Stable Enterotoxin Research Articles

Lack of Restorationin Vivoby K+-Channel Modulators of Jejunal Fluid Absorption after Heat StableEscherichia coliEnterotoxin (STa) Challenge

Enhanced potassium ion permeability at the enterocyte basolateral membrane is assumed to facilitate sustained chloride ion and fluid secretion into the intestinal lumen during episodes of secretory diarrhoeal disease. To examine this concept in vivo, two potassium ion channel blockers and a channel opener were coperfused with E. coli heat stable STa enterotoxin to determine whether such compounds improved or worsened the inhibited fluid absorption. In the STa (80 ng/mL) challenged jejunal loop, the fluid absorption rate of 28.6 ± 5.8 (14) μL/cm/hr was significantly below (P < .001) the normal rate of 98.8 ± 6.2 (17) μL/cm/hr. Intraluminal (300 uM) glibenclamide added to STa perfused loops failed to improve the inhibited fluid absorption rate, which was 7.4 ± 3.2 (6) μL/cm/hr on coperfusion with STa. Similarly, on coperfusion with 30 uM clotrimazole, the fluid absorption rate with STa present remained inhibited at 11.4 ± 7.0 (4) μL/cm/hr. On coperfusion with intraluminal 1 uM cromakalim, STa reduced fluid absorption significantly (P < .02) to 24.7 ± 8.0 (10) μL/cm/hr, no different from STa challenge in the absence of cromakalim. Infusion i.v. with these agents also failed to restore fluid absorption after STa challenge. These observations do not support the proposed potassium ion permeability event as a necessary corollary of enterotoxin-mediated secretion. This makes it unlikely that modulators of such permeability prevent enterocyte secretion in diarrhoeal disease.

Loss of PDZ-adaptor protein NHERF2 affects membrane localization and cGMP- and [Ca2+]- but not cAMP-dependent regulation of Na+/H+exchanger 3 in murine intestine

Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport.

Multiplex Polymerase Chain Reaction Assay for Detection of Nonserotypable Shiga Toxin–ProducingEscherichia coliStrains of Serogroup O147

Nonserotypable Shiga toxin-producing Escherichia coli (STEC) strains (n = 72) from the collection of the E. coli Reference Center were O typed by a polymerase chain reaction (PCR)-restriction fragment length polymorphism method, and those that exhibited similar profiles (n = 17) were chosen for the study. These isolates, derived from pigs, carried genes for Shiga toxin variant 2e (100%), heat stable enterotoxins STa and STb (70% and 76%, respectively), and F107 (F18) fimbriae (82%). DNA sequencing and analysis of the O-antigen gene cluster of one of the nonserotypable strains exhibited 100% homology with O-antigen cluster of E. coli O147 although the lipopolysaccharide profiles differed significantly between the nonserotypable strains and O147 reference control strain normally used for antibody production. Scanning electron micrographs of the nonserotypable strains showed altered morphology as compared to E. coli O147. Therefore, nonserotypable strains may share 100% homology with O-antigen gene cluster of a certain serogroup but may not express that specific O-antigen. Highly specific multiplex PCR for detecting the nonserotypable STEC of serogroup O147 was developed targeting virulence genes stx2, stb, and fedA encoding for F107 fimbriae, and wzx and wzy of the O147 O-antigen cluster genes. The multiplex PCR method will allow identifying potentially pathogenic subgroup of STEC important in porcine and human health.

Recovery of Salmonella and Escherichia coli from commercial egg shells and effect of translucency on bacterial penetration in eggs

This experiment was conducted to study the prevalence of Salmonella and Escherichia coli ( E. coli). from the surface of egg shells, egg shell membranes or pores, and internal contents from unwashed eggs collected from commercial caged layer farms in Australia. Egg shell swabs, shell crush and egg internal contents (yolk and albumen) of an individual egg were processed for bacteriological examination. Salmonella spp. were not detected from any of the egg shell surfaces, egg shell crush or egg internal contents. Thirty five E. coli isolates were isolated from the egg shell surface. Ten E. coli strains were also isolated from shell crush. However, the internal contents of eggs appeared to be sterile. Polymerase chain reaction was performed on forty-five E. coli isolates using primers for heat stable enterotoxin genes A and B ( STa and STb) and also for colicin V gene ( cvaC). STa gene was detected in four E. coli isolates isolated from egg shell surfaces. All the E. coli isolates were negative for STb and cvaC genes. These data provide useful information regarding the prevalence of virulent E. coli and Salmonella spp. on and in unwashed eggs collected from layer farms. These data also suggest that unwashed eggs collected from caged layer farms are unlikely to be sources of Salmonella outbreaks. Egg shell translucency could be due to changes in the mammillary layer and mamillary cores during the early phases of egg shell formation and has the potential to increase the incidence of microcracks in egg shells, and hence, may be responsible for bacterial penetration. There was a significant correlation between egg shell translucency and egg shell penetration by Salmonella Infantis and E coli. Both strains of bacteria were able to penetrate the translucent egg shells even at very low doses. The penetration, however, was hindered in both translucent and non translucent eggs at 4 °C, as compared with room temperature which highlights the importance of storage of eggs at refrigerated temperatures.

Evaluation for the Effect of Heat Stable Enterotoxin (a) Produced by Enterotoxigenic Escherichia coli on Different Cancer Cells In Vitro

This study was conducted for evaluating the cytotoxic effect of heat stable enterotoxin a (STa) produced by enterotoxigenic Escherichia coli on the proliferation of primary cancer cell cultures, obtained from tumor samples that were collected from (13) cancer patients and as follows: (five colon cancer patients, two bladder cancer patients, two breast cancer patients, two stomach cancer patients and two lung cancer patients), and on normal cell line (rat embryonic fibroblast / REF) (in vitro) with the use of different concentrations starting from (1) mg/ml and ending with (0.0002) mg/ml by making two fold serial dilutions by using the 96- well microtiter plate, and in comparison with negative (PBS) and positive (MMC, at concentration of 10 µg/ml) controls . Results showed that, after (24) hours of exposure to STa, the growth of all primary cancer cell cultures obtained from colon cancer patients was inhibited by STa treatment and this inhibition was concentration dependent. Also it was shown that the cytotoxic effect of the high concentration of STa was close to that seen after MMC treatment. While no differences were seen in the growth of all primary cancer cell cultures that were obtained from the other cancer patients, which mean that STa treatment neither inhibit nor enhanced their growth. At the same time STa did not show or has any cytotoxic effect on the normal cell line (REF).

Diagnosis and Treatment of Acute or Persistent Diarrhea

Studies of microbial pathogens and the toxins they produce are important for determining the mechanisms by which they cause disease and spread throughout a population. Some bacteria produce secretory enterotoxins (such as cholera toxin or the heat-labile or stable enterotoxins produced by Escherichia coli) that invade cells directly. Others invade cells or produce cytotoxins (such as those produced by Shigella, enteroinvasive E coli, or Clostridium difficile) that damage cells or trigger host responses that cause small or large bowel diseases (such as enteroaggregative or enteropathogenic E coli or Salmonella). Viruses (such as noroviruses and rotaviruses) and protozoa (such as Cryptosporidium, Giardia, or Entamoeba histolytica) disrupt cell functions and cause short- or long-term disease. Much epidemiologic data about these pathogens have been collected from community- and hospital-acquired settings, as well as from patients with traveler's or persistent diarrhea. These studies have led to practical approaches for prevention, diagnosis, and treatment.

Is an increase in duodenal bicarbonate concentration after STa really enhanced bicarbonate ion secretion?

I was interested in the paper of Sellers et al. reporting on the ability of heat stable enterotoxin from E. coli allegedly to stimulate duodenal bicarbonate anion secretion (1). However, I believe the main conclusion is not supported by the data because the chosen physiological techniques do not prove that enhanced bicarbonate anion secretion occurred after the duodenum was exposed to the enterotoxin. Enhanced appearance of bicarbonate anion might arise by enhanced bicarbonate ion secretion but the more likely cause is cessation of hydrogen ion secretion, a known effect of STa that is likely to be the only effect of that enterotoxin (2, 3). The chosen physiological technique was in vivo perfusion followed by measurement of bicarbonate appearance in the lumen of the perfused anaesthetised mouse. Samples were taken and the bicarbonate ion concentration assayed indirectly by titration. The technique involves adding an amount of hydrochloric acid to the sample that will consume the bicarbonate that was present. Back titration with sodium hydroxide to the original sample pH, or even beyond it, estimates the amount of titratable buffer that was initially present but that was consumed by the hydrochloric acid by conversion into carbon dioxide. Any difference between the amount of HCl added and NaOH needed for the back titration allows calculation of the total amount of bicarbonate that was present. In order for this to be assayed directly and with even greater accuracy, any bicarbonate in the buffer that was converted into carbon dioxide could be measured as carbon dioxide. This was measured by the authors and there is no doubt that bicarbonate was converted into carbon dioxide, as sensed by the CO2 gas-sensing electrode. This means that the reader can be confident that bicarbonate in solution was accurately measured. However, the claim that titratable bicarbonate secretion was true bicarbonate secretion and not altered hydrogen ion secretion (Materials and Methods, “Measurement of HCO3 secretion in vivo,” paragraph 3, line 13) is a non-sequitur. The conversion of bicarbonate into carbon dioxide and the establishing that carbon dioxide was indeed formed does not by itself rule out changes in luminal bicarbonate concentration arising because of reduced hydrogen ion secretion. The authors perfused isotonic saline through the duodenum and over time, because of the concentration gradient between the lumen and the interstitial fluid or perhaps because of putative cellular bicarbonate secretion, the bicarbonate concentration increased from zero to the values recorded. In other experiments, the inclusion of STa enterotoxin in the luminal perfusate caused the bicarbonate concentration to increase still further. However, it is known that Na /H exchange occurs in the duodenum, where NHE:3 is present. Bicarbonate that enters the duodenum might be expected to react in part with secreted hydrogen ion so that the eventual bicarbonate concentration that is achieved is the result of appearance of bicarbonate anion and its removal by chemical reaction with secreted hydrogen ion. If the amount of secreted hydrogen ion is reduced by STa, a likely occurrence because it does stop luminal acidification, then it is to be expected that the bicarbonate ion concentration will be higher than normal in the perfused duodenum, as the authors probably showed. This is likely to be the explanation for the higher rates of bicarbonate appearance, given the known effects of STa. It is still possible that bicarbonate secretion is enhanced after STa exposure through the mechanism that the authors invoke but this is not something they have shown in their paper. I believe they have shown that STa reduces hydrogen ion secretion into the lumen and this is manifested by higher concentrations of bicarbonate ion. What they have not shown is that STa stimulates duodenal bicarbonate secretion, certainly not by simply verifying that bicarbonate is convertible into carbon dioxide, as their methods section states. This is a conclusion that goes beyond what the data can reasonably support. A further pharmacological argument in favour of hydrogen ion secretion not being involved is that some of experiments were done in the presence of amiloride. With amiloride inhibiting any hydrogen ion secretion, it might safely be concluded that any subsequent action of guanylin and STa might be restricted to enhanced secretion of bicarbonate ion. It is the case that amiloride might inhibit duodenal NHE:3, but evidence suggests that amiloride and its derivatives are only effective in moderate to low sodium ion containing perfusates (4). Amiloride at 1 mM concentration fails to affect the mucosal surface pH in rat proximal jejunum (5) and 100 uM ethyl-iso-propyl-amiloride (EIPA) fails to inhibit water absorption, whilst STa does inhibit water absorption in high sodium ion containing perfusates (6). Only when the sodium ion concentration is low can

Structural Characterisation of the E. coli Heat Stable Enterotoxin STh

E. coli heat stable enterotoxin STa is an agonist of the membrane guanylate cyclase C whose endogenous ligands are the peptide hormones guanylin and uroguanylin. Whereas these peptides contain only two disulfide bonds, STa is stabilized by one additional disulfide bridge. We chemically synthesized the enterotoxin STh that originates from the E. coli strain found in humans, and we determined its structure and its dynamics by nuclear magnetic resonance spec- troscopy and molecular dynamics calculations. Chemical synthesis clearly proved successful and resulted in the formation of the native disulfide bonds. The endogenous ligands guanylin and uroguanylin show the same general structural features and dynamics properties as the enterotoxin.

Enterocyte chloride and water secretion into the small intestine after enterotoxin challenge: Unifying hypothesis or intellectual dead end?

Many forms of diarrhoeal disease, particularly so called "secretory" diarrhoeal disease are thought to arise by the active secretion of chloride ion from the enterocytes, creating an osmotic gradient for fluid movement into the small intestinal lumen. This model implies that normally occurring intestinal secretion is catastrophically enhanced by bacterial enterotoxins. This review advocates that neither normal nor abnormal intestinal secretion from the enterocytes occurs and that no competent proof for chloride secretion exists. Prior to 1970, the physiological evidence failed to support the concept of the formation of intestinal juice as a normal intestinal event. The concept was later revived to explain the high rate of fluid entry into the lumen after exposure to cholera toxin. Much evidence has been advanced for the chloride secretion hypothesis, the dominant secretory paradigm after 1974, but is the evidence sufficiently compelling for it to be regarded as proving the chloride secretory model? The evidence falls into four categories and a fifth conjectural argument that proposes that an abnormal chloride ion channel in cystic fibrotic sufferers confers a natural selective advantage by preventing diarrhoeal disease. Secretion is putatively demonstrated by 1) showing that mass transfer of fluid is into the lumen (secretion) and not merely a failure to transport out of the lumen (failed absorption). Support is offered by 2) chloride ion flux measurements in vitro in Ussing chambers and by 3) short-circuit current measurements that are consistent with and purport to show chloride ion movement into the lumen. In addition, 4) pharmacological agents are identified that affect short-circuit current and these are assumed to be anti-secretory, consistent with the biochemical mechanism for secretion, confirmed wherever possible by mouse knock-out models. Finally, the proxy methods used to study water movement such as elevated short-circuit current measurements show these to be absent in cystic fibrotic patients. The enterocyte secretion hypothesis is challenged here on the basis of an examination of the methods used to show secretion, particularly after exposing the small intestine to heat stable enterotoxin (STa) from E. coli. STa is thought to be secretory because fluid entry into the lumen is claimed, enhanced isotopic flux of chloride ion towards the lumen occurs, an increase in short-circuit current is found, preventable by various drugs that are deemed likely to be anti-secretory and also because the short-circuit current changes after STa are not seen in cystic fibrotic patients. Using volume recovery in vivo, STa is found not to be secretory but only anti-absorptive. Hence, other techniques used to show secretion are not fit for that purpose. If STa is identified as secretory and yet no secretion occurs, how reliable is the evidence for other toxins being secretory when these methods are used? This review concludes that chloride ion secretion is unproven. A review of the literature indicates that secretion occurs not because epithelial cells actively pump water but by interdiction of fluid absorption, increased conductivity through tight junctions and an increased hydrostatic driving force through elevated capillary pressure. The exclusive focus on chloride secretion may explain the failure to develop antisecretory drugs over the last three decades.

Lack of evidence in vivo for a remote effect of Escherichia coli heat stable enterotoxin on jejunal fluid absorption

On contact with the mucosa, heat stable (STa) enterotoxin from Escherichia coli reduces fluid absorption in vivo in the perfused jejunum of the anaesthetized rat. The question of whether it also has a vagally mediated remote action on jejunal absorption, when instilled into the ileum, was re-examined, given contradictory findings in the literature. A standard perfused loop preparation was used to measure luminal uptake of fluid in vivo by means of volume recovery. STa in the ileum was found to have no effect on jejunal absorption, regardless of cervical or sub-diaphragmatic vagotomy and also regardless of the nature of the perfusate anion. The batches of toxin were shown in parallel experiments to reduce fluid absorption directly in the jejunum and also in the ileum. Similarly, vagal nerves prior to section had demonstrable in vivo physiological function. There was therefore no evidence for an indirect, vagally mediated ileal effect of STa on proximal fluid absorption.

Fenofibrate inhibits intestinal Cl− secretion by blocking basolateral KCNQ1 K+ channels

Fibrates are peroxisome proliferator-activated receptor-alpha (PPARalpha) ligands in widespread clinical use to lower plasma triglyceride levels. We investigated the effect of fenofibrate and clofibrate on ion transport in mouse intestine and in human T84 colonic adenocarcinoma cells through the use of short-circuit current (I(sc)) and ion flux analysis. In mice, oral administration of fenofibrate produced a persistent inhibition of cAMP-stimulated electrogenic Cl(-) secretion by isolated jejunum and colon without affecting electroneutral fluxes of (22)Na(+) or (86)Rb(+) (K(+)) across unstimulated colonic mucosa. When applied acutely to isolated mouse intestinal mucosa, 100 microM fenofibrate inhibited cAMP-stimulated I(sc) within 5 min. In T84 cells, fenofibrate rapidly inhibited approximately 80% the Cl(-) secretory responses to forskolin (cAMP) and to heat stable enterotoxin STa (cGMP) without affecting the response to carbachol (Ca(2+)). Both fenofibrate and clofibrate inhibited cAMP-stimulated I(sc) with an IC(50) approximately 1 muM, whereas other PPARalpha activators (gemfibrozil and Wy-14,643) were without effect. Membrane permeabilization experiments on T84 cells indicated that fenofibrate inhibits basolateral cAMP-stimulated K(+) channels (putatively KCNQ1/KCNE3) without affecting Ca(2+)-stimulated K(+) channel activity, whereas clofibrate inhibits both K(+) pathways. Fenofibrate had no effect on apical cAMP-stimulated Cl(-) channel activity. Patch-clamp analysis of HEK-293T cells confirmed that 100 microM fenofibrate rapidly inhibits K(+) currents associated with ectopic expression of human KCNQ1 with or without the KCNE3 beta-subunit. We conclude that fenofibrate inhibits intestinal cAMP-stimulated Cl(-) secretion through a nongenomic mechanism that involves a selective inhibition of basolateral KCNQ1/KCNE3 channel complexes. Our findings raise the prospect of fenofibrate as a safe and effective antidiarrheal agent.

Downregulation of human colon carcinoma cell (COLO‐205) proliferation through PKG‐MAP kinase mediated signaling cascade by E. coli heat stable enterotoxin (STa), a potent anti‐angiogenic and anti‐metastatic molecule

It was reported earlier that Escherichia coli heat stable enterotoxin (STa), a major causative agent of secretory diarrhea, can also inhibit the proliferation of colon carcinoma cells with the involvement of cGMP mediated calcium influx. In the present study it is shown that E. coli STa inhibits cell proliferation in the colonic carcinoma cell line COLO-205 by the PKG-ERK44/42 mediated signaling pathway. This enterotoxin negatively regulates cell proliferation by downregulating the activity of ERK44/42(MAPK) and subsequently the activity of a transcription regulatory protein cMyc. The antiproliferative effect of STa was reversed by LY83583, a guanylate cyclase (GC) inhibitor and KT5823, a PKG inhibitor. Thus suggesting the involvement of cGMP dependent protein kinase (PKG) in the downregulation of ERK44/42 and subsequent inactivation of cMyc activity. Moreover, it has been shown that a specific ERK44/42 inhibitor, PD98059, also inhibits cMyc activation and cell proliferation, which further confirms the involvement of ERK44/42 in the activation of cMyc. It is also shown that E. coli STa significantly inhibits the vascular endothelial growth factor (VEGF, a potent angiogenic factor) expression in COLO-205 cells and also downregulates vascular cell adhesion molecule-1 (VCAM-1, a potent metastatic factor) expression on the COLO-205 cell surface. So it is reported for the first time that E. coli STa inhibits the proliferation of the colonic carcinoma cell line COLO-205 by the PKG-ERK44/42 mediated pathway and it may have a role against the development of colon carcinoma.

Lack of evidence in vivo for nitrergic inhibition by Escherichia coli (STa) enterotoxin of fluid absorption from rat proximal jejunum

Fluid absorption from the proximal jejunum of the anaesthetised rat was measured in vivo by fluid recovery. As expected, heat stable (STa) enterotoxin from E. coli reduced fluid absorption. Neither intraperitoneal L-NAME, thought to inhibit a putative neurally mediated action of STa, nor similar doses of D-NAME, ameliorated the inhibitory effect on jejunal fluid absorption of STa. Luminally perfused 10 mM sodium nitroprusside (SNP) had no effect on fluid absorption when expressed per gram dry weight per hour but reduced fluid absorption when expressed per cm length per hour. Similarly, 80 but not 40 mg/Kg of L-NAME reduced fluid absorption when expressed per cm length per hour, while the same dose of D-NAME did not. L-NAME and SNP significantly increased the wet weight to dry weight and the length to dry weight ratio of perfused loops. We conjecture that smooth muscle relaxation caused by these compounds increases interstitial fluid volumes that can be misconstrued as changes in absorption when this is expressed per cm length or per tissue wet weight. When fluid absorption is expressed per gram dry weight of tissue, there is no evidence for a role of nitric oxide in normal or STa inhibited fluid absorption.

Defective CFTR Apical Endocytosis and Enterocyte Brush Border in Myosin VI‐Deficient Mice

In polarized epithelial cells such as those that line the inner ear, kidney and gut, myosin VI has been localized to the intermicrovillar domains where it is proposed to regulate clathrin-dependent endocytosis; however, a direct role for myosin VI in apical endocytosis has not been shown. We examined the apical membrane distribution and endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in myosin VI-deficient Snell's Waltzer Myo6((sv/sv)) mice. Confocal microscopy and cell-surface biotinylation confirmed that surface levels of CFTR in the intestine of Myo6((sv/sv)) mice were markedly higher, and CFTR internalization from the apical plasma membrane was reduced compared with heterozygous controls. Consistent with a defect in CFTR endocytosis and accumulation at the cell surface, exaggerated CFTR-mediated fluid secretion was observed in Myo6((sv/sv)) mice following treatment of isolated jejunum with the cyclic GMP-activated heat stable enterotoxin. These data establish that myosin VI modulates apical endocytosis and may be an important physiological modulator of CFTR function and CFTR-associated secretory diarrhea in the gut.

Interaction ofEscherichia coliheat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn kids

The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.

CLOTH‐BASED HYBRIDIZATION ARRAY SYSTEM FOR DETECTION OF TOXIN GENES ASSOCIATED WITH MAJOR FOODBORNE PATHOGENIC BACTERIA

ABSTRACT A simple cloth‐based hybridization array system (CHAS) was developed for the identification of toxin genes associated with major foodborne pathogenic bacteria, including toxigenic Escherichia coli, Vibrio cholerae and Salmonella spp. Bacterial isolates were subjected to a multiplex polymerase chain reaction incorporating digoxigenin (DIG)–deoxyuridine triphosphate and primers targeting a variety of toxin genes (verotoxin, Salmonella enterotoxin, cholera toxin and heat‐labile/stable enterotoxin), followed by hybridization of the amplicons with an array of probes immobilized on polyester cloth and subsequent immunoenzymatic assay of the bound DIG label. This system provided sensitive and specific detection of the different target toxin gene markers in a variety of bacterial isolates, exhibiting the expected patterns of reactivity with a panel of bacteria having defined toxigenicity profiles. The CHAS is a cost‐effective tool facilitating the determination of the potential toxigenic profile of bacteria in the food microbiology laboratory, thus contributing valuable information to the risk‐assessment process in the microbiological analysis of foods.

Escherichia coli Heat Stable (STa) Enterotoxin and the Upper Small Intestine: Lack of Evidence in Vivo for Net Fluid Secretion

Heat stable (STa) enterotoxin from E. coli reduced fluid absorption in vivo in the perfused jejunum of the anaesthetized rat in Krebs-phosphate buffer containing lactate and glucose (nutrient buffer), in glucose saline and in glucose free saline. Bicarbonate ion enhanced fluid absorption of 98 +/- 7 (6) microl/cm/h was very significantly (P < 0.0001) reduced by STa to 19 +/- 4 (6) microl/cm/h, but net secretion was not found. When impermeant MES substituted for bicarbonate ion, net fluid absorption of 29 +/- 3 (6) microl/cm/h was less (P < 0.01) than the values for phosphate buffer and bicarbonate buffer. With STa in MES buffer, fluid absorption of 3 +/- 2 (6) microl/cm/h was less than (P < 0.001) that in the absence of STa and not significantly different from zero net fluid absorption. E. coli STa did not cause net fluid secretion in vivo under any of the above circumstances. Neither bumetanide nor NPPB when co-perfused with STa restored the rate of fluid absorption. In experiments with zero sodium ion-containing perfusates, STa further reduced fluid absorption modestly by 20 microl/cm/h. Perfusion of ethyl-isopropyl-amiloride (EIPA) with STa in zero sodium ion buffers prevented the small increment in fluid entry into the lumen caused by STa, indicating that the STa effect was attributable to residual sodium ion and fluid uptake that zero sodium-ion perfusates did not eradicate. These experiments, using a technique that directly measures mass transport of fluid into and out of the in vivo proximal jejunum, do not support the concept that E. coli STa acts by stimulating a secretory response.

STa and cGMP stimulate CFTR translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase G

The cystic fibrosis transmembrane conductance regulator (CFTR) is critical to cAMP- and cGMP-activated intestinal anion secretion and the pathogenesis of secretory diarrhea. Enterotoxins released by Vibrio cholerae (cholera toxin) and Escherichia coli (heat stable enterotoxin, or STa) activate intracellular cAMP and cGMP and signal CFTR on the apical plasma membrane of small intestinal enterocytes to elicit chloride and fluid secretion. cAMP activates PKA, whereas cGMP signals a cGMP-dependent protein kinase (cGKII) to phosphorylate CFTR in the intestine. In the jejunum, cAMP also regulates CFTR and fluid secretion by insertion of CFTR from subapical vesicles to the surface of enterocytes. It is unknown whether cGMP signaling or phosphorylation regulates the insertion of CFTR associated vesicles from the cytoplasm to the surface of enterocytes. We used STa, cell-permeant cGMP, and cAMP agonists in conjunction with PKG and PKA inhibitors, respectively, in rat jejunum to examine whether 1) cGMP and cGK II regulate the translocation of CFTR to the apical membrane and its relevance to fluid secretion, and 2) PKA regulates cAMP-dependent translocation of CFTR because this intestinal segment is a primary target for toxigenic diarrhea. STa and cGMP induced a greater than fourfold increase in surface CFTR in enterocytes in association with fluid secretion that was inhibited by PKG inhibitors. cAMP agonists induced a translocation of CFTR to the cell surface of enterocytes that was prevented by PKA inhibitors. We conclude that cAMP and cGMP-dependent phosphorylation regulates fluid secretion and CFTR trafficking to the surface of enterocytes in rat jejunum.

Effects of tetrodotoxin and ion replacements on the short-circuit current induced by Escherichia coli enterotoxin STa across the colon of the gerbil ( Gerbillu cheesmani) in different dietary states

The effects of mucosally added Escherichia coli heat stable enterotoxin (STa, 30 ng mL −1) on the basal short-circuit current (Isc in μA cm −2) across stripped and unstripped sheets of proximal, mid and distal colon were investigated. Samples were taken from fed, starved (4 days, water ad lib) and undernourished (50% of control food intake for 21 days) gerbils ( Gerbillus cheesmani). The effect of neurotoxin tetrodotoxin (TTX 10 μM) and the effect of replacing chloride by gluconate or the effect of removing bicarbonate from bathing buffer on the maximum increase in Isc induced by E. coli heat stable enterotoxin STa were also investigated. The results showed that there is a segmental difference both in the basal and STa-induced Isc. Also, STa is more effective in the proximal than distal colon in the three feeding conditions. Undernourishment raised the STa-induced Isc in the three regions of the colon. In fed and starved gerbils part of STa-induced Isc in the proximal colon was chloride-dependent, while the other was bicarbonate-dependent; in the mid colon, the STa-induced Isc was bicarbonate-dependent only. In the three regions of the colon taken from undernourished gerbil, the STa-induced Isc was both chloride-and bicarbonate-dependent. The increase in STa-induced Isc as a results of undernourishment in proximal and mid colon was chloride-dependent, while in the distal colon, it was both chloride- and bicarbonate-dependent.

Involvement of phospholipase C in Yersinia enterocolitica heat stable enterotoxin (Y-STa) mediated rise in intracellular calcium level in rat intestinal epithelial cells

In response to Yersinia enterocolitica heat stable enterotoxin (Y-STa) intracellular calcium level was increased with a prolong sustained phase in presence of calcium chloride in extracellular environment in rat intestinal epithelial cells. Chelation of extracellular calcium with EGTA (extracellular calcium chelator) and suspension of cells in calcium free buffer demonstrated a rapid but transient rise in calcium level, which suggested that Y-STa induced rise in intracellular calcium concentration was the combination of both intracellular calcium store depletion and calcium influx from extracellular environment. Moreover, in response to Y-STa phosphoinositide specific phospholipase C activity and inositol tri phosphate (IP 3) level was increased and U73122, a phospholipase C inhibitor could completely inhibit Y-STa induced calcium rise. However, treatment of rat enterocytes with dantrolene IP 3, a mediated calcium release inhibitor from intracellular store resulted partial inhibition of Y-STa induced rise in intracellular calcium level. Similar observation was noted with IP 3 receptor antagonist 2ABP (2-amino-ethoxydiphenylborate). These results suggested that beside phospholipase C IP 3 pathway, phospholipase C might have an independent role in Y-STa induced calcium influx. Rise in phospholipase Cγ isoform activity in response to Y-STa suggested that γ isoform of phospholipase C might have a role in Y-STa mediated rise in intracellular calcium level.