Involvement of the EnteroaggregativeEscherichia coli Plasmid-Encoded Toxin in Causing Human Intestinal Damage

ABSTRACT Enteroaggregative Escherichia coli (EAEC) strains have been shown to adhere to human intestinal tissue in an in vitro organ culture (IVOC) model, and certain strains manifest mucosal toxicity. We have recently described the EAEC plasmid-encoded toxin (Pet), a member of a specific serine protease subclass of the autotransporter proteins. When injected into rat ileal loops, Pet both elicited fluid accumulation and had cytotoxic effects on the mucosa. Furthermore, the Pet protein caused rises in short circuit current from rat jejunal tissue mounted in a Ussing chamber and rounding of intestinal epithelial cells in culture. We therefore hypothesized that the mucosal pathology induced by EAEC strains in the IVOC model was related to expression of the Pet protein. Here, we have examined the effects of EAEC strain 042 and its isogenic pet mutant in the IVOC model. 042-infected colonic explants exhibited dilation of crypt openings, increased cell rounding, development of prominent intercrypt crevices, and absence of apical mucus plugs. Colonic tissue incubated with the pet mutant exhibited significantly fewer mucosal abnormalities both subjectively and as quantitated morphometrically by measurement of crypt aperture diameter. Mucosal effects were restored upon complementation of the pet mutation intrans. Interestingly, we found that the ability of 042 to damage T84 cells was not dependent upon Pet. The data suggest that the Pet toxin is active on the human intestinal mucosa but that EAEC may have other mechanisms of eliciting mucosal damage.

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Cellular pathways mediating tachykinin-evoked secretomotor responses in guinea pig ileum

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1997.273.5.g1127 ◽

1997 ◽

Vol 273 (5) ◽

pp. G1127-G1134 ◽

Cited By ~ 9

Author(s):

W. MacNaughton ◽

B. Moore ◽

S. Vanner

Keyword(s):

Guinea Pig ◽

Receptor Agonist ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Neurokinin A ◽

Guinea Pig Ileum ◽

Cellular Pathways ◽

20 Nm

This study characterized tachykinin-evoked secretomotor responses in in vitro submucosal and mucosal-submucosal preparations of the guinea pig ileum using combined intracellular and Ussing chamber recording techniques. Superfusion of endogenous tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B depolarized single submucosal neurons and evoked increased short-circuit current ( I sc) responses in Ussing chamber preparations. The NK1-receptor agonist [Sar9,Met(O2)11]SP [50% effective concentration (EC50) = 2 nM] depolarized all submucosal neurons examined. The NK3-receptor agonist senktide (EC50 = 20 nM) depolarized ∼50% of neurons examined, whereas the NK2-receptor agonist [Ala5,β-Ala8]NKA-(4—10) had no effect on membrane potential. [Sar9,Met(O2)11]SP and senktide evoked similar increases in I sc that were tetrodotoxin sensitive (91 and 100%, respectively) and were selectively blocked by the NK1antagonist CP-99,994 and the NK3antagonist SR-142801, respectively. Capsaicin-evoked increases in I sc were significantly inhibited (54%, P < 0.05) by CP-99,994 but not by SR-142801. Neither antagonist inhibited slow excitatory postsynaptic potentials. These findings suggest that tachykinin-evoked secretion in guinea pig ileum is mediated by NK1 and NK3 receptors on submucosal secretomotor neurons and that capsaicin-sensitive nerves release tachykinin(s) that activate the NK1 receptors.

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Antisecretory effect of prostaglandin D2 in rat colon in vitro: action sites

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1991.260.6.g904 ◽

1991 ◽

Vol 260 (6) ◽

pp. G904-G910 ◽

Cited By ~ 2

Author(s):

K. J. Goerg ◽

C. Diener ◽

M. Diener ◽

W. Rummel

Keyword(s):

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Rat Colon ◽

Prostaglandin D2 ◽

Cyclic Monophosphate ◽

Heat Stable ◽

Antisecretory Effect ◽

Flux Measurements

The effect of prostaglandin D2 (PGD2) on colonic ion transport was studied in the Ussing chamber. PGD2 (10(-6) M) decreased baseline short-circuit current (Isc) in two preparations of rat colon descendens, a mucosa-submucosa preparation with and a mucosa preparation without the submucosal plexus. In both preparations, PGD2 inhibited the neuronally mediated secretory responses to electric field stimulation, the sea anemone toxin ATX II, and different cholinergic agents. Unidirectional flux measurements revealed that PGD2 diminished the secretagogue-induced increase in the serosal-to-mucosal flux of Cl- and thereby inhibited net Cl- secretion. PGD2, however, had no effect on the adenosine 3',5'-cyclic monophosphate-mediated response to forskolin or vasoactive intestinal peptide or on guanosine 3',5'-cyclic monophosphate-mediated secretion induced by the heat-stable enterotoxin of Escherichia coli. The PGD2 also blocked the increase in Isc evoked by two neuronally acting inflammatory mediators, i.e., bradykinin and PGI2 in the mucosa-submucosa preparation, but had no effect on the response to PGE2. Consequently, PGD2 exerts an indirect antisecretory effect caused by an inhibition of enteric secretomotor neurons of both the submucosal and the mucosal plexus.

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Cytoskeletal Effects Induced by Pet, the Serine Protease Enterotoxin of Enteroaggregative Escherichia coli

Infection and Immunity ◽

10.1128/iai.67.5.2184-2192.1999 ◽

1999 ◽

Vol 67 (5) ◽

pp. 2184-2192 ◽

Cited By ~ 92

Author(s):

Fernando Navarro-García ◽

Cynthia Sears ◽

Carlos Eslava ◽

Alejandro Cravioto ◽

James P. Nataro

Keyword(s):

Escherichia Coli ◽

Serine Protease ◽

Protease Activity ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

T84 Cells ◽

Ussing Chambers ◽

E Coli ◽

Cytopathic Effects

ABSTRACT We have previously described enteroaggregative Escherichia coli (EAEC) strains that induce cytotoxic effects on T84 cells, ligated rat ileal loops, and human intestine in culture. Such strains secrete a 104-kDa protein termed Pet (for plasmid-encoded toxin). We have also shown previously that the Pet toxin induces rises in short-circuit current and decreases the electrical resistance in rat jejunum mounted in an Ussing chamber. The nucleotide sequence of thepet gene revealed that Pet is a member of the autotransporter class of secreted proteins. Here we show that a concentrated supernatant of E. coli HB101 harboring the minimal pet clone pCEFN1 induces temperature-, time- and dose-dependent cytopathic effects on HEp-2 cells and HT29 C1 cells in culture. The effects were characterized by release of the cellular focal contacts from the glass substratum, followed by complete rounding of the cells and detachment from the glass. Staining of the Pet-treated cells with Live/Dead viability stain revealed that >90% of rounded cells were viable. Pet-intoxicated HEp-2 and HT29 cells stained with fluorescein-labeled phalloidin revealed contraction of the cytoskeleton and loss of actin stress fibers. However, the effects of Pet were not inhibited by cytoskeleton-altering drugs, including colchicine, taxol, cytochalasin D, and phallicidin. The Pet protein induced proteolysis in zymogram gels, and preincubation with the serine protease inhibitor phenylmethylsulfonyl fluoride resulted in complete abrogation of Pet cytopathic effects. We introduced a mutation in a predicted catalytic serine residue and found that the mutant (Pet S260I) was deficient in protease activity and did not produce cytopathic effects, cytoskeletal damage, or enterotoxic effects in Ussing chambers. These data suggest that Pet is a cytoskeleton-altering toxin and that its protease activity is involved in each of the observed phenotypes.

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Investigation of effects ofGiardia duodenalison transcellular and paracellular transport in enterocytes usingin vitroUssing chamber experiments

Parasitology ◽

10.1017/s0031182014001772 ◽

2014 ◽

Vol 142 (5) ◽

pp. 691-697 ◽

Cited By ~ 5

Author(s):

KRISTOFFER R. TYSNES ◽

LUCY J. ROBERTSON

Keyword(s):

Epithelial Transport ◽

Short Circuit ◽

Ussing Chamber ◽

Field Isolate ◽

Giardia Duodenalis ◽

Short Circuit Current ◽

Paracellular Transport ◽

Detailed Knowledge ◽

Transport Mechanisms

SUMMARYThe mechanisms by which different genotypes ofGiardia duodenalisresult in different symptoms remain unresolved. In particular, we lack detailed knowledge on which transport mechanisms (transcellular or paracellular) are affected by differentGiardiaisolates. Using horse radish peroxidase (HRP) and creatinine as transcellular and paracellular probes, respectively, we developed a robust assay that can be used with an Ussing chamber to investigate epithelial transport, as well as short-circuit current as an indicator of net ion transport. We investigated 2Giardiaisolates, both Assemblage A, one a lab-adapted strain and the other a field isolate. Results indicate that products from sonicatedGiardiatrophozoites increase both transcellular and paracellular transport. A non-significant increase in transepithelial electrical resistance (TEER) and short-circuit current were also noted. The paracellular transport was increased significantly more in the field isolate than in the lab-adapted strain. Our results indicate that while both transcellular and paracellular transport mechanisms may be increased following exposure of cells toGiardiatrophozoite sonicate, perhaps by inducing non-specific increases in cellular traffic, it is important thatin vitrostudies ofGiardiapathophysiology are conducted with differentGiardiaisolates, not just lab-attenuated strains.

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Dietary flavonol quercetin induces chloride secretion in rat colon

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1998.275.5.g1166 ◽

1998 ◽

Vol 275 (5) ◽

pp. G1166-G1172 ◽

Cited By ~ 7

Author(s):

Rainer Cermak ◽

Ursula Föllmer ◽

Siegfried Wolffram

Keyword(s):

Short Circuit ◽

Ussing Chamber ◽

Phosphodiesterase Inhibitor ◽

Short Circuit Current ◽

Distal Colon ◽

Chloride Secretion ◽

Proximal Colon ◽

Rat Colon ◽

Quercetin Glycoside

The aim of this study was to investigate the possible effects of the flavonol quercetin, the most abundant dietary flavonoid, on the intestinal mucosa. In vitro experiments were performed with various segments of the rat intestine, using the Ussing chamber technique. Quercetin increased the short-circuit current ( I sc) in the jejunum, ileum, and proximal and distal colon. Additional experiments were performed using preparations of the proximal colon. The maximum effective dose of quercetin was found to be ∼100 μM. The quercetin-induced increase in I sc was inhibited by the Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid. Adding blockers of the Na+-K+-2Cl−cotransporter to the serosal compartment diminished the increase of I sc due to quercetin. Ion substitution and flux measurements indicated that the effect of quercetin was due to electrogenic Cl− and[Formula: see text] secretion. In contrast to the aglycone, the quercetin glycoside rutin had no effect. The effect of quercetin on I scwas additive to the I sc increase induced by forskolin, but the flavonoid diminished the I sc evoked by carbachol. The phosphodiesterase inhibitor theophylline blocked the effect of quercetin. Genistein, a related isoflavone, did not alter the I sc evoked by quercetin. These findings demonstrate that the dietary flavonol quercetin induces Cl−secretion and most likely [Formula: see text]secretion in rat small and large intestine. The effects are restricted to the flavonol aglycone.

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Activation of PPARγ by rosiglitazone attenuates intestinal Cl− secretion

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.90640.2008 ◽

2009 ◽

Vol 297 (1) ◽

pp. G82-G89 ◽

Cited By ~ 15

Author(s):

Poonam J. Bajwa ◽

Jimmy W. Lee ◽

Daniel S. Straus ◽

Christian Lytle

Keyword(s):

Short Circuit ◽

Short Circuit Current ◽

Intestinal Epithelial ◽

Secretory Function ◽

Ht29 Cells ◽

Human Intestinal Epithelial Cells ◽

Positive Modulator ◽

Transient Loss ◽

Pparγ Agonists

The thiazolidinedione (TZD) drugs rosiglitazone (Ro) and pioglitazone (Po) are PPARγ agonists in widespread clinical use as insulin-sensitizing agents in Type 2 diabetes. On the basis of recent evidence implicating PPARγ as a positive modulator of intestinal epithelial differentiation, we hypothesized that TZD drugs might attenuate intestinal secretory function. To evaluate this possibility, we examined the effects of Ro and Po on electrogenic Cl− secretion [short-circuit current ( Isc)] in mouse intestinal segments and in cultured human intestinal epithelial cells (HT29-Cl.19A). As hypothesized, oral administration of Ro (20 mg·kg−1·day−1) to mice for 8 days markedly reduced intestinal Isc responses to cAMP (forskolin)- and Ca2+ (carbachol)-dependent stimuli. In these Ro-treated mice, cholera toxin-induced intestinal fluid accumulation was reduced 65%. With continued Ro treatment, the Isc response to carbachol recovered significantly, whereas that to forskolin remained attenuated. Treatment of HT29 cells for 5 days with 10 μM Ro or Po in vitro brought about a similar hyposecretory state. In HT29 cells, the loss of cAMP-dependent Cl− secretion was attributable to a reduced expression of CFTR Cl− channel, KCNQ1 K+ channel, and Na-K-2Cl cotransporter-1 proteins. The transient loss of Ca2+-dependent Cl− secretion involved an impairment of basolateral Ca2+-stimulated K+ channel activity without a detectable loss of KCa3.1 channel protein. Our results establish TZD drugs as important modulators of intestinal Cl− secretory function.

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Human colonic epithelial cells express galanin-1 receptors, which when activated cause Cl− secretion

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.276.1.g64 ◽

1999 ◽

Vol 276 (1) ◽

pp. G64-G72 ◽

Cited By ~ 5

Author(s):

Richard V. Benya ◽

Jorge A. Marrero ◽

Denis A. Ostrovskiy ◽

Athanasia Koutsouris ◽

Gail Hecht

Keyword(s):

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Peptide Hormone ◽

Human Colon ◽

Smooth Muscle Contraction ◽

Dependent Manner ◽

Gi Tract ◽

T84 Cells ◽

Concentration Dependent Manner

Galanin is a peptide hormone widely expressed in the central nervous system and gastrointestinal (GI) tract. Within the GI tract galanin is present in enteric nerve terminals where it is known to modulate intestinal motility by altering smooth muscle contraction. Recent studies also show that galanin can alter intestinal short-circuit current ( I sc) but with differing results observed in rats, rabbits, guinea pigs, and pigs. In contrast, nothing is known about the ability of galanin to alter ion transport in human intestinal epithelial tissues. By RT-PCR, we determined that these tissues express only the galanin-1 receptor (Gal1-R) subtype. To evaluate Gal1-R pharmacology and physiology, we studied T84 cells. Gal1-R expressed by these cells bound galanin rapidly (half time 1–2 min) and with high affinity (inhibitor constant 0.7 ± 0.2 nM). T84 cells were then studied in a modified Ussing chamber and alterations in I sc, a measure of all ion movement across the tissue, were determined. Maximal increases in I sc were observed in a concentration-dependent manner around 2 min after stimulation with peptide, with 1 μM galanin causing I sc to rise more than eightfold and return to baseline occurring within 10 min. The increase in galanin-induced I sc was shown by125I efflux studies to be due to Cl− secretion, which occurred independently of alterations in cAMP and phospholipase C. Rather, Cl− secretion is mediated via a Ca2+-dependent, pertussis toxin-sensitive mechanism. These data suggest that galanin released by enteric nerves may act as a secretagogue in the human colon by activating Gal1-R.

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Coconut milk regulates the allergic response in Balb/c mice sensitized with whey proteins

South Asian Journal of Experimental Biology ◽

10.38150/sajeb.8(1).p19-25 ◽

2018 ◽

Vol 8 (1) ◽

pp. 19-25

Author(s):

Yamina Benaissa ◽

Samia Addou ◽

Wafaa Dib ◽

Hadria Grar ◽

Omar Kheroua ◽

...

Keyword(s):

Clinical Symptoms ◽

Ex Vivo ◽

Short Circuit ◽

Ussing Chamber ◽

Whey Proteins ◽

Milk Proteins ◽

Short Circuit Current ◽

Coconut Milk ◽

Intestinal Damage

In this work the effect of coconut milk on mice sensitized to cow's milk proteins was evaluated. Balb/c mice treated orally for twenty eight days with coconut milk were sensitized intraperitoneally with β-lactoglobulin (β-Lg) or α-Lactoglobulin (α-Lac). We used Ussing chamber to analyze ex vivo electrical parameters characterizing the intestinal tissue of mice by measuring the variations of the short current circuit Isc (μA/cm2) as well as the epithelial conductance (G). Jejunal fragments of sensitized and treated mice were mounted in Ussing chamber and stimulated by the deposit of β-Lg or α-Lac. Symptom scores were determined after in vivo challenge to β-Lg or α-Lac. Intestinal damage was assessed by histological analysis. Coconut milk influ-enced the electrophysiological parameters by significantly decreasing the short-circuit current (Isc) (p < 0.001) and the epithelial conductance ((p< 0.01 and (p < 0.001, respectively). Moreover, in coconut milk-treated mice, no significant clinical symptoms were observed. Analysis of histological sections revealed that coconut milk reduced the microscopic lesions induced by β-Lg or α-Lac sensitization. We speculate that the administration of coconut milk could prevent the systemic and anaphylactic responses in sensitized mice.

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Vibrio choleraeACE stimulates Ca2+-dependent Cl−/HCO3−secretion in T84 cells in vitro

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.3.c567 ◽

2000 ◽

Vol 279 (3) ◽

pp. C567-C577 ◽

Cited By ~ 33

Author(s):

Michele Trucksis ◽

Timothy L. Conn ◽

Steven S. Wasserman ◽

Cynthia L. Sears

Keyword(s):

Short Circuit ◽

Short Circuit Current ◽

Fluid Secretion ◽

Apical Surface ◽

Rabbit Ileum ◽

T84 Cells ◽

Ussing Chambers ◽

Intracellular Ca ◽

Vitro Model

ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current ( Isc) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in Iscand potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl−secretory cell. ACE added to the apical surface alone stimulated a rapid increase in Iscand PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl−and HCO3−. ACE acted synergistically with the Ca2+-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca2+but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca2+as a second messenger and that this toxin stimulates a novel Ca2+-dependent synergy.

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Pet, an Autotransporter Enterotoxin from Enteroaggregative Escherichia coli

Infection and Immunity ◽

10.1128/iai.66.7.3155-3163.1998 ◽

1998 ◽

Vol 66 (7) ◽

pp. 3155-3163 ◽

Cited By ~ 163

Author(s):

Carlos Eslava ◽

Fernando Navarro-García ◽

John R. Czeczulin ◽

Ian R. Henderson ◽

Alejandro Cravioto ◽

...

Keyword(s):

Escherichia Coli ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Nucleotide Sequence Analysis ◽

E Coli ◽

Eaec Strain ◽

Heat Labile ◽

Tissue Resistance

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a β-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenicE. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects.

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