STb Toxin Research Articles

Impact of early inoculation of probiotics to suckling piglets on postweaning diarrhoea – A challenge study with Enterotoxigenic E. Coli F18

Postweaning diarrhoea caused by Enterotoxigenic Escherichia coli (ETEC) is a threat to the pig industry. With an intensified focus on finding alternatives to the use of medical zinc oxide and antibiotics in newly weaned pigs, the objective of this study was to investigate the effect of early inoculation of probiotics to suckling piglets on subsequently ETEC faecal shedding and immune parameters in ETEC F18-challenged weaned piglets. Sixty pigs weaned on day 28 of age were assigned to three treatment groups: (i) Negative Control (non-challenged), (ii) Positive Control (challenged) and (iii) Probiotic (challenged and inoculated with a multi-species probiotic product during suckling). On days 1 and 2 postweaning, pigs in the Positive Control and Probiotic groups were challenged with 5 × 108 colony-forming unit ETEC F18/pig/day, whereas pigs in the Negative Control group were provided with NaCl. Growth and diarrhoea incidence were not significantly affected by ETEC challenge or probiotic administration. ETEC F18 shedding and C-reactive protein concentration in plasma were significantly lower in the Negative Control group, confirming a successful challenge model. Pigs in the Probiotic group had a significantly reduced number of pigs shedding ETEC F18 and STb toxin in faeces compared with the Positive Control group. Probiotic application did not significantly impact the concentration of C-reactive protein, haptoglobin and cytokines in plasma nor haematology numbers. In conclusion, weaned pigs administered with a multi-species probiotic product early in life had a more rapid response towards the pathogen challenge and a faster clearance of ETEC compared with the Positive Control group. Administration of a multi-species probiotic to newborn piglets may thus promote resilience in the newly weaned pig. However, further studies with pigs subjected to a more severe pathogen challenge are needed to confirm these results and to investigate the mechanism of action of the probiotic intervention.

Enterotoxigenic Escherichia coli Heat-Stable Toxin and Ebola Virus Delta Peptide: Similarities and Differences.

Enterotoxigenic Escherichia coli (ETEC) STb toxin exhibits striking structural similarity to Ebola virus (EBOV) delta peptide. Both ETEC and EBOV delta peptide are enterotoxins. Comparison of the structural and functional similarities and differences of these two toxins illuminates features that are important in induction of pathogenesis by a bacterial and viral pathogen.

Evaluation of Escherichia coli STb toxin in an in vitro organ culture model (IVOC) / Avaliação de toxina STb de Escherichia coli em modelo de cultura de órgãos in vitro (IVOC)

ETEC pathogenesis comprises adhesion to the small intestine, colonization and enterotoxin production. STb is one of the heat-stable toxins produced by ETEC. Previous reports, using culture supernatants of STb-positive ETEC strains, indicated loss of villous absorptive cells causing mild atrophy and microscopic alterations in jejunum mucosa. These culture supernatants contained many other compounds beside STb toxin and in addition the quantity of toxin in these supernatants was not determined. Thus, this study was undertaken to evaluate the effect of pure STb toxin on piglet jejunum explants in an in vitro organ culture (IVOC) model. Tissues of piglets of 11 weeks were used and put into culture. Morphometric analysis of tissues revealed that villous epithelial area was reduced in tissues treated with pure STb after 3, 4 and 5 hours (p 0.0001) compared to untreated tissues. STb-treated tissues presented atrophic villi due to loss of villi tip. Our data shows that, in piglet jejunum explants, pure STb toxin can lead to cell modification and consequently to destruction, seen as villi atrophy. These changes result in a reduced absorptive area and could be partly responsible for the diarrhea observed in the animal following STb intoxication.

The fecal presence of enterotoxin and F4 genes as an indicator of efficacy of treatment with colistin sulfate in pigs

BackgroundEnterotoxigenic Escherichia coli (ETEC) strains producing multiple enterotoxins are important causes of post-weaning diarrhea (PWD) in pigs. The aim of the present study was to investigate the fecal presence of ETEC enterotoxin as well as F4 and F18 genes as an indicator of colistin sulfate (CS) efficacy for treatment of PWD in pigs. Forty-eight piglets were weaned at the age of 21 days, and were divided into four groups: challenged treated, challenged untreated, unchallenged treated, and unchallenged untreated. Challenge was performed using 109 CFU of an ETEC: F4 strain, and treatment was conducted using oral CS at the dose of 50,000 IU/kg. The fecal presence of genes encoding for STa, STb, LT, F4 and F18 was detected using PCR.ResultsThe PCR amplification of ETEC virulence genes showed that nearly 100% of pigs excreted genes encoding for STa and STb toxins in the feces before the challenge. These genes, in the absence of the gene encoding F4, were considered as a marker for F4-negative ETEC. One day after ETEC: F4 oral challenge pigs in the two challenged groups excreted the genes encoding LT and F4 in the feces. These genes were considered as a marker for F4-positive ETEC. However, the gene encoding F18 was not detected in any fecal samples of the 4 groups throughout the experiment. After only 3 days of successive oral treatment with CS, a significant reduction in both the F4-positive and negative ETEC populations was observed in the challenged treated group compared to the challenged untreated group (p < 0.0001).ConclusionsOur study is among the first to report that under controlled farming conditions, oral CS treatment had a significant effect on both fecal F4-positive and F4-negative ETEC in pigs. However, CS clinical efficiency was correlated with non-detection of F4-positive ETEC in the feces. Furthermore the fecal presence of F4-negative ETEC was not associated with clinical symptoms of post-weaning diarrhea in pigs.

Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STaP12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LTR192G), two toxoid MEFAs, ‘LTR192G-STb-Stx2e-STaP12F’ and ‘LTR192G-STb-Stx2e-3xSTaP12F’ which carried three copies of STaP12F, were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with ‘LTR192G-STb-Stx2e-3xSTaP12F’ were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs.

Analysis and application of a neutralizing linear epitope on liable toxin B of enterotoxin Escherichia coli.

Heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) is one of the major virulence factors for causing diarrhea in piglets, and LT is a strong immunogen. Thus, LT represents an important target for development of vaccines and diagnostic tests. In this study, bioinformatic tools were used to predict six antigenic B cell epitopes in the B subunit of LT protein (LTB) of ETEC strains. Then, seven antigenic B cell epitopes of LTB were identified by polyclonal antisera (polyclonal antibody (PAb)) using a set of LTB-derived peptides expressed as maltose-binding protein (MBP) fusion protein. In addition, one LTB-specific monoclonal antibody (MAb) was generated and defined its corresponding epitope as mentioned above. This MAb was able to specifically bind with native LT toxin and has no cross-reaction with LT-II (type II heat-labile enterotoxin), Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), STa (heat-stable enterotoxin I), and STb (heat-stable enterotoxin II) toxins. Further, this MAb was able to interrupt LT toxin specific binding to GM1 receptor, indicating that the corresponding epitope is the specific binding region to GM1 receptor. Moreover, in vitro and in vivo assay showed that the MAb was able to neutralize the native LT toxin. Diarrheal suckling pigs challenged with LT-positive ETEC strain recovered when an enema with this purified MAb was administered. This study will provide the foundation for further studies about the interaction between LT toxin and GM1 receptor and about the developing of epitope-based vaccines and specific therapeutic agent.

Escherichia coli STb enterotoxin dislodges claudin-1 from epithelial tight junctions.

Enterotoxigenic Escherichia coli produce various heat-labile and heat-stable enterotoxins. STb is a low molecular weight heat-resistant toxin responsible for diarrhea in farm animals, mainly young pigs. A previous study demonstrated that cells having internalized STb toxin induce epithelial barrier dysfunction through changes in tight junction (TJ) proteins. These modifications contribute probably to the diarrhea observed. To gain insight into the mechanism of increased intestinal permeability following STb exposure we treated human colon cells (T84) with purified STb toxin after which cells were harvested and proteins extracted. Using a 1% Nonidet P-40-containing solution we investigated the distribution of claudin-1, a major structural and functional TJ protein responsible for the epithelium impermeability, between membrane (NP40-insoluble) and the cytoplasmic (NP-40 soluble) location. Using immunoblot and confocal microscopy, we observed that treatment of T84 cell monolayers with STb induced redistribution of claudin-1. After 24 h, cells grown in Ca++-free medium treated with STb showed about 40% more claudin-1 in the cytoplasm compare to the control. Switching from Ca++-free to Ca++-enriched medium (1.8 mM) increased the dislodgement rate of claudin-1 as comparable quantitative delocalization was observed after only 6 h. Medium supplemented with the same concentration of Mg++ or Zn++ did not affect the dislodgement rate compared to the Ca++-free medium. Using anti-phosphoserine and anti-phosphothreonine antibodies, we observed that the loss of membrane claudin-1 was accompanied by dephosphorylation of this TJ protein. Overall, our findings showed an important redistribution of claudin-1 in cells treated with STb toxin. The loss of phosphorylated TJ membrane claudin-1 is likely to be involved in the increased permeability observed. The mechanisms by which these changes are brought about remain to be elucidated.

Virulence profiles of enterotoxigenic, shiga toxin and enteroaggregative Escherichia coli in South African pigs

Enterotoxigenic Escherichia coli (ETEC) and shiga toxin E. coli (STEC) are important causes of colibacillosis in piglets. Recently, enteroaggregative E. coli heat-stable enterotoxin 1 (EAST-1) has been implicated in pig diarrhoea. This study investigated the prevalence of enterotoxin [heat-labile toxins (LT), heat-stable toxin a (STa), heat-stable toxin b (STb)], shiga toxins (Stx1, Stx2, Stx2e), enteroaggregative heat-stable E. coli (EAST-1), associated fimbriae (F4, F5, F6, F41, F18ab, F18ac) and non-fimbrial adhesins [adhesin involved in diffuse adherence 1 (AIDA-1), attaching and effacing factor, porcine attaching- and effacing-associated factor] in South African pigs. A total of 263 E. coli strains were isolated from Landrace (n = 24), Large White (n = 126), Duroc (n = 28) and indigenous (n = 85) breeds of piglets aged between 9 and 136 days. PCR was used in the analysis. Virulent genes were detected in 40.3% of the isolates, of which 18.6, 0.4 and 17.5% were classified as ETEC, STEC and enteroaggregative E. coli (EAEC), respectively. Individual genes were found in the following proportions: STb (19.01%), LT (0.4%), STa (3.4%), St2xe (1.1%) and EAST-1 (20.2%) toxins. None of the tested fimbriae were detected in ETEC and STEC isolates. About one third of the ETEC and STEC isolates was tested negative for both fimbrial and non-fimbrial adhesins. Twenty-five pathotypes from ETEC-, EAEC- and STEC-positive strains were identified. Pathotypes EAST-1 (30.2%), STb (13.2%) and STb/AIDA-1 (10.4%) were most prevalent. The study provided insight on possible causes of colibacillosis in South African pigs.

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines

A previous study conducted in our laboratory demonstrated that cells having internalized Escherichia coli STb toxin display apoptotic-like morphology. We therefore investigated if STb could induce programmed cell death in both a human and an animal intestinal epithelial cell lines. HRT-18 (Human Colon Tumor) and IEC-18 (Rat Ileum Epithelial Cells) cell lines were used. As STb is frequently tested in a rat model, the IEC-18 cell line was most relevant to our work. The cell lines were treated with various amounts of purified STb (nanomole range) for a period of 24 h after which cells were harvested and examined for apoptotic characteristics. Caspase-9, the initiator of mitochondrion-mediated apoptosis, and caspase-3, an effector of caspase-9, were both activated following STb intoxication of HRT-18 and IEC-18 cells whereas caspase-8, the initiator caspase of the extrinsic pathway, was not activated. For both cell lines, agarose gel electrophoresis of the cell DNA content reveals laddering of DNA, resulting from DNA fragmentation, a characteristic of apoptosis. Hoechst 33342-stained DNA of STb-treated cell lines, observed using fluorescence microscopy, revealed condensation and fragmentation of the nuclei. Apoptotic indexes calculated from fragmented nuclei of Hoechst 33342-stained DNA for HRT-18 and IEC-18 cells showed an STb dose-dependent response. Overall, these data indicate that STb toxin induces a mitochondrion-mediated caspase-dependent apoptotic pathway.

Biochemical and biological characterization of Escherichia coli STb His12 to Asn variant

We identified a variant of Escherichia coli STb toxin by PCR amplification of clinical isolates obtained from diseased pigs. The variant differed by only one amino acid at position 12 from His to Asn. This change was observed in 23 of the 100 randomly selected enterotoxigenic E. coli (ETEC) isolates tested. There was a positive correlation between the presence of the STa enterotoxin and the STb variant. As the variant represented a high percentage of the ETEC strains tested, we were interested in determining if the single amino acid change results in altered biological characteristics of the toxin. Circular dichroism analysis revealed that the secondary structure of the variant was similar to wildtype and that their thermal stabilities were similar. Surface plasmon resonance showed that the variant and the wildtype toxins possessed similar binding affinities for sulfatide but the variant exhibited a reduced binding capacity. A flow cytometry-based internalization assay showed that the variant toxin is more internalized into epithelial intestinal cells than the wildtype strain. However, this difference was minor. Overall, our results indicate that while wildtype STb and the variant share similar structural properties, modest differences exist in their internalization.

Prevalence of enterotoxigenic Escherichia coli virulence genes from scouring piglets in Zimbabwe

World-wide, enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC)-induced diarrhea are economically important for porcine producers. Our aim was to investigate the prevalence of toxin and fimbrial genes among E. coli isolated from diarrheic piglets from randomly selected piggeries in Zimbabwe. We used multiplex PCR for screening STa, STb, LT, and Stx-2e toxins. Subsequently F4, F5, F6, F18 and F41 fimbriae genes were screened in toxin positive isolates. Toxin positive strains lacking tested fimbriae genes were characterized using transmission electron microscopy, agglutination and agglutination inhibition tests. Approximately 32% of the 1,984 isolates tested positive for STa, STb, LT or Stx-2e genes. Of these, approximately 81% had F4, F5, F6, F18 or F41 fimbriae genes. The remaining toxin positive strains lacked tested fimbriae genes and appeared to either express F1-like fimbriae, or lacked fimbriae. The data constitute an important framework for implementation of prevention measures, such as using relevant fimbriae-based vaccines against ETEC induced diarrhea or VTEC-induced edema.

Effect ofEscherichia coliSTb toxin on NIH-3T3 cells

Previous studies have shown that STb causes microscopic histological alterations in animal intestinal models. Disrupted intestinal epithelium at the villous tips could be the result of an altered physiological cell state induced by the toxin. As a cellular model we used NIH-3T3 cells, a mouse fibroblast cell line, previously shown to be capable of internalizing the STb toxin. Using various probes specific for the cellular physiological state or cell organelles, we investigated STb activity using flow cytometry and confocal microscopy. In NIH-3T3 cells, labelled with propidium iodide and carboxyfluorescein diacetate, STb permeabilized the plasma membrane but the cellular esterases remained active. Confocal microscopy showed that fluorescein isothiocyanate (FITC)-labelled STb toxin molecules were internalized and were found scattered in the cytoplasm. Moreover, important clusters of FITC-STb were observed inside the cells after 6 h and these clusters matched with mitochondria labelling. After cell treatment with STb, using a fluorescent mitochondrial potential sensor, we observed mitochondria hyperpolarization, as an early event of intoxication. This phenomenon increased linearly with the dose of STb. The cell population treated with STb showed histological alterations such as membrane budding, granular cytoplasm and enlarged nucleus. Altogether, these results provide new information, at the cellular level, on the effect of the STb toxin.

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

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 lambda-carragenan but not by galactose, 4-SO(4)-galactose or 6-SO(4)-galactose. Inhibition of STb binding to its receptor was achieved using lambda-carragenan at picomolar concentrations. Then, using IPEC-J2 cells in culture and flow cytometry, we showed that lambda-carragenan was able to inhibit the permeabilization process associated with STb.

Escherichia coliSTb toxin and colibacillosis: knowing is half the battle

Expression of both adherence and enterotoxin expression are required for enterotoxigenic Escherichia coli (ETEC) strains to cause colibacillosis. ETEC strains are responsible for diarrhea in humans and animals by production of various enterotoxins. For many years, the role of the heat-stable E. coli enterotoxin STb as a diarrhea-causing toxin in animals, and in particular in swine, has been controversial. In fact, although the presence of STb-positive E. coli strains and diarrhea in animals is frequently observed, the difficulty of reproducing the pathology in an animal model was interpreted as a lack of toxicity. Recently, new light was shed on the activity of STb in intestinal ligated loops and in pigs orally inoculated with STb-positive E. coli strains. This minireview revisits the effects of STb on the intestinal epithelium and enlightens the significance of STb in swine colibacillosis. The interaction of STb toxin with other E. coli enterotoxins and dual ETEC/enteropathogenic E. coli or ETEC/attaching effacing E. coli infections are also discussed.

Serotypes, virulence genes, and PFGE profiles of Escherichia coliisolated from pigs with postweaning diarrhoea in Slovakia

BackgroundPostweaning diarrhoea (PWD) in pigs is usually the main infectious problem of large-scale farms and is responsible for significant losses worldwide. The disease is caused mainly by enterotoxigenic E. coli (ETEC) and Shiga-toxin producing E. coli (STEC). In this study a total of 101 E. coli isolated from pigs with PWD in Slovakia were characterized using phenotypic and genotypic methods.ResultsThese 101 isolates belonged to 40 O:H serotypes. However, 57% of the isolates belonged to only six serotypes (O9:H51, O147:H-, O149:H10, O163:H-, ONT:H-, and ONT:H4), including two new serotypes (O163:H- and ONT:H4) not previously found among porcine ETEC and STEC isolated in other countries. Genes for EAST1, STb, STa, LT and Stx2e toxins were identified in 64%, 46%, 26%, 20%, and 5% of isolates, respectively. PCR showed that 35% of isolates carried genes for F18 colonization factor, and further analyzed by restriction endonuclease revealed that all of them were F18ac. Genes for F4 (K88), F6 (P987), F17, F5 (K99), F41, and intimin (eae gene) adhesins were detected in 19 %, 5%, 3%, 0.9%, 0.9%, and 0.9% of the isolates, respectively. The study of genetic diversity, carried out by PFGE of 46 representative ETEC and STEC isolates, revealed 36 distinct restriction profiles clustered in eight groups. Isolates of the same serotype were placed together in the dendrogram, but high degree of polymorphism among certain serotypes was detected.ConclusionSeropathotype O149:H10 LT/STb/EAST1/F4 (14 isolates) was the most commonly detected followed by O163:H- EAST1/F18 (six isolates), and ONT:H4 STa/STb/Stx2e/F18 (five isolates). Interestingly, this study shows that two new serotypes (O163:H- and ONT:H4) have emerged as pig pathogens in Slovakia. Furthermore, our results show that there is a high genetic variation mainly among ETEC of O149:H10 serotype.

Serotypes, virulence genes, and PFGE patterns of enteropathogenic Escherichia coli isolated from Cuban pigs with diarrhea.

Thirty-six enteropathogenic Escherichia coli strains isolated from Cuban pigs with diarrhea were serotyped and screened by PCR for the presence of virulence genes. The 36 isolates belonged to 11 O serogroups and 14 O:H serotypes, with 53% of the isolates belonging to only two serotypes: O141:H- (13 isolates) and O157:H19 (6 isolates). Genes coding for STb, STa, VT2e, and LT toxins were identified in 69, 61, 53, and 6% of the isolates, respectively. The most prevalent fimbrial adhesin was F18, detected in 22 (61%) isolates. The gene encoding F6 (P987) colonization factor was identified in three (8%) isolates. None of the 36 isolates assayed contained genes encoding F4 (K88), F5 (K99), or F41. The seropathotype O141:H-:STa/STb/VT2e/F18 (13 isolates) was the most frequently detected, followed by O157:H19:VT2e/F18 (5 isolates). A genetic diversity study, carried out by pulsed-field gel electrophoresis (PFGE) of 24 representative isolates, revealed 21 distinct restriction patterns clustered in 18 groups (I-XVIII). Isolates of the same serotype were placed together in a dendrogram, but isolates of serotype O157:H19 showed a high degree of polymorphism. The results of this study demonstrate the presence in Cuba of different clusters among one of the most prevalent serotypes isolated from pigs with diarrhea. Further experiments are needed to determine whether some of these clusters have appeared recently; if so, their evolution, as well as their possible association with pathogenicity in farms should be studied.

Escherichia coli heat-stable enterotoxin b (STb) in vivo internalization within rat intestinal epithelial cells.

Heat-stable enterotoxin b (STb) is a low molecular weight toxin known to bind sulfatide, its receptor. The fate of STb bound to rat intestinal epithelium cells was followed using an anti-toxin gold labeled assay and transmission electron microscopy. The data suggest that STb toxin and the fusion protein maltose binding protein (MBP)-STb were internalized whereas its mutant I41 E-M42R with reduced hydrophobicity did not show internalization. There was a significant difference in the mean of gold particles per field between rat intestine incubated with STb or the fusion protein MBP-STb and the negative control consisting of intestine incubated with PBS alone. No subcellular compartment seems to be particularly aimed by the toxin as gold particles were randomly distributed within the cell.

Binding to sulfatide and enterotoxicity of various Escherichia coli STb mutants.

Binding of the 48 amino acid polypeptide of the mature heat-stable Escherichia coli enterotoxin b (STb) to the functional receptor sulfatide (SFT) constitutes the first step in inducing secretory diarrhoea in the intestinal lumen of animals. The NMR structure of this toxin dictated the choice of amino acids for site-directed mutagenesis to delineate the binding site of STb to SFT. Amino acids facing the solvent either in the loop or the hydrophobic alpha-helix were selected. Seventeen site-specific mutants of STb toxin were produced and purified by high-pressure liquid chromatography. Enterotoxicity of the 17 mutants was determined using a rat loop assay and binding was evaluated using a microtitre plate binding assay. Both hydrophobic and electrostatic interactions are important for STb attachment. When mutations (F37K, I41S and M42S) were introduced into the hydrophobic alpha-helix to lessen hydrophobicity, binding activity and enterotoxicity decreased by more than sixfold. The loop defined by C21 and C36 also made specific contributions. Mutants generated at basic residues (K22, K23 and R29) within this region exhibited both reduced binding activities and reduced toxic activities. For all STb mutants constructed and analysed, when binding to SFT was reduced, a reduction in toxicity equivalent or greater was noted, indicating that binding to SFT is a step that precedes the toxic effect observed for STb toxin. Significantly, when the negatively charged D30 was substituted for either alanine or valine, the binding to SFT was about twice that of native STb, whereas the enterotoxicity was reduced by half.

Trypan blue uptake by chinese hamster ovary cultured epithelial cells: a cellular model to study Escherichia coli STb enterotoxin

The thermostable enterotoxin b (STb) produced by enterotoxigenic Escherichia coli strains is responsible for diarrheal diseases mainly in weaning piglets. For now, the only available assay for biological activity of STb toxin was in the animal host (i.e. piglet) or in an animal model (i.e. rat, mouse). In this study, we developed a cellular model for the study of the biological activity of STb enterotoxin. Using a trypan blue vital stain method, we showed that STb-treated cells of three out of the five cell lines tested absorbed more vital stain than their controls. Of all the cell lines tested, the chinese hamster's ovary derivated cells (CHO) were the most sensitive, absorbing 50% more trypan blue than their control. Maximal stain uptake was observed after 2 h. We then evaluated the trypan blue uptake for 16 STb mutants, produced in a previous work, on the CHO cell lines in order to compare it with the in vivo rat loop assay data. Interestingly, we observed a good correlation between the two bioassays. In fact, the biological activity observed in the rat could be correlated with the trypan blue uptake by the CHO cells ( R 2=0.78) for STb toxin and the 16 mutants. Using the variance analysis statistical test, we determined that the correlation between the two bioassays is significant ( F c≥ F 0.005). These results suggest that the trypan blue uptake bioassay could represent a new method to evaluate the biological activity and facilitate the elucidation of the mechanism of action of E. coli STb enterotoxin.

Oligomerization of Escherichia coli enterotoxin b through its C-terminal hydrophobic α-helix

Using a chemical cross-linker and gel electrophoresis or a dot blot overlay assay, we studied protein–protein interaction of STb toxin, a 48-residue amphiphilic polypeptide causing intestinal disorders. For the first time, we report on the oligomerization property of STb. This enterotoxin forms hexamers and heptamers in a temperature-independent fashion in presence or absence of its receptor (sulfatide) anchored in a 50-nm liposome or as a free molecule. Full STb structure integrity is necessary for its oligomerization as this process is not observed under reducing conditions in the presence of β-mercaptoethanol. STb treatment with tetramethylurea (TMU) and different detergents prevented oligomerization. Site-directed mutagenesis decreasing overall STb hydrophobicity in the hydrophobic α-helix resulted in the incapacity to form oligomers. Taken together, these data suggest that the C-terminal hydrophobic α-helix corresponds to the domain of STb–STb inter-binding where hydrophobic interaction is involved.