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Escherichia coli STb toxin binding to sulfatide and its inhibition by carragenan

Carina Gonçalves, Frédéric Berthiaume, Michaël Mourez, J. Daniel Dubreuil
DOI: http://dx.doi.org/10.1111/j.1574-6968.2008.01075.x 30-35 First published online: 1 April 2008


Escherichia coli heat-STb is an important cause of diarrhea in piglets. STb was shown to interact specifically with sulfatide (3′-sulfogalactosyl-ceramide) present on the surface of epithelial cells of piglet jejunum. Basic data are lacking on STb binding to sulfatide in solution and more precisely on the possible inhibition of this interaction. Using surface plasmon resonance technology, we compare binding of STb to sulfatide and other glycoshingolipids previously shown, with a multiplate-binding assay, to also interact to various degrees with the enterotoxin. In addition, inhibition of STb-sulfatide binding was studied using free galactose, galactose-sulfate residues and a polymer of sulfated galactans known as carragenan. We determined a dissociation constant of 2.4±0.61 nM for the STb-sulfatide interaction. These data indicated that STb was binding to sulfatide with greater affinity than previously determined using radiolabeled toxin. Much lower affinities were observed for lactoceramide and glucoceramide. The binding of STb to sulfatide was clearly inhibited by λ-carragenan but not by galactose, 4-SO4-galactose or 6-SO4-galactose. Inhibition of STb binding to its receptor was achieved using λ-carragenan at picomolar concentrations. Then, using IPEC-J2 cells in culture and flow cytometry, we showed that λ-carragenan was able to inhibit the permeabilization process associated with STb.

  • Escherichia coli
  • STb toxin
  • receptor
  • glycosphingolipid
  • toxin inhibition


Heat-stable toxin b (STb) is produced by enterotoxigenic Escherichia coli strains. This enterotoxin is responsible for diarrhea in piglets but the toxin has also been detected in E. coli isolates from human (Lortie et al., 1991; Handl & Flock, 1992). STb is a cytotonic toxin that was shown to bind to sulfatide, a glycosphingolipid, found on the epithelial cells of the pig jejunum (Rousset et al., 1998a). Binding was observed for glycosphingolipids containing terminal β-galactose with a significant preference for a galactose with a sulfate group in position 3. Additionally, STb was shown to bind significantly to glucoceramide (Beausoleil & Dubreuil, 2001). Using I125-radiolabelled STb and a microtiter plate-binding assay (MPBA), a Kd of 2–6±1.5 μM was determined for the STb-sulfatide interaction. Lipidic extracts of pig jejunum, when examined by high-performance thin-layer chromatography, confirmed the presence of sulfatides in this tissue. Hydroxylated sulfatides with fatty acid chains of 16, 22 and 24 carbons and a sulfatide with saturated fatty acid chains of 16 carbons were present in the extract. The major sulfatide found corresponded to a hydroxylated molecule harbouring a ceramide comprising 16 carbons (Beausoleil et al., 2002b). We recently showed, using brush border membrane vesicles from pig jejunum, that STb could permeabilize these vesicles by forming pores (Gonçalves et al., 2007). The toxin was also shown to allow the entry of Trypan blue into cells in culture of various origins (Beausoleil et al., 2002a). The dye uptake was dose-dependent but the cells were not killed.

Carragenans are sulfated galactans extracted from marine red algae (seaweed). These polysaccharides possess a linear structure alternating 3-linked β-d-galactopyranose and 4-linked α-d-galactopyranose units. These compounds represent highly charged anionic polymers with three sulfate groups per repeat unit of dissacharide, as one galactopyranose 2-sulfate and one galactopyranose 2,6-disulfate (Michel et al., 2006). Compared with other types of carragenans, λ-carragenan is a nongelling compound.

This study was conducted in order to quantitatively evaluate, using surface plasmon resonance technology (SPR), the interaction between STb and its receptor, as well as other glycosphingolipids on which terminal galactose and/or glucose are present. Free galactose, sulfated galactoses and a sulfated galactan (carragenan) were tested for their possible binding inhibition potential. Then, λ-carragenan was tested, using IPEC-J2 cells in culture, to determine its efficacy to inhibit the permeabilization process described for STb.

Materials and methods


Sulfatide, lactoceramide, glucoceramide, galactose, 4-SO4-galactose, 6-SO4-galactose, λ-carragenan, N-octyl-β-d-glucopyranoside and bovine serum albumin (BSA) were purchased from Sigma Chemical (Oakville, ON, Canada). Carboxyfluorescein diacetate and propidium iodine were purchased from Invitrogen Life Technologies (Burlington, ON, Canada).

Production and purification of STb

STb toxin was produced according to a previously described method (Gonçalves et al., 2007). The purified STb toxin was lyophilized and kept at −20 °C until use. The identity and purity of the toxin was verified by N-terminal sequence analysis using Edman degradation (Model 494 CLC Procise Sequencer, Applied Biosystems) as described by Ménard (2004). STb was quantified spectrophotometrically at 214 nm using aprotinin as the reference protein.

SPR experiments

Biosensor experiments were carried out on a BIAcore 3000 system (GE Healthcare, Baie D'Urfé, QC, Canada) using HPA sensor chips (Biacore AB, Uppsala, Sweden) using the protocol described by Mozsolits (2001).

Sulfatide, lactoceramide or glucoceramide in 10 mM NaHPO4, 10 mM Na2HPO4, pH 5.8 (PB) (100 μL, 0.1 mM) was applied to the chip surface at a flow rate of 2 μL min−1. To remove multilamellar structures from the lipid surface, NaOH (50 μL, 10 mM) was injected at a flow rate of 50 μL min−1. BSA was used (25 μL, 0.1 mg mL−1 in PB) to block free sites on the chip, preventing nonspecific binding. Excess BSA was removed from the chip surface by injection of NaCl (25 μL, 1 M) at a flow rate of 50 μL min−1. The glycosphingolipidic monolayer linked to the chip surface was then used as a cell surface membrane model to study toxin binding.

Various concentrations of the analyte (a volume of 30 μL STb in PB; concentrations ranging between 0 and 100 ng mL−1) were injected at 5 μL min−1 at 25 °C over the glycosphingolipid-covered sensor chip followed by PB for 10 min to allow dissociation. The alkanethiol surface on the HPA chip was cleaned by injection of 40 mM N-octyl-β-d-glucopyranoside (25 μL) at a flow rate of 5 μL min−1. Regeneration of the chip surface was done using 10 mM NaOH/10 mM dithiothreitol at 50 μL min−1. Sensorgrams were obtained by plotting the SPR angle against time. The STb–glycosphingolipid-binding events were analyzed from a series of sensorgrams collected for six different toxin concentrations. All experiments were performed at least three times.

Data analysis

Sensorgram data were analyzed during the binding and dissociation processes using biaevaluation 4.1 software (Biacore, GE Healthcare). Concerning the saturation curve, the average of the maximal response (RUmax) for each toxin concentration was plotted as a function of STb concentration. Using nonlinear regression (one binding site equation), the curve was fitted and residuals were concomitantly calculated. Analyses were performed using graphpad prism version 4.03 for windows (GraphPad software, San Diego, CA). For the carragenan inhibition experiments, averages of the RUmax for each carragenan concentration were expressed as function of carragenan concentration.

IPEC-J2 cell line

IPEC-J2 cells were derived from porcine jejunal epithelial cells and were kindly supplied by Dr A. Blikslager (North Carolina State University, Raleigh). The cells were grown and maintained in 50% Dulbecco's Modified Eagle Medium and 50% Nutrient Mixture F12 (Ham) (DMEM-F12) supplemented with 5% fetal bovine serum and penicillin/streptomycin (1%). Cells were maintained by serial passages in 75 cm2 flasks at 37 °C in a 5% CO2 atmosphere. Medium and supplements for cell culture were from Invitrogen Life Technologies.

Permeabilization assay

To conduct the permeabilization experiments, six-well plates were seeded at a density of 100 000 cells per well and grown for 2 days. Cells were washed with DMEM–100 mM HEPES (pH 7.4) and then incubated in the presence or absence of STb (10 μg) and/or λ-carragenan (5 and 10 μg mL−1) at 37 °C. Carragenan was added first and left for 10 min before STb was added. After 1 h of incubation, the treatment solution was substituted by complete DMEM-F12 medium and the cells were grown for 24 h at 37 °C. Cells were trypsinized using a 0.05% trypsin–EDTA solution (Invitrogen) collected at a concentration of 106 cells mL−1 and treated with carboxyfluorescein diacetate (CFDA) at 0.4 μg mL−1 and propidium iodine (PI) at 2 μg mL−1.

Samples were analyzed on a FACS Vantage SE (Becton Dickinson, Oakville, ON, Canada). The instrument is equipped with an argon laser for excitation of the fluorescent dye at 488 nm. CFDA fluorescent emission was detected in the FL1 channel (530±20 nm) while PI was detected in the FL3 channel (630±22 nm). Analysis was done on, at least, 10 000 cells. Data were collected with cell quest pro program (Becton Dickinson) and further analyzed with winmdi program (version 2.8; Joseph Trotter, Salk Institute for Biological Studies, La Jolla, CA). At least three independent experiments were conducted for each cell treatment. A Student's t-test was done on the final normalized data.

Results and discussion

SPR analyses of STb binding to glycosphingolipids

Sulfatide was shown by Rousset (1998a) to represent a functional receptor for STb toxin. Beausoleil & Dubreuil (2001), using a MPBA assay, have quantitatively shown that STb toxin has a high specificity for sulfatide. The binding was dose-dependent and saturable. In the present study, STb toxin binding to various glycosphingolipids was assessed, on a lipid monolayer, using SPR technology. As Rousset (1998b) suggested, using pig jejunum treated with specific glycosidases, that a glucose and/or galactose containing molecule could probably act as a receptor, glucoceramide and lactoceramide were included in this study for comparison purposes.

Immobilization of the various glycosphingolipids on the HPA chips were the following: sulfatide (2000 RU), glucoceramide (525 RU) and lactoceramide (1225 RU). Under these conditions, STb toxin (80 ng mL−1) binding to sulfatide and other selected glycosphingolipids indicated important differences in their affinities (Fig. 1). A marked lower response was noted for glucoceramide (RUmax of 10.5) and lactoceramide (RUmax of 53.5) compared with sulfatide (RUmax of 985), than expected from a previous study. In fact, using MPBA and pure glycosphingolipids, Beausoleil & Dubreuil (2001) had confirmed the observation of Rousset (1998a) and have shown that STb was binding to galactosyl ceramide (27% compared to sulfatide) and lactosylceramide (24%), two molecules sharing terminal β-galactose in their structure. Interestingly, binding to glucoceramide was more important than to glycosphingolipids with terminal galactose: 63% compared with sulfatide (Beausoleil & Dubreuil, 2001). This molecule containing a terminal glucose residue was included in the study to dispute or confirm previous data. As the data obtained using SPR did not correlate with the MPBA assay, it probably indicates that in the MPBA assay the immobilized glycosphingolipid molecules present the sugar(s) moieties differently than on a lipid monolayer as it is the case for SPR. Thus, data obtained using SPR technology are probably more indicative of the situation encountered in nature.

Figure 1

Binding of STb to sulfatide, lactoceramide and glucoceramide immobilized on HPA chips. Thirty microliters of STb toxin (80 ng mL−1) was injected at 5 μL min−1 over the glycosphingolipid-covered sensor chip. The experiment was repeated three times. A representative sensorgram is presented but the dissociation phase is not shown.

STb binding to sulfatide

Kinetic experiments with various concentrations of STb toxin (0–100 ng mL−1) were realized. A typical sensorgram of STb binding to sulfatide (RUmax plotted against time) in Fig. 2, shows that the binding is dependent of the amount of toxin added to the system. The binding is rapid and saturation is reached rapidly. At 5 and 10 ng mL−1, the curves are practically superposed over the 0 ng mL−1 (data not shown). Saturation of the reaction was observed at 40 ng mL−1. If we consider only one binding site per STb molecule, maximum binding at equilibrium was plotted as RUmax in function of the concentration of STb. From the saturation curve, data transformation indicated a Kd of 2.4±0.61 nM (Fig. 3). Using MPBA technology, a Kd of 2–6±1.5 μM was determined previously (Beausoleil & Dubreuil, 2001). This value is 1000 × higher than what is observed with SPR technology. Such discrepancy is difficult to explain but could be due to incomplete radiolabeling of STb with I125 and/or because the radiolabeling of STb toxin affected the binding to its receptor. Thus, addition of unlabeled or inactive STb toxin would have affected the Kd deduced from the previous data or the iodination of a small molecule like STb could result in binding interference with its receptor. The Kd determined for STb and sulfatide with SPR indicates a greater affinity between these molecules than previously determined. In fact, it compares well with other known interactions between a toxin and a receptor of glycolipidic nature. For example, the affinities of E. coli LT (heat-labile) toxin and Vibrio cholerae CT for GM1 are 0.57 and 0.73 nM, respectively (MacKenzie et al., 1997). Binding of E. coli STa toxin to guanylate cyclase type C, a proteic receptor, is also in the nanomolar order (nM) (Guarino et al., 1987). Thus, it appears that, at least for a small toxin like STb, determination of the affinity for a receptor is probably more rigorous when done on a lipid monolayer using SPR technology compared with monitoring of the binding of I125-labelled toxin to a receptor immobilized on a multiwell plate.

Figure 2

SPR analyses of binding of various STb concentrations to sulfatide immobilized on HPA chips. The figure is a representative sensorgram. Three independent experiments were done for each STb concentration tested. At 5 and 10 ng mL−1, the curves are practically superposed over the 0 ng mL−1 (data not shown). Saturation of binding was observed at a concentration of 40 ng mL−1.

Figure 3

Saturation curve of STb binding to sulfatide. The plotted data are averages from three independent experiments. Insert shows the calculated residuals for various STb concentrations taking into account one binding site for STb. The curve was obtained using nonlinear regression. The Kd was determined from the fitted curve.

Inhibitory effect of simple sugar, sulfated sugars and carragenan

In an attempt to observe the effect on STb binding to sulfatide immobilized on a HPA chips, potentially inhibitory molecules were preincubated with STb toxin. Galactose (up to 0.1 mg mL−1), free 4-SO4-galactose and 6-SO4-galactose, at the same concentration, did not result in any observable inhibition of STb binding to sulfatide (data not shown). Because, in a previous study, c. 50% of binding was inhibited at high concentrations (1 mg mL−1) of glucose sulfate (position 3) and of galactose sulfate (position 4 or 6) (Beausoleil & Dubreuil, 2001), polymeric molecules containing sulfated sugars but most specifically galactose were considered. As λ-carragenan is a polymer molecule containing sulfated galactose and does not form a gel compared with other types of carragenans, it was investigated with the intent of blocking the recognition process of STb with its receptor.

When tested, λ-carragenan was capable of effectively preventing binding of STb to sulfatide. This phenomenon was observed at picomolar concentrations (Fig. 4a). Binding inhibition was dose-dependent and a complete inhibition was observed for this molecule at 15 μg mL−1λ-carragenan (Fig. 4b).

Figure 4

Inhibitory assay of STb binding to sulfatide using various concentrations of λ-carragenan. STb was preincubated for 10 min with λ-carragenan before the experiment was carried out. (a) Sensorgram data averages obtained with 0, 1, 5, 15 and 30 μg mL−1 of λ-carragenan. (b) Inhibition curve for the corresponding amount of λ-carragenan added to the system. RUmax for each sensorgram is expressed as RU in function of the concentration of λ-carragenan. Total inhibition of the reaction is observed at 15 μg mL−1.

Cell permeabilization inhibition observed with λ-carragenan

Previous studies had shown that STb toxin could permeabilize pig jejunum brush border membrane vesicles (Gonçalves et al., 2007) and cells of various origins (Beausoleil et al., 2002a). In the case of animal cells, Trypan blue was used to observe the dose-response uptake of this vital dye, due to the action of STb. However, it was also determined that STb did not have a lethal effect on the cells. Here, to track cell permeabilization, we used PI, used to evaluate cell death. This dye, binding to double-stranded DNA, can only be taken up by cells if the cell membrane is affected in its integrity. CFDA, a lipophylic substrate, is readily taken up by cells. Intracellular esterases, in live cells, cleave the diacetate from the molecule and produce a fluorescent carbofluorescein molecule. In our experiments, a CFDA and PI-positive result indicated STb permeabilization, a process that allows PI to be internalized, without killing of the cells.

Using IPEC-J2 cells, a study was conducted to evaluate the capacity of λ-carragenan to prevent cell permeabilization. As seen in Fig. 5, addition of 5 or 10 μg mL−1 of λ-carragenan to cell treated with STb resulted in an appreciable inhibition of the permeabilization process observed for STb alone. In fact, inhibitions of 72% and 80% were observed, respectively, for 5 and 10 μg mL−1 of λ-carragenan. However, there was no significant inhibitory difference between the two quantities of λ-carragenan tested.

Figure 5

Effect of λ-carragenan on IPEC-J2 cells permeabilization due to STb. STb and λ-carragenan were added at 5 and 10 μg mL−1 to IPEC-J2 cells after preincubation of these molecules for 30 min. After treatment for 1 h, cells were transferred into fresh medium and grown for 24 h at 37°C. Then, PI and CFDA fluorophores were added. Fluorescence was evaluated in a FACS Vantage SE (Becton Dickinson) on, at least, 10 000 cells. Permeabilization percentages are shown with the SD. A Student's t-test was performed on the data (*P<0.05). No statistical difference was observed between the two quantities of λ-carragenan tested.

Thus, it seems that although λ-carragenan does not possess 3-SO4-galactose residue, known to be responsible for attachment of STb toxin to sulfatide (Beausoleil & Dubreuil, 2001), the multivalent galactose sulfate groups presents (2-sulfate and 2,6-disulfate galactose) could be responsible for the inhibition observed. Galactose 6-sulfate at 1 mg mL−1 had already been shown to inhibit binding of STb to sulfatide (Beausoleil & Dubreuil, 2001). Overall, multivalent compared with univalent compounds are known to inhibit the binding of various bacterial toxins (Rousset & Dubreuil, 2000; Bovin et al., 2004) and this could explain the inhibition observed for λ-carragenan using SPR technology and cells in culture.

Carragenans are used in the food industry, in important quantities, as a thickener but are also found in many products ranging from lubricants to infant feeding formulas. In this study, we demonstrated that added in picomolar amount it can completely block the recognition process of STb with its receptor. Carragenan has been shown to block viral infectivity of herpes simplex virus and HIV (Konlee, 1998; Carlucci et al., 2004; Talarico et al., 2004). Sulfated polysaccharides are thought to block viral infection by chemical mimicking of heparin sulfate, thereby competing against initial virion attachment to cell surface. Recently, carragenan was also shown to be a potent inhibitor of papillomavirus infection (Buck et al., 2006).

The present study cannot explain at the molecular level the discrepancies observed for STb binding to various glycosphingolipids using two distinct technologies. However, it clearly revealed a strong affinity of STb for sulfatide that is comparable to other bacterial toxins sharing a receptor of glycolipidic nature. On the other hand, the inhibition observed with λ-carragenan cannot be explained by the presence of 3-SO4-galactose residues, as found on sulfatide but perhaps by two to six SO4-galactose residues. Nevertheless, this study indicated that SPR technology represents an interesting way to screen rapidly for inhibitory molecules. In fact, IPEC-J2 cells when used as a cellular model confirmed the inhibitory potential of this compound on STb permeabilization process. The identified inhibitors could then be tested in the more fastidious animal models to ascertain their efficacy.

For now, no commercial vaccine is available for STb toxin. It is thus tempting to suggest that λ-carragenan could be used as a prophylactic agent to control STb diarrhea in pigs. For example, λ-carragenan could be added to the feed of animals, in relatively small amounts, during weaning, the period for which the STb intoxication is most noted, and for some weeks thereafter (Fairbrother et al., 2005). This polysaccharide, readily available, will have to be tested in animals before a conclusion can be drawn as to its usefulness in preventing STb diarrhea in the field.


This work was supported by grants to J.D. Dubreuil (#139070-01) from the Natural Sciences and Engineering Research Council of Canada and by the Fonds Québecois de la Recherche sur la Nature et les Technologies (#2007-PR-114426). The authors thank P. Vincent from the Centre de Recherche en Reproduction Animale de l'Université de Montréal for the flow cytometry analyses.


  • Editor: Mark Schembri


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