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The Escherichia coli Enterotoxin STb Permeabilizes Piglet Jejunal Brush Border Membrane Vesicles

Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec J2S 7C6,¹ Groupe d'Étude des Protéines Membranaires, Université de Montréal, Montreal, Quebec H3C 3J7, Canada²

Received 17 November 2006/ Returned for modification 2 January 2007/ Accepted 12 February 2007

	ABSTRACT

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The membrane-permeabilizing ability of the Escherichia coli enterotoxin STb was evaluated using brush border membrane vesicles isolated from piglet jejunum and a membrane-potential-sensitive fluorescent probe, 3,3'-dipropylthiadicarbocyanine iodide. A strong membrane potential was generated by the efflux of K⁺ ions from the vesicles in the presence of the potassium ionophore valinomycin. Under these conditions, preincubation of the vesicles with STb efficiently depolarized the membrane in a dose-dependent and saturable manner. This activity was independent of pH, however, at least between pH 5.5 and 8.0. On the other hand, in the absence of valinomycin, STb had no significant influence on the measured fluorescence levels, indicating that it was unable to modify the ionic selectivity of the intact membrane. In agreement with the fact that the integrity of the disulfide bridges of STb is known to be essential for its biological activity, a reduced and alkylated form of the toxin was unable to depolarize the membrane in the presence of valinomycin. Furthermore, two previously described poorly active STb mutants, M42S and K22A-K23A, showed no membrane-permeabilizing capacity. These results demonstrate for the first time that STb can permeabilize its target membrane and suggest that it does so by forming nonspecific pores.

	INTRODUCTION

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Enterotoxigenic Escherichia coli strains cause severe diarrhea in humans and animals. In large-scale farms, this disease brings about significant losses in pig herds, mainly in newborn and recently weaned animals (29). Diarrhea depends on the production of a variety of toxins belonging to two different groups, the heat-labile toxins (LTI and LTII) and the heat-stable toxins (STa and STb). The heat-stable enterotoxins share no homology and appear to differ in their mechanisms of action (11, 12). STb is synthesized as a 71-amino-acid precursor that undergoes cleavage of its signal sequence of 23 residues (24). A nuclear magnetic resonance study revealed that STb is composed of two antiparallel -helices connected by a glycine-rich loop (39). Its tertiary structure is stabilized by two disulfide bridges (Cys10-Cys48 and Cys21-Cys36) (9), and both bridges are necessary for secretion and toxicity (2, 9, 30).

Until now, the interaction of STb with the intestinal epithelium has not been studied in detail. An acidic glycosphingolipid, sulfatide [Gal(3-SO₄)ß1Cer], has nevertheless been identified as a functional receptor for STb at the level of the luminal surface of pig jejunal epithelial cells (34). The fact that this toxin is composed of an amphipathic (Cys10-Lys22) and a hydrophobic (Gly38-Ala44) -helix suggests that it could possibly insert into the cell membrane and form a pore. This possibility is further suggested by our recent demonstration that STb undergoes oligomerization in vitro (26), a process that constitutes a well-documented characteristic of many pore-forming bacterial toxins (1) and antimicrobial peptides (44).

In the present study, the ability of STb to permeabilize brush border membrane vesicles isolated from piglet jejunum was demonstrated by using a fluorescent membrane potential-sensitive probe, 3,3'-dipropylthiadicarbocyanine iodide [diS-C₃(5)], and a procedure that was recently developed to study the properties of bacterial pore-forming toxins in membrane vesicles (23).

	MATERIALS AND METHODS

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Purification of STb. Unless specified otherwise, all chemicals were from the Sigma Chemical Co. (Oakville, Ontario, Canada). The method used to prepare wild-type and mutated (M42S and K22A-K23A) (25) STb toxins was modified from a previously described protocol (7). An E. coli HB101 strain harboring the plasmid pMal-STb, which is responsible for the expression of a maltose-binding protein-mature STb fusion protein, with or without the selected mutation, was grown in Luria broth containing 50 µg of ampicillin/ml until the optical density at 600 nm (OD₆₀₀) reached 0.5. Then, 0.3 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) was added to induce the synthesis of the fusion protein. Three hours after induction, cells were harvested by centrifugation at 4,000 x g for 10 min at 4°C. The pellet was gently resuspended in 500 ml of 30 mM Tris-HCl (pH 8.0) containing 20% (wt/vol) sucrose and 1 mM EDTA. After centrifugation at 8,000 x g for 10 min at 4°C, the cells were resuspended in 500 ml of 5 mM MgSO₄ and incubated at 4°C for 10 min. After centrifugation at 8,000 x g for 10 min at 4°C, the fusion protein was affinity purified from the supernatant representing the osmotic shock fluid by using an amylose resin (New England Biolabs, Pickering, Ontario, Canada). The amylose-purified fusion protein was dialyzed against Xa buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM CaCl₂) and then cleaved with protease factor Xa (GE Healthcare, Baie d'Urfé, Quebec, Canada). The cleaved material was loaded onto a C₈ reversed-phase microbore column (Applied Biosystems, Foster City, CA) using an Ákta Purifier10 system (GE Healthcare) and eluted with a linear gradient of acetonitrile (5 to 100%) in a water solution containing 0.1% trifluoroacetic acid. Wild-type and mutant STb preparations were quantified at 214 nm using aprotinin as the reference. 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 earlier (28).

Preparation of reduced and alkylated STb. Purified STb toxin was modified by reduction and alkylation with the method described by William et al. (42). Briefly, STb was reduced with 45 mM dithiothreitol in 100 mM NH₄HCO₃ at 60°C for 30 min and alkylated with 100 mM iodoacetamide in 100 mM NH₄HCO₃ for 30 min at room temperature in the dark. The reduced and alkylated STb toxin was dialyzed (membrane cutoff of 3.5 kDa) against 50 mM NH₄HCO₃ (pH 8.5) for 2 h and against water for another 2 h.

Preparation of brush border membrane vesicles. Four-week-old weaned piglets from a conventional herd were obtained from a local producer. The animals were killed the day they arrived at the university. In the meantime, the piglets were cared for in accordance with the Guidelines of the Canadian Council for Animal Care. They were sedated by intramuscular injection of a mixture of 10 mg of ketamine hydrochloride (Biomeda-MTC, Cambridge, Ontario, Canada) per kg of body weight and 20 mg of xylazine (Bayer, Toronto, Ontario, Canada) per kg of body weight. Euthanasia was carried out by an intracardiac injection of 540 mg of sodium pentobarbital (Pharmacy, Faculté de médecine vétérinaire, Saint-Hyacinthe)/ml, and 30 cm of jejunum was recovered, rinsed thoroughly with ice-cold physiological buffer, and stored at –80°C until use.

Brush border membrane vesicles were prepared with a modification of the MgCl₂-precipitation method (18). All manipulations were carried out on ice or in a centrifuge refrigerated at 4°C. Frozen jejunum segments were thawed and scraped by using a spatula, weighed, and homogenized in 50 mM mannitol-5 mM EGTA-2 mM Tris-HCl (pH 7.0) (20 ml for each g of tissue). The mixture was stirred for 10 min after the addition of MgCl₂ to a final concentration of 10 mM and centrifuged at 7,700 x g for 15 min. The supernatant was centrifuged for 30 min at 20,000 x g. The pellet was resuspended in 250 mM KCl-125 mM mannitol-0.1 mM MgSO₄-50 mM Tris-HEPES (pH 7.5). The preparation was then centrifuged for 15 min at 1,900 x g, and the resulting supernatant was centrifuged for 15 min at 30,900 x g. The final membrane preparation was resuspended in 250 mM KCl-0.1 mM MgSO₄-50 mM MES (morpholineethanesulfonic acid)-Tris (pH 5.8) and stored at –80°C until use. In preparation for the experiments, the vesicles were diluted to 2.0 mg of protein/ml with this same solution.

Protein concentrations were estimated by using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL) and bovine serum albumin as the standard. Leucine aminopeptidase activity, assayed with L-leucine p-nitroanilide as the substrate (16), was enriched 10-fold (±1; average of seven independent preparations) relative to the epithelium homogenate.

Fluorescence measurements. The membrane-permeabilizing effects of STb were monitored with a membrane potential-sensitive fluorescent probe, diS-C3(5) (Molecular Probes, Eugene, OR) (23). Experiments were carried out at room temperature in polystyrene cuvettes containing 1.5 µM diS-C₃(5), from a 1 mM stock solution in dimethyl sulfoxide, in 2 ml of the appropriate buffer solution, using a Spex Fluorolog CM-3 spectrofluorometer (Jobin Yvon Horiba, Edison, NJ) at a frequency of 10 Hz, an excitation wavelength of 620 nm, and an emission wavelength of 670 nm. Before each experiment, the mixture was stirred for 10 min in the dark to allow fluorescence to reach a steady level (22, 36). Vesicles were preincubated for 15 min in the presence or absence of STb at room temperature. A few seconds after the beginning of the recording, vesicles were injected into the cuvette at a final concentration of 5 µg of protein/ml. In some experiments, 7.5 µM valinomycin was added to the cuvette from a 5 mM stock solution in ethanol. Fluorescence values were normalized relative to the level measured before addition of the vesicles. For each experimental condition tested, the fluorescence level was measured in the absence of a membrane potential by injecting vesicles into the same solution as that with which they were loaded.

Statistical analyses. Unless specified otherwise, values presented in the text and figures are means ± the standard error of the mean (SEM). Statistical analyses were carried out by using the linear model of the SAS package (version 9.0; SAS Institute, Cary, NC). We also performed a priori contrasts to examine differences between levels of the different factors.

	RESULTS AND DISCUSSION

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Generation and reduction of a membrane potential in piglet jejunal brush border membrane vesicles. The membrane-permeabilizing ability of STb was analyzed with a fluorescence assay (23) using diS-C₃(5), a membrane-potential-sensitive probe (40). In this assay, vesicles loaded with 250 mM KCl and preincubated with or without STb were first injected in the presence of the fluorescent probe and the potassium ionophore valinomycin into cuvettes containing the same solution as that with which they were loaded (Fig. 1). This procedure allowed the measurement of the fluorescence level in the absence of ionic gradients and therefore in the absence of a membrane potential. The slight decrease in fluorescence was due to the association of some of the fluorescent probe with the membrane vesicles. Under these conditions, STb had no influence on the fluorescence level attained after the injection, an indication that the toxin does not interact directly with the probe. When vesicles were injected into cuvettes in which the KCl solution was replaced by an isotonic solution of N-methyl-D-glucamine hydrochloride, fluorescence decreased to a much greater extent, a finding indicative of the presence of an inside-negative membrane potential generated by the preferential efflux of potassium ions induced by valinomycin. The resulting concentration and aggregation of the positively charged probe within the vesicles and their membranes caused a strong quenching of the fluorescence (33, 36, 41). The fact that a strong membrane potential can be generated under these conditions in the presence of valinomycin demonstrates that the vesicles used in the present study were tightly sealed. In the presence of STb, less quenching of the fluorescence signal, indicative of a lower membrane potential, demonstrated an increased permeability of the membrane to at least one of the other ions present, N-methyl-D-glucamine and chloride.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Effect of the potassium transmembrane gradient, valinomycin, and STb on the membrane potential generated in brush border membrane vesicles isolated from piglet jejunum epithelial cells. Vesicles loaded with 250 mM KCl-0.1 mM MgSO4-50 mM MES-Tris (pH 5.8) were incubated for 15 min with or without 12.5 nmol of STb/mg of membrane protein. Fluorescence was monitored in a Spex Fluorolog spectrofluorometer as described in Materials and Methods. Five seconds after the beginning of the recording, vesicles were injected into cuvettes containing 1.5 µM diS-C3(5), 7.5 µM valinomycin, and either the same solution as that with which they were loaded (KCl) or a solution of similar composition in which KCl was replaced by N-methyl-D-glucamine hydrochloride (NMDGCl). Each curve corresponds to a representative experimental record.

View larger version (9K): [in this window] [in a new window]   FIG. 2. Calibration of the fluorescence signal as a function of the transmembrane potassium concentration ratio. Fluorescence was monitored by using a protocol similar to that illustrated in Fig. 1. Vesicles loaded with 250 mM KCl-0.1 mM MgSO4-50 mM MES-Tris (pH 5.8) were injected into cuvettes containing the appropriate concentration of KCl, enough N-methyl-D-glucamine hydrochloride to maintain osmolarity and ionic strength constant, 0.1 mM MgSO4, 50 mM MES-Tris (pH 5.8), 1.5 µM diS-C3(5), and 7.5 µM valinomycin. Values are means ± the SEM for nine experiments, each performed with a different membrane preparation.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Effect of STb on the membrane potential generated in the presence of various electrolyte solutions. Vesicles loaded with 250 mM KCl-0.1 mM MgSO4-50 mM MES-Tris (pH 5.8) were incubated for 15 min in the presence () or absence () of 12.5 nmol of STb/mg of membrane protein. Fluorescence was monitored by using a protocol similar to that illustrated in Fig. 1. The vesicles were injected in cuvettes containing 1.5 µM diS-C3(5) and either the same solution as that with they were loaded (KCl) or a solution of similar composition in which 250 mM KCl was replaced by the same concentration of either NaCl, N-methyl-D-glucamine hydrochloride (NMDGCl), LiCl, potassium gluconate (KGlu), tetramethylammonium chloride (TMACl), or tetraethylammonium chloride (TEACl), in the absence (A) or presence (B) of 7.5 µM valinomycin. Values are means ± the SEM for five experiments, each performed with a different membrane preparation. Means labeled with different letters are significantly different (P < 0.05).

Evidence for pore formation by STb. To test the possibility that STb forms pores, its permeabilizing effect was measured in vesicles preincubated with various concentrations of the toxin and diluted in a solution of N-methyl-D-glucamine hydrochloride in the presence of valinomycin (Fig. 4). Fluorescence levels increased rapidly at the lower STb concentrations but soon leveled off as the toxin concentration was further increased. The data presented in Fig. 4 were well fitted with the Michaelis-Menten equation, with relative fluorescence levels reaching a plateau at 0.81 ± 0.01 and an apparent half-saturation constant of 1.8 ± 0.4 nmol STb/mg membrane protein (corresponding to 1.1 ± 0.2 µM). This value matches reasonably well the earlier published estimates of 2 to 6 µM for the dissociation constant of STb binding to its receptor, sulfatide, obtained with an enzyme-linked immunosorbent assay (3).

View larger version (9K): [in this window] [in a new window]   FIG. 4. Effect of STb concentration on membrane potential. Experiments were carried out by injecting KCl-loaded vesicles, previously incubated with the indicated concentrations of STb, in an isotonic solution of N-methyl-D-glucamine hydrochloride as described in the legend of Fig. 1. The fluorescence level measured in the absence of a membrane potential was 0.963 ± 0.008. Values are means ± the SEM for four experiments, each performed with a different membrane preparation. The datum points were fitted with the Michaelis-Menten equation using the software Origin (version 7.5; OriginLab Corp., Northampton, MA).

Lack of effect of pH on the membrane-permeabilizing ability of STb. Because somewhat contrasting results have been published regarding the effect of pH on the binding of STb to the piglet jejunal luminal membrane (35) and to its receptor (3), vesicles incubated in the presence or absence of toxin were injected in solutions of KCl or N-methyl-D-glucamine hydrochloride at various pH values ranging from 5.5 to 8.0 in the presence of valinomycin (Fig. 5). This range comprises the pH values reported for the jejunal contents of piglets, 2 weeks after weaning (pH 5.5 to 7.0) (37). Fluorescence levels were not affected significantly by pH in the absence of a membrane potential. However, in the N-methyl-D-glucamine hydrochloride solution, both in the presence and in the absence of STb, fluorescence levels decreased gradually as pH was increased. This result is consistent with earlier studies indicating that, although the fluorescence of diS-C₃(5) is independent of pH in solution, it is strongly pH dependent in the presence of cells or cell membranes (13, 21, 23, 41). The pattern illustrated in Fig. 5 can be simply explained by the fact that, as pH is increased, an increasing number of amino groups belonging to membrane proteins lose their charge, thus rendering the membrane more negatively charged and causing an increase in the number of positively charged probe molecules interacting with the vesicles.

View larger version (11K): [in this window] [in a new window]   FIG. 5. Membrane-permeabilizing ability of STb at various pH values. Experiments were carried out as described in the legend of Fig. 1 except that the KCl solutions were buffered with a 50 mM concentration of either MES-Tris at between pH 5.5 and 6.5, HEPES-Tris at pH 7.0 and 7.5, or Tris-HCl at pH 8.0. Values are means ± the SEM for four experiments, each performed with a different membrane preparation.

Only functional STb displays a membrane-permeabilizing ability. The membrane-depolarizing capacity of STb was compared to those of three inactive forms of the toxin (Fig. 6). STb in which disulfide bonds have been chemically removed by reduction and alkylation has long been shown to be nonactive (9). Similarly, the single mutant M42S and the double mutant K22A-K23A have been shown to bind poorly to sulfatide and to be poorly active in the ligated rat jejunal loop assay (25). In contrast to intact STb, none of the three inactive forms of the toxin was able to increase the fluorescence level above that measured in the absence of toxin, even when they were added at twice the concentration used for STb (Fig. 6). The ability of STb to depolarize the brush border membrane therefore correlates well with its enterotoxicity.

View larger version (10K): [in this window] [in a new window]   FIG. 6. Comparison of the permeabilizing ability of STb with that of its reduced and alkylated derivative (STb/AR) and of two of its mutants (M42S and K22A-K23A). Experiments were carried out by injecting KCl-loaded vesicles, previously incubated with either 12.5 nmol of STb/mg of membrane protein or 25 nmol of either one of the other toxins/mg of membrane protein, in an isotonic solution of N-methyl-D-glucamine hydrochloride (NMDGCl) as described in the legend of Fig. 1. The fluorescence level measured in the absence of a membrane potential was 0.948 ± 0.008. Values are means ± the SEM for three experiments each performed with a different membrane preparation. The asterisk indicates a significant difference with the corresponding value measured in the absence of toxin (P < 0.05).

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the contribution of the following coworkers from the Faculté de Médecine Vétérinaire, Saint-Hyacinthe, Quebec, Canada: John M. Fairbrother and Francis Girard for providing piglet jejunal tissue and Guy Beauchamp for help with the statistical analyses.

This study was supported by Natural Sciences and Engineering Research Council research grant 139070-01 to J.D.D., by a Fonds de la Recherche en Santé du Québec research group grant to J.-L.S., and by the Groupe d'Étude des Protéines Membranaires (grant 5252).

	FOOTNOTES

 
* Corresponding author. Mailing address: Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte Street, Saint-Hyacinthe, Quebec J2S 7C6, Canada. Phone: (450) 773-8521, ext. 18433. Fax: (450) 778-8108. E-mail: daniel.dubreuil{at}umontreal.ca

Published ahead of print on 16 February 2007.

Editor: J. F. Urban, Jr.

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