Selection of Escherichia coli Heat-Labile Toxin (LT) Inhibitors Using Both the GM1-ELISA and the cAMP Vero Cell Assay

Abstract

Weaned piglets are very susceptible to diarrhea caused by enterotoxigenic Escherichia coli. In the past, various natural components were proposed to have beneficial effects by reducing the effects of diarrheal infectious diseases in humans and animals, and thus may represent an alternative for the use of (prophylactic) antibiotics. Alternatives may inactivate enterotoxigenic Escherichia coli heat-labile toxin (LT) by interfering with toxin binding to the cellular receptor GM1. In this study, various plants and other natural substances were tested for inhibitory properties, in the GM1 binding assay, and in the LT-induced cAMP production in Vero cells. The toxic dose of each compound was determined in a cell viability assay, and the highest nontoxic concentrations were used in the GM1 and cAMP assays. Results demonstrated that only d-(+)-galactose, lactose, N-acetyl-d-galactosamine, and two tea extracts were able to inhibit the binding of LT to its GM1 receptor. In the cAMP assay, only the two tea extracts showed inhibitory activity. This shows that d-(+)-galactose, lactose, and N-acetyl-d-galactosamine can indeed inhibit LT binding to GM1 based on structural homology with GM1 in the absence of living cells. However, in the cAMP assay, d-(+)-galactose, and lactose, N-acetyl-d-galactosamine are apparently metabolized to below their effective inhibitory concentration, likely predicting limited practical applicability in vivo. Both tea extracts maintained their activity in the presence of cells. The active compounds in both are probably polyphenols, which are not easily metabolized, and most likely work by aggregating the toxin. In conclusion, the combination of methods used here is a convenient and fast method for preselecting natural substances containing potentially toxin-binding compounds. Furthermore, if antidiarrhea activity is attributed to compounds found inactive here, their activity is unlikely based on interference with toxin binding.

Introduction

I nfectious diarrhea is a major cause of mortality, morbidity, and a decreased growth rate in weaned piglets, as well as in children. Enterotoxigenic Escherichia coli (ETEC) strains are the most common etiological agent of piglet enteric infections and diarrhea (Nagy and Fekete, 2005; Turner et al., 2006). After adhesion, ETEC bacteria start producing several toxins, of which heat-labile toxin (LT) is the most important. LT is very similar to the toxin produced by Vibrio cholerae (CT) and has a similar mode of action (Bagley et al., 2002; Holmner et al., 2011). The B subunit of these AB5 enterotoxins binds to the oligosaccharide part of the GM1 ganglioside in epithelium. LT binds to the terminal glycosyl sequence Galβ1-3GalNAcβ1-4(NeuAca2-3)Gal, of which β-galactose and N-acetyl-d-galactosamine are the most important for toxin receptor recognition (Turner et al., 2006). After endocytosis by epithelial cells, the A subunit activates adenyl cyclase, increasing intracellular cyclic adenosyl monophosphate (cAMP) levels, leading to secretory diarrhea. Antibiotics have been extensively used to reduce pathogen levels in piglets, but since increasing numbers of bacteria are becoming resistant to antibiotics (McEwen and Fedorka-Cray, 2002; Smith et al., 2002), there is an urgent need for alternatives to in-feed antibiotics as antibacterial growth promoters. This quest for alternatives has intensified after the recent full ban of antibiotic use as growth promoters within the European Union. What is clear is that complete inhibition of LT in vitro and in vivo can be achieved using specific antibodies against LT from, for example, porcine plasma (Niewold et al., 2007), but they are too costly (Harmsen et al., 2005). Natural components and plant extracts have been claimed to have antidiarrheal effects. Apart from antibacterial effects, compounds could render the ETEC LT inactive. Inactivation could occur on several different levels, by blocking the receptor, by offering competing LT binding sites (Becker et al., 2010), or by direct inactivation of the toxin (Verhelst et al., 2010).

Blocking the receptor

Lectins are sugar-binding proteins that are highly specific for their specific sugar moieties. Several lectins are known to bind to the galactose and N-acetyl-d-galactosamine sugars of the GM1 receptor (Kamiya et al., 2011). Lectins were also suggested to significantly reduce the LT caused fluid accumulation in rabbit's ileal loops (Galvan et al., 1999).

Competing LT binding sites

Galactose-containing or -related oligosaccharides

Because galactose is so important for toxin receptor binding recognition, it is hypothesized that galactose-like sugars could be an alternative binding site for the toxin instead of the GM1 ganglioside (Fukuta et al., 1988; Holmner et al., 2011).

Yeast cells and yeast cell extracts

These cells and extracts contain components such as manno-oligosaccharides, which could bind enterotoxin. The nonpathogenic yeast Saccharomyces boulardii is used in many countries for the treatment of gastrointestinal disorders (Schellenberg et al., 1994).

Direct inactivation

Tea extracts are traditional herbal medications used in China and Japan for many centuries to treat diarrhea. They contain polyphenols, which can form aggregates with enterotoxin, rendering them inactive (Verhelst et al., 2010).

Here, several natural products from each group were tested for activity in a GM1 binding assay, and for the capacity to reduce LT-induced cAMP accumulation in the Vero cell line. To exclude aspecific toxic effects, products were first tested for their toxicity in the Vero cell line; the highest nontoxic concentration or a 2 mg/mL concentration was used for toxin inactivation in both assays.

Materials and Methods

Products

GM1 ganglioside, d-(+)-galactose, d-galacto-d-mannan from Ceratonia siliqua, lactose, lactulose, N-acetyl-d-galactosamine, yeast extract, yeast autolysate, lectin from Abrus precatorius (specificity for Galα1-3/4Gal, and Gal-GalNAc), lectin from Arachis hypogaea (specificity for Galβ1-3GalNAc), concanavalin A lectin from Canavalia ensiformis (specificity for α-d-mannosyl and α-d-glucosyl residues) extract from black tea, Polyphenon 60 from green tea, and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MO). Synblock (ready to use solution) was purchased from AbD Serotec (Kidlington, UK). Monoclonal mouse anti-E. coli heat labile toxin antibodies (anti-LT) and horseradish peroxidase–labeled rabbit anti-mouse immunoglobulin G antibodies were purchased from Abcam (Cambridge, MA). E. coli LT was a gift from Dr. Clements (University of Tulane, LA) (Clements and Finkelstein, 1979). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), MTT solvent (0.1 N HCl in isopropanol, ready to use), and ABTS (2,2-azino-di(3-ethyl-benzthiazolydine-6-sulfonic acid), and 96-well plates (Nunclon Microwell, flat bottom) were obtained from Sigma-Aldrich (St. Louis, MO).

GM1-enzyme-linked immunosorbent assay (ELISA)

All steps were performed at room temperature. Twenty-five microliters of GM1 in ethanol (10 μg/mL) was added to each well of a 96-well plate. Thereafter, the plate was incubated for 2 h. All unattached GM1 was removed by washing the plate three times with phosphate-buffered saline Tween (PBST, pH 7.2, 0.010 mg/ml Tween) and the remaining binding sites of the surface were blocked for 1 hour with Synblock followed by washing with PBST. Then 50 μL of heat-labile toxin (0.5 μg/mL) with 50 μL of a test compound was added. After 1-h incubation, the unbound toxins were removed by washing three times with PBST. Primary anti-LT antibodies (100 μL, 1:500) were added to each well and incubated for 1 h. Before the secondary antibodies (rabbit anti-immunoglobulin G) (100 μL, 1:1000) were added, the plate was washed three times with PBST. After 1 h, the ABTS substrate (100 μL) was added to each well and absorbance was measured at 405 nm (Morinaga et al., 2005).

Vero cell culture

Stock cultures of Vero cells (ATCC) were grown in Eagle's MEM medium supplemented with 10% fetal calf serum (FCS) in 6% CO₂ at 37°C and were trypsinized and centrifugated (400×g) for 5 min. Media was removed and cells were counted and diluted to a concentration of 75,000 cells/mL medium. In a 96-well plate, 100 μL of cells was added to each well and left for incubation at 37°C overnight, before use in either the cell viability assay or the cAMP assay.

Cell viability assay (MTT assay)

Vero cells were treated with serially twofold-diluted compounds starting from 10 mg/mL in culture medium. After 24 h, 20 μL of 5 mg/mL MTT was added to each well and left for incubation for 3.5 h at 37°C. Living cells reduce yellow MTT to insoluble purple formazan. The latter is solubilized by adding 150 μL of MTT solvent by shaking for 15 min. Absorbance was read at 590 nm with a reference filter of 620 nm (Mossmann, 1983).

cAMP assay

The natural components (at the noncytotoxic concentrations indicated in the results section) were incubated with LT (50 nM, in phosphate-buffered saline pH 7.2) to evaluate reduction of LT-induced cAMP accumulation with phosphate-buffered saline as a negative control. These mixtures (100 μL) were then added for 20 min to the Vero cells and cAMP accumulation was quantified using a cAMP-Glo Assay (Promega, Madison, WI) according to the manufacturer's instructions.

Statistics

Statistical software program Graphpad Prism 5.0 for Windows (Graphpad Software, San Diego, CA) was used to carry out all statistics. The data sets did not follow a normal distribution according to the Kolmogorov-Smirnov normality test. Therefore, the nonparametric Mann–Whitney U test of significance (significance level was set p<0.05) was used to compare values of treatments versus control.

Results

Determining the highest nontoxic concentration

A wide range of natural components concentrations was screened to verify if they could affect Vero cell viability. Among those screened, the highest nontoxic concentration of each natural component (defined as MTT values higher than 75% survival) was subsequently used to investigate the protective activity against LT binding and LT-induced cAMP production. Yeast and yeast extract appeared to have no toxicity in this range, and a 2 mg/mL concentration was used for toxin inactivation in both assays. The highest nontoxic concentration was for galactose, N-acetyl-d-galactosamine, galacto-mannan, lactose, and lactulose 5 mg/mL, for Polyphenon 60 and black tea extract 150 μg/mL. The lowest nontoxic concentrations were found for the lectins, for concanavalin A lectin and A. hypogaea lectin 7.5 μg/mL, and for A. precatorius lectin 10 μg/mL.

Evaluation of natural components inhibiting LT binding to GM1

LT (0.5 μg/mL) was preincubated for 60 min before being applied to the GM1-ELISA. The data indicate that some but not all natural compounds were able to prevent the binding of LT to its receptor GM1. d-(+)-Galactose, lactose, N-acetyl-d-galactosamine, extract from black tea and Polyphenon 60 from green tea were able to inhibit the binding of LT to GM1 when compared to the control, LT only. The other components tested did not exert any inhibitory activity (Fig. 1).

FIG. 1.

Significant inhibition of heat-labile toxin binding to GM1 by d-(+)-galactose, lactose, N-acetyl-d-galactosamine, black tea extract, and Polyphenon 60. Data are means±standard error of the mean of values of three experiments. Significant differences (p<0.05) from control are indicated with an asterisk. Con A, concanavalin A; O.D., optical density.

Effect of active compounds on LT-induced cAMP concentration

Because the above results suggested that several natural components were able to prevent the binding of LT to its receptor, we wanted to assess whether they are also capable of preventing the LT-induced cAMP production in Vero cells. In Vero cells, co-incubation of LT with the active natural compounds revealed that extract from black tea and Polyphenon 60 from green tea were able to significantly lower the cAMP concentration to 56% and 61% of the control, respectively. d-(+)-Galactose, lactose, and N-acetyl-d-galactosamine did not lower the concentration of cAMP in Vero cells. The compounds without activity in the GM1 ELISA did not significantly lower the cell cAMP concentration compared to the control (Fig. 2).

FIG. 2.

Significant inhibition of heat-labile toxin (LT)–induced cyclic adenosyl monophosphate (cAMP) concentration in Vero cells by black tea extract, and Polyphenon 60. Data are expressed as a percentage of the cAMP level induced by LT alone in the control. Data are means±standard error of the mean of values of three experiments. Significant differences (p<0.05) from control are indicated with an asterisk. Con A, concanavalin A.

Discussion

Because LT is the major virulence factor in ETEC diarrhea, natural inhibitors of LT could show promise as an in-feed alternative for antibiotics. Our results show that black tea extract, Polyphenon 60, galactose, N-acetyl-d-galactosamine, and lactose were able to inhibit the binding of LT to GM1 in the GM1 ELISA. Earlier, binding assays with GM1 gangliosides have demonstrated the importance of galactose as a recognition molecule for the binding site of the toxin (Becker et al., 2010). Here we find inhibition by galactose and lactose in the GM1 ELISA at identical (5 mg/mL) inclusion levels as described by Becker et al. (2010) using the same method. In contrast to their results, we did find clear activity for N-acetyl-d-galactosamine at the 5 mg/mL level as could be expected from the structure of GM1 (Kamiya et al., 2011). Furthermore, lactulose was not active, whereas it was in the test of Becker et al. (2010). This could be due to differences between the procedures used such as the type, source, and concentration of LT.

Based on the results of the GM1 ELISA, a similar result was anticipated in the Vero cell cAMP assay. This was true for black tea extract and Polyphenon 60. However, to our surprise, galactose, galactosamine, and lactose did not lower LT-induced cAMP production in the Vero cell assay. The most likely explanation is that a large part of these compounds was quickly metabolized in the cell culture, and at least galactose was described to be rapidly metabolized by Vero cells (Mendonça et al., 2002). Because the same may happen in vivo by intestinal cells, this means that either it is very unlikely that these compounds are active in vivo, or that in order to maintain activity the already very high concentrations used here should even be increased. The only natural compounds that were able to both prevent the binding of LT to GM1 and to lower cAMP concentration in Vero cells were the tea extracts. The chewing of tea leaves is well known as a folk medicine to reduce diarrhoea. Tea leaves are characterized by high concentrations of polyphenols (Bruins et al., 2006). Previous research in intestinal loops of mice demonstrated that catechins and black tea extract were able to reduce ETEC-induced fluid secretion in an animal model (Bruins et al., 2006). Polyphenols have been reported to inhibit the binding of CT to GM1 and also reduce CT-caused cAMP production in Vero cells through aggregation (Saito et al., 2002; Morinaga et al., 2005; Morinaga et al., 2010). Using the same system with LT and purified polyphenols from tea, we recently demonstrated that only polyphenols with two or more galloyl moieties in the molecule are responsible for the toxin-inactivating properties (Verhelst et al., 2013). Furthermore, polyphenols have a low absorption in the small intestine (Biasi et al., 2011). The latter fact, and the lower effective concentrations means that polyphenols can be used at realistic and commercially feasible inclusion levels in feed.

In conclusion, our data show that combining the GM1 ELISA with the Vero cell assay is a powerful method for selecting effective inhibitors, because it includes a cell culture that better mimics the in vivo situation. It also shows that blocking of LT-receptor interactions forms a promising avenue for therapeutic intervention and can lead to a wide new range of natural products to be used as an alternative for in-feed antibiotics.

Footnotes

Acknowledgments

This work was carried out within the framework of the IWT project 60680 sponsored by the Flemish Institute for the Promotion of Scientific and Technological Research in the Industry (IWT).

Disclosure Statement

No competing financial interests exist.

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