secreted from enteropathogenic Escherichia coli via the type III export pathway.
FEMS Microbiol Lett
166:
71-78,
1998[ISI][Medline].
34.
McNamara, B,
Koutsouris A,
O'Connell C,
Nougayrede JP,
Donnenberg M,
and
Hecht G.
Translocated EspF protein of enteropathogenic Escherichia coli (EPEC) disrupts host intestinal barrier function.
J Clin Invest
107:
621-629,
2001
35.
Moss, J,
Fisher P,
Vick B,
Groisman E,
and
Zychlinsky A.
The regulatory protein PhoP controls susceptibility to the host inflammatory response in Shigella flexneri.
Cell Microbiol
2:
443-452,
2000[ISI][Medline].
36.
Mun, E,
Tally K,
and
Matthews J.
Characterization and regulation of adenosine transport in T84 intestinal epithelial cells.
Am J Physiol Gastrointest Liver Physiol
274:
G261-G269,
1998
37.
Nash, S,
Parkos C,
Nusrat A,
Delp C,
and
Madara J.
In vitro model of intestinal crypt abscess: a novel neutrophil derived secretagogue activity.
J Clin Invest
87:
1474-1477,
1991[ISI][Medline].
38.
Nataro, J,
Steiner T,
and
Guerrant R.
Enteroaggregative Escherichia coli.
Emerg Infect Dis
4:
251-261,
1998[ISI][Medline].
39.
Nataro, JP,
Deng Y,
Cookson S,
Cravioto A,
Savarino SJ,
Guers LD,
Levine MM,
and
Tacket CO.
Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers.
J Infect Dis
171:
465-468,
1995[ISI][Medline].
40.
Nataro, JP,
Hicks S,
Phillips AD,
Vial PA,
and
Sears CL.
T84 cells in culture as a model for enteroaggregative Escherichia coli pathogenesis.
Infect Immun
64:
4761-4768,
1996[Abstract].
41.
Navarro-Garcia, F,
Sears C,
Eslava C,
Cravioto A,
and
Nataro JP.
Cytoskeletal effects induced by Pet, the serine protease enterotoxin of enteroaggregative Escherichia coli.
Infect Immun
67:
2184-2192,
1999
42.
Philpott, DJ,
McKay DM,
Sherman PM,
and
Perdue MH.
Infection of T84 cells with enteropathogenic Escherichia coli alters barrier and transport functions.
Am J Physiol Gastrointest Liver Physiol
270:
G634-G645,
1996
43.
Pier, GB,
Grout M,
Zaidi T,
Meluleni G,
Mueschenborn SS,
Banting G,
Ratcliff R,
Evans MJ,
and
Colledge WH.
Salmonella typhi uses CFTR to enter intestinal epithelial cells.
Nature
393:
79-82,
1998[ISI][Medline].
44.
Rabinowitz, RP,
Lai LC,
Jarvis K,
McDaniel TK,
Kaper JB,
Stone KD,
and
Donnenberg MS.
Attaching and effacing of host cells by enteropathogenic Escherichia coli in the absence of detectable tyrosine kinase mediated signal transduction.
Microb Pathog
21:
157-171,
1996[ISI][Medline].
45.
Roman, R,
Feranchak A,
Salter K,
Wang Y,
and
Fitz J.
Endogenous ATP release regulates Cl secretion in cultured human and rat biliary epithelial cells.
Am J Physiol Gastrointest Liver Physiol
276:
G1391-G1400,
1999
46.
Roman, R,
and
Fitz J.
Emerging roles of purinergic signaling in gastrointestinal epithelial secretion and hepatobiliary function.
Gastroenterology
116:
964-979,
1999[ISI][Medline].
47.
Rothbaum, R,
McAdams A,
Giannella R,
and
Partin J.
A clinicopathologic study of enterocyte-adherent Escherichia coli: a cause of protracted diarrhea in infants.
Gastroenterology
83:
441-454,
1982[ISI][Medline].
48.
Shaw, R,
Daniell S,
Ebel F,
Frankel G,
and
Knutton S.
EspA filament-mediated protein translocation into red blood cells.
Cell Microbiol
3:
213-222,
2001[ISI][Medline].
49.
Smitham, J,
and
Barrett K.
Differential effects of apical and basolateral uridine triphosphate on intestinal epithelial chloride secretion.
Am J Physiol Cell Physiol
280:
C1431-C1439,
2001
50.
Stein, MA,
Mathers DA,
Yan H,
Baimbridge KG,
and
Finlay BB.
Enteropathogenic Escherichia coli markedly decreases the resting membrane potential of Caco-2 and HeLa human epithelial cells.
Infect Immun
64:
4820-4825,
1996[Abstract].
51.
Strohmeier, GR,
Lencer WI,
Patapoff TW,
Thompson LF,
Carlson SL,
Moe SJ,
Carnes DK,
Mrsny RJ,
and
Madara JL.
Surface expression, polarization, and functional significance of CD73 in human intestinal epithelia.
J Clin Invest
99:
2588-2601,
1997
52.
Strohmeier, GR,
Reppert SM,
Lencer WI,
and
Madara JL.
The A2b adenosine receptor mediates cAMP responses to adenosine receptor agonists in human intestinal epithelia.
J Biol Chem
270:
2387-2394,
1995
53.
Stutts, MJ,
Lazarowski ER,
Paradiso AM,
and
Boucher RC.
Activation of CFTR Cl conductance in polarized T84 cells by luminal extracellular ATP.
Am J Physiol Cell Physiol
268:
C425-C433,
1995
54.
Sugita, M,
Yue Y,
and
Foskett J.
CFTR Cl channel and CFTR-associated ATP channel: distinct pores regulated by common gates.
EMBO J
17:
898-908,
1998
55.
Tai, YH,
Gage T,
McQueen C,
Formal S,
and
Boedeker E.
Electrolyte transport in rabbit cecum. I. Effect of RDEC-1 infection.
Am J Physiol Gastrointest Liver Physiol
256:
G721-G726,
1989
56.
Taylor, C,
Hart A,
Batt R,
McDougall C,
and
McLean L.
Ultrastructural and biochemical changes in human jejunal mucosa associated with enteropathogenic Escherichia coli (O111) infection.
J Pediatr Gastroenterol Nutr
5:
70-73,
1986[ISI][Medline].
57.
Ulshen, M,
and
Rollo J.
Pathogenesis of Escherichia coli gastroenteritis in man-another mechanism.
N Engl J Med
302:
99-101,
1980[ISI][Medline].
58.
Warawa, J,
Finlay B,
and
Kenny B.
Type III-secretion dependent hemolytic activity of enteropathogenic Escherichia coli.
Infect Immun
67:
5538-5540,
1999
59.
Zalkin, H,
and
Nygaard P.
Biosynthesis of purine nucleotides. In: Escherichia coli and Salmonella (2nd ed), edited by Neidhardt F.. Washington, DC: ASM, 1996, p. 575-576.