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ABSTRACT: 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.
Toxicon 12/2011; 59(2):300-5. · 2.51 Impact Factor
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ABSTRACT: Previous studies have suggested that internalization of the Escherichia coli STb enterotoxin in human and rat intestinal epithelial cells is involved in STb pathogenesis, but toxin uptake in porcine jejunum epithelium, the in vivo target tissue, still remains elusive. Using flow cytometry, we studied the internalization of fluorescein isothiocyanate-labelled STb in porcine intestinal epithelial IPEC-J2 and murine fibroblast NIH-3T3 cell lines. In contrast to the selective pronase resistance of STb in NIH-3T3 cells at 37 °C, but not at 4 °C, indicative of toxin internalization, most of the toxin was pronase-sensitive at both temperatures in IPEC-J2 cells, indicating reduced uptake, but significant cell surface binding. Actin reorganization is required for STb internalization by NIH-3T3 cells, confirming STb endocytosis in these cells. The toxin receptor, sulfatide, could not explain these internalization differences because both cell lines possessed surface sulfatide and internalized antisulfatide antibodies over time at 37 °C. Inhibition of lipid rafts endocytosis, known to contain sulfatide, with methyl-β-cyclodextrin or genistein, did not influence toxin uptake by either cell line. STb internalization is therefore differentially regulated depending on the cell type, possibly by factors other than sulfatide. Although a small STb fraction could be internalized by porcine intestinal epithelial cells, our findings suggest the ability of STb to induce, from the cell surface, intracellular signalling leading to fluid secretion in porcine intestinal epithelium.
FEMS Immunology & Medical Microbiology 03/2011; 61(2):205-17. · 2.44 Impact Factor
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The Journal of Infectious Diseases 06/2010; 201(11):1775-6; author reply 1776-7. · 6.41 Impact Factor
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J Daniel Dubreuil
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ABSTRACT: Escherichia coli enterotoxigenic strains produce one or more toxins which action result in production of diarrhea in animals including Man. One of these toxins, STb, has been mainly associated with colibacillosis in swine. Although highly prevalent in pigs with diarrhea, a relation between STb and disease was arduous to establish. With the recent recognition of a new adhesin, originally found in human E. coli isolates, named AIDA (adhesin involved in diffuse adherence) and its association with new E. coli pathotypes to which STb is linked, new light was shed on STb toxic potency. In this review, the association of STb and AIDA is examined according to the recent knowledge gained with newly described E. coli pathotypes.
Critical Reviews in Microbiology 04/2010; 36(3):212-20. · 6.27 Impact Factor
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ABSTRACT: In our ongoing efforts to develop a vaccine against Streptococcus suis infection, we tested the potential of S. suis enolase (SsEno), a recently described S. suis adhesin with fibronectin-binding activity, as a vaccine candidate in a mouse model of S. suis-induced septicemia and meningitis. Here, we show that SsEno is highly recognized by sera from convalescent pigs and is highly immunogenic in mice. Subcutaneous immunization of mice with SsEno elicited strong immunoglobulin G (IgG) antibody responses. All four IgG subclasses were induced, with IgG1, IgG2a and IgG2b representing the highest titers followed by IgG3. However, SsEno-vaccinated and nonvaccinated control groups showed similar mortality rates after challenge infection with the highly virulent S. suis strain 166'. Similar results were obtained upon passive immunization of mice with hyperimmunized rabbit IgG anti-SsEno. We also showed that anti-SsEno antibodies did not increase the ability of mouse phagocytes to kill S. suis in vitro. In conclusion, these data demonstrate that although recombinant SsEno formulated with Quil A triggers a strong antibody response, it does not confer effective protection against infection with S. suis serotype 2 in mice.
FEMS Microbiology Letters 06/2009; 294(1):82-8. · 2.04 Impact Factor
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ABSTRACT: 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.
FEMS Immunology & Medical Microbiology 03/2009; 55(3):432-41. · 2.44 Impact Factor
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ABSTRACT: Streptococcus suis is an important swine pathogen that causes meningitis, endocarditis, arthritis and septicaemia. As a zoonotic agent, S. suis also causes similar diseases in humans. Binding of pathogenic bacteria to extracellular matrix components enhances their adhesion to and invasion of host cells. In the present study we isolated and identified a novel fibronectin-binding protein from S. suis. The native protein (designated SsEno) possessed not only high homology with other bacterial enolases but also enolase activity. We cloned, expressed and purified SsEno and showed that it is ubiquitously expressed by all S. suis serotypes and we identified its surface localization using immunoelectron microscopy. ELISA demonstrated that SsEno binds specifically to fibronectin and plasminogen in a lysine-dependent manner. Additional surface plasmon resonance assays demonstrated that SsEno binds to fibronectin or plasminogen with low nanomolar affinity. Inhibition experiments with anti-SsEno antibodies also showed that bacterial SsEno is important for the adhesion to and invasion of brain microvascular endothelial cells by S. suis. Overall, the present work is the first study, to our knowledge, to demonstrate a fibronectin-binding activity of a bacterial enolase, and shows that, similar to other bacterial fibronectin-binding proteins, SsEno may contribute to the virulence of S. suis.
Microbiology 10/2008; 154(Pt 9):2668-79. · 3.06 Impact Factor
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ABSTRACT: Environmental phosphate is an important signal for microorganism gene regulation, and it has recently been shown to trigger some key bacterial virulence mechanisms. In many bacteria, the Pho regulon is the major circuit involved in adaptation to phosphate limitation. The Pho regulon is controlled jointly by the two-component regulatory system PhoR/PhoB and by the phosphate-specific transport (Pst) system, which both belong to the Pho regulon. We showed that a pst mutation results in virulence attenuation in extraintestinal pathogenic Escherichia coli (ExPEC) strains. Our results indicate that the bacterial cell surface of the pst mutants is altered. In this study, we show that pst mutants of ExPEC strains display an increased sensitivity to different cationic antimicrobial peptides and vancomycin. Remarkably, the hexa-acylated 1-pyrophosphate form of lipid A is significantly less abundant in pst mutants. Among differentially expressed genes in the pst mutant, lpxT coding for an enzyme that transfers a phosphoryl group to lipid A, forming the 1-diphosphate species, was found to be downregulated. Our results strongly suggest that the Pho regulon is involved in lipid A modifications, which could contribute to bacterial surface perturbations. Since the Pho regulon and the Pst system are conserved in many bacteria, such a lipid A modification mechanism could be widely distributed among gram-negative bacterial species.
Journal of bacteriology 09/2008; 190(15):5256-64. · 3.94 Impact Factor
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ABSTRACT: To investigate the presence and frequency of estB variant(s), a collection of 100 STb-positive enterotoxigenic Escherichia coli (ETEC) strains isolated from 1980 to 2007 inclusively and randomly selected from diseased pigs in Québec, Canada, was analysed. A wide diversity of virulence gene profiles (virotypes) was detected in the strain collection. The estB gene was amplified by PCR using primers designed from the signal sequence and the C-terminal end, and the amplified fragment was sequenced using the forward primer. The translated DNA sequence revealed a His(12)-->Asn change in 23 of the 100 ETEC isolates tested. The STb-variant strains were observed throughout the sampling period covered in the study. No other STb-variant type was found in this study. All 23 variant strains were also positive for the STa enterotoxin and were resistant to tetracycline, as for strain 2173. The STb variant was associated with Stx2-positive strains (5/6) and STa : STb strains that did not harbour any of the tested porcine fimbrial adhesins (13/17). The remaining variant strains were associated with fimbriae F4 (1/40), F5 (1/6), F6 (1/1) and F18 (2/7; excluding F18 : Stx2 strains).
Journal of Medical Microbiology 07/2008; 57(Pt 7):887-90. · 2.50 Impact Factor
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ABSTRACT: 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.
FEMS Microbiology Letters 05/2008; 281(1):30-5. · 2.04 Impact Factor
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ABSTRACT: EAST1 (EnteroAggregative heat-Stable Toxin 1) is a 4.1kDa toxin that was first detected in the enteroaggregative Escherichia coli (EAEC) strain 17-2 (O3:H2) isolated from the stools of a Chilean child with diarrhoea. Accordingly, EAST1 is thought to play a role in the pathogenicity of EAEC. The goal of this study was to obtain purified biologically active forms of two EAST1 variants (17-2 and O 42). Purified toxin samples were treated with protein disulfide isomerase (PDI) to ascertain the integrity of the disulfide bridges. Since EAST1 is often compared to STa (heat-Stable Toxin a), both purified EAST1 variants were tested for biological activity using the suckling mouse assay, the reference test for STa. A positive gut to body (G/B) weight ratio was not observed for any of the EAST1 preparations tested, although STa was active. Exposure of the purified toxins to T84 cell monolayers, an epithelial cell line derived from a human colon carcinoma, in modified Ussing flux chambers resulted in a rapidly attained and prolonged increase in short circuit current, a sensitive measure of net ion transport. Responses to 17-2 and O 42 variants were comparable in magnitude and inhibitable by bumetanide and DASU-02, indicating net anion secretion. The results demonstrate that EAST1 toxin stimulates anion secretion by T84 cell monolayers and it is sustained for the duration of toxin exposure.
Comparative immunology, microbiology and infectious diseases 03/2008; 31(6):567-78. · 2.99 Impact Factor
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J Daniel Dubreuil
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ABSTRACT: 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.
FEMS Microbiology Letters 02/2008; 278(2):137-45. · 2.04 Impact Factor
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Microbiology-sgm. 01/2008; 154(9):2668-2679.
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ABSTRACT: Sao is a Streptococcus suis surface protein recently identified as a potential vaccine candidate. In this study, recombinant Sao in combination with Quil A provided cross-protection against S. suis serotype 2 disease in mouse and pig vaccination protocols. Subcutaneous immunization of mice elicited strong immunoglobulin G (IgG) antibody responses. All four IgG subclasses were induced, with the IgG2a titer being the highest, followed by those of IgG1, IgG2b, and IgG3. Challenge of the mice with S. suis strain 31533 resulted in a mortality rate of 80% for the control group, which received Quil A only. In contrast, all of the mice immunized with Sao survived. In a pig vaccination protocol, intramuscular immunization with Sao also elicited significant humoral antibody responses, and both the IgG1 and IgG2 subclasses were induced, with a predominance of IgG2 production. In vitro assay showed that Sao-induced antibodies significantly promoted the ability of porcine neutrophils in opsonophagocytic killing of S. suis. An aerosol challenge of the pigs with S. suis strain 166 resulted in clinical signs characteristic of S. suis infection in diseased pigs. The vaccine group showed significantly better survival, lower clinical scores, and less S. suis recovery from postmortem tissue samples than did the control group. Furthermore, this study also revealed that although challenge S. suis strains express Sao size variants, recombinant Sao conferred cross-protection. These data demonstrate that recombinant Sao formulated with Quil A triggers strong opsonizing antibody responses which confer efficient immunity against challenge infection with heterologous S. suis type 2.
Clinical and Vaccine Immunology 09/2007; 14(8):937-43. · 2.55 Impact Factor
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ABSTRACT: 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.
Infection and Immunity 06/2007; 75(5):2208-13. · 4.16 Impact Factor
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ABSTRACT: A Streptococcus suis surface protein reacting with convalescent-phase sera from pigs clinically infected by S. suis type 2 was identified. The apparent 110-kDa protein, designated Sao, exhibits typical features of membrane-anchored surface proteins of gram-positive bacteria, such as a signal sequence and an LPVTG membrane anchor motif. In spite of high identity with the partially sequenced genomes of S. suis Canadian strain 89/1591 and European strain P1/7, Sao does not share significant homology with other known sequences. However, a conserved avirulence domain that is often found in plant pathogens has been detected. Electron microscopy using an Sao-specific antiserum has confirmed the surface location of the Sao protein on S. suis. The Sao-specific antibody reacts with cell lysates of 28 of 33 S. suis serotypes and 25 of 26 serotype 2 isolates in immunoblots, suggesting its high conservation in S. suis species. The immunization of piglets with recombinant Sao elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are intratracheally challenged with a virulent strain in our conventional vaccination model.
Infection and Immunity 02/2006; 74(1):305-12. · 4.16 Impact Factor
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ABSTRACT: Escherichia coli O78 strains are frequently associated with extraintestinal diseases, such as airsacculitis and septicemia, in poultry, livestock, and humans. To understand the influence of the pst operon in the virulence of E. coli, we introduced mutations into the pst genes of the avian pathogenic E. coli (APEC) O78:K80 strain chi7122 by allelic exchange. The mutation of pst genes led to the constitutive expression of the Pho regulon. Furthermore, the virulence of APEC strain chi7122 in a chicken infection model was attenuated by inactivation of the Pst system. The pst mutant caused significantly fewer extraintestinal lesions in infected chickens, and bacterial numbers isolated from different tissues after infection were significantly lower for the mutant than for the wild-type strain. Moreover, resistance to the bactericidal effects of rabbit serum and acid shock was impaired in the pst mutant, in contrast to the wild-type strain. In addition, the MIC of polymyxin was twofold lower for the mutant than for the wild-type strain. Although the pst mutant demonstrated an increased susceptibility to rabbit serum, this strain was not killed by chicken serum, suggesting the presence of differences in host innate immune defenses and complement-mediated killing. In APEC O78 strain chi7122, a functional Pst system is required for full virulence and resistance to acid shock and polymyxin. Our results suggest that the mutation of pst genes induces a deregulation of phosphate sensing and changes in the cell surface composition that lead to decreased virulence, indicating the importance of the Pst system for the virulence of pathogenic E. coli strains from different hosts.
Infection and Immunity 08/2005; 73(7):4138-45. · 4.16 Impact Factor
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ABSTRACT: Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) is a 4.1k Da protein originally discovered in EAEC but known to be scattered in other diarrheagenic E. coli as well, possibly causing diarrhea in humans and animals. We report for the first time a method to express and purify EAST1 using the Glutathione S-transferase (GST) fusion system. The gst and astA genes were fused together on a pGEX-2T plasmid vector to produce a GST-EAST1 fusion protein. Using Glutathione Sepharose affinity chromatography and C(8) reverse phase high pressure liquid chromatography, EAST1 was purified to homogeneity with a yield of 0.29 mg/L of culture. The protein purified by this method was confirmed to be EAST1 by NH(2)-terminal sequencing and mass spectrometry. The molecular weight of EAST1 is 4104.0 Da, confirming a 38 amino acid peptide as predicted by the astA gene sequence. Mass spectrometry analysis of EAST1 and of two generated peptides established the presence and suggested the position of two disulfide bridges of EAST1 in the conformations C1-C2 and C3-C4. Polyclonal antibodies were raised against EAST1 in New Zealand white rabbits to a titer of 1:8000 using affinity-purified GST-EAST1 fusion protein and to a titer of 1:100 using HPLC-purified EAST1. The biological activity of various EAST1 preparations was tested using the suckling mouse assay with CD-1 and CFW mice strains, but failed to produce fluid accumulation in the intestine.
Protein Expression and Purification 03/2004; 33(2):223-31. · 1.59 Impact Factor
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ABSTRACT: The gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, a disease that causes important economic losses to the swine industry worldwide. In general, the initial step of bacterial colonization is attachment to host cells. The purpose of the present study was to evaluate the binding of A. pleuropneumoniae serotype 1 to phospholipids, which are the major constituents of biological membranes. Phospholipids serve as receptors for several bacteria, including respiratory pathogens. To study this effect, we used thin-layer chromatography overlay binding assays to test commercial phospholipids such as phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine (PE). Our results indicate that A. pleuropneumoniae serotype 1 binds to PE but not to the other phospholipids tested. Serotypes 5b and 7, which, along with serotype 1, are the most prevalent serotypes of A. pleuropneumoniae in North America, share the ability to bind PE. Inhibition of binding with a monoclonal antibody against A. pleuropneumoniae serotype 1 O antigen and the use of isogenic lipopolysaccharide (LPS) mutants of A. pleuropneumoniae serotype 1 showed that the O antigen seems to be implicated in the binding to PE, at least for A. pleuropneumoniae serotype 1. A. pleuropneumoniae was also shown to bind to a phospholipid extracted from swine lungs by using the method of Folch. Chemical staining with molybdenum blue and ninhydrin, migration with neutral, acidic, and basic solvent systems, and mass spectrometry analysis all indicated that this lipid is PE. This study is, to the best of our knowledge, the first description of A. pleuropneumoniae binding to phospholipids. Our data also suggest that LPS O antigens could be involved in binding to PE.
Infection and Immunity 09/2003; 71(8):4657-63. · 4.16 Impact Factor
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ABSTRACT: STb, a 48-amino acid thermostable enterotoxin is produced by enterotoxigenic Escherichia coli strains and is responsible for diarrheal diseases in many animals, including man. Our laboratory recently identified a family of molecules, from a lipid extract of porcine intestinal epithelial cells, that could bind to STb. These molecules were identified as sulfatides as they reacted with a monoclonal antibody raised against this family of molecules. However, as the epitope recognized by this monoclonal antibody was the galactose 3-sulfate, a doubt could remain as to the exact nature of the identified receptors. The goal of this study was thus to confirm the chemical nature of the STb-binding molecule as sulfatides or as distinctive molecules comprising a sulfated galactosyl residue. Using a thin-layer chromatography-overlay method we confirmed using antibodies to STb that STb recognizes the commercial sulfatides and a band migrating at the same level from the intestinal tissue lipid extract obtained from an 8-week-old piglet. The compounds recovered from the silica gel plates were analyzed by mass spectrometry in electrospray negative-ionization mode. The most abundant ions observed had m/z values of 779, 795, 879 and 907. For commercial bovine brain sulfatides the ions 795, 879 and 907 have been attributed to hydroxylated sulfatides with a saturated fatty acid chain containing 16, 22 and 24 carbons, while the 779 ion contained a saturated fatty acid chain of 16 carbons. The general profile of the ions observed was similar to the already described commercial bovine brain sulfatides.
FEMS Microbiology Letters 05/2002; 209(2):183-8. · 2.04 Impact Factor
