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PubMed2005

Humoral immune response to rotavirus NSP4 enterotoxin in Spanish children.

The rotavirus non-structural protein 4 (NSP4) has been shown to play a crucial role in rotavirus-induced diarrhea, acting as a viral enterotoxin. It has also been demonstrated that antibody to NSP4 can reduce the severity of rotavirus-induced diarrhea in newborn mice. Two recombinant baculoviruses, expressing the NSP4 protein from the SA11 and Wa rotavirus strains, genotypes A and B, respectively, were used to produce and purify these glycoproteins, which were applied as antigen in an enzyme-linked immunosorbent assay (ELISA) to test the specific antibody response to NSP4 in human sera. Serum samples from 30 children convalescing from a rotavirus infection, from 54 healthy children under 5-years-old, and from 49 adults were tested to determine the presence of antibodies to the viral enterotoxin and to rotavirus structural proteins. Seventy percent of the sera from rotavirus-infected children showed an IgG antibody response to either one or both NSP4 proteins used in this study, although the response was weak. However, IgG antibodies towards either one or both NSP4 proteins were only detected in 26% of the non-convalescent healthy children and in only 18% of the adults. No serum IgA antibodies towards NSP4 were found in this study. IgG antibody recognition of the NSP4 protein from the SA11 and Wa rotavirus strains was not always heterotypic.

Adult↗

PubMed2004

Systematic survey on the prevalence of genes coding for staphylococcal enterotoxins SElM, SElO, and SElN.

Staphylococcus aureus remains a leading cause of food-poisoning with substantial impact on public health. Using a multiplex polymerase chain reaction-DNA enzyme immunoassay (PCR-DEIA), we studied the presence of genes encoding staphylococcal enterotoxin-like (SEl) superantigens sem, sen, and seo, associated with the enterotoxin gene cluster (egc), in 429 clinical Staphylococcus aureus isolates. 294 (68.5%) isolates tested positive for at least one of the three SEl genes. In contrast to the fixed gene combination seg/sei also located on egc, a substantial number of isolates (n = 108) were found to bear only one or two of the genes encoding SElM, SElN, and SElO. Regarding the origin of the S. aureus isolates, a significant difference (P = 0.022) was found for the possession of seo (61.2% of blood isolates versus 42.9% of nasal strains). Also sem (not significantly) was found more common in blood isolates (52.1% versus 40.5%). The survey of the newly described SEl genes sem-seo supports the concept that most clinical S. aureus isolates harbor subsets of pyrogenic toxin superantigens. The potential contribution of seo and sem to the pathogenic potential of S. aureus has to be further evaluated.

Bacteremia↗

PubMed1993

Purification of staphylococcal enterotoxin E.

Staphylococcal enterotoxin E has been purified by a combination of Red-A ligand chromatography and Mono-S (cation-exchange) chromatography. Commencing with 30 litres of fermenter-grown culture the method has been used to purify 17 mg of toxin, giving a purification yield of around 40%. The purity of the toxin was over 98% when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, but demonstrated three isoelectric forms by isoelectric focusing, with pl values of 7.6, 7.4, and 7.0. This method adds to the versatility of the Red-A matrix system which can also be used for the purification of staphylococcal enterotoxins A, B, and C2.

Enterotoxins↗

PubMed1996

Tumor marker disaccharide D-Gal-beta 1, 3-GalNAc complexed to heat-labile enterotoxin from Escherichia coli.

Heat-labile enterotoxin (LT) is part of the cholera toxin (CT) family and consists of a catalytic A subunit and a B pentamer that serves to recognize the oligosaccharide part of the GM1 ganglioside receptor. We report here the crystal structure of heat-labile enterotoxin in complex with the disaccharide portion of the Thomsen-Friedenreich (T-antigen) tumor marker. The toxin:carbohydrate complex is determined to 2.13 A resolution, yielding an R-factor of 18.5%. The T-antigen disaccharide, D-Gal-beta 1,3-GalNAc-Ser/Thr, is present in more than 85% of human carcinomas and monitoring its autoimmune response is used for the early detection of tumors. Insight into the molecular recognition of this tumor antigen by sugar binding proteins can benefit the development of a diagnostic tool for human carcinomas as well as a T-antigen directed anticancer drug delivery system.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed1997

Crystal structure of heat-labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit.

Cholera toxin (CT) produced by Vibrio cholerae and heat-labile enterotoxin (LT-I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP-ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti-diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT-I are known to be relatively temperature sensitive. To improve the thermostability of LT-I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT-I has been determined to 2.0 A resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT-I by 6 degrees C. The enhancement in thermostability could make this disulfide bond variant of LT-I of considerable interest for the design of enterotoxin-based vaccines.

Bacterial Toxins↗

PubMed1997

A receptor-based immunoassay to detect Staphylococcus enterotoxin B in biological fluids.

A rapid, simple, and inexpensive sandwich enzyme-linked receptor based immunodot assay was developed for the detection of staphylococcal enterotoxin B (SEB) in human fluids by using purified glycosphingolipid digalactosylceramide (diGalCer) receptor for SEB. Three micrograms of diGalCer was immobilized on a polyvinyledene difluoride membrane and the membrane was subsequently incubated with primary and secondary alkaline-phosphatase-labeled antibodies. A positive reaction was discerned as a blue spot. As little as 1 ng/ml of SEB could be detected in the assay. SEB did not bind to structurally related glycosphingolipids, such as glucosylceramide, galactosylceramide, and lactosylceramide in this assay. Of five monoclonal anti-SEB antibodies and commercial anti-SEB antiserum tested, the latter was the most sensitive in our assay. The specificity of SEB assay was assessed by comparison with structurally related toxins, for example, staphylococcal enterotoxin A, and toxic shock syndrome toxin 1 (TSST-1). TSST-1 was not detected in the assay. This was because these toxins were not recognized by the anti-SEB antibody and did not bind to diGalCer. In conclusion, we believe that this assay may be widely applicable because it is highly specific for SEB, it does not require special equipment, and the results can be obtained within few hours with the naked eye. Since the receptor for SEB has a long shelf life, it can be easily stored and used for a long time.

Antibodies↗

PubMed1996

Induction of interstitial pneumonia in autoimmune mice by intratracheal administration of superantigen staphylococcal enterotoxin B.

The pathogenesis of lung complications in autoimmune disease remains unclear. To examine whether superantigens participate in the development of interstitial pneumonia in autoimmune disease, we instilled the bacterial superantigen staphylococcal enterotoxin B (SEB) into the tracheas of autoimmune and nonautoimmune mice. The intratracheal administration of SEB resulted in the induction of interstitial pneumonia manifested by infiltration of mononuclear cells into the alveolar septal walls and into the periarterial space and an increase in pulmonary interstitial collagen fibers in the autoimmune mouse strains. In the nonautoimmune strains, AKR, but not BALB/c and B10BR, mice also developed interstitial pneumonia after the intratracheal administration of SEB, although the degree of severity was milder than that induced in three autoimmune mouse strains. Although the intratracheal administration of another bacterial superantigen staphylococcal enterotoxin A also induced interstitial pneumonia, protein A which is a staphylococcal product but not a superantigen induced no remarkable change in lungs of MRL-+/+ mice. Immunohistologic studies revealed that not only SEB-reactive Vbeta8+ T cells, but also SEB-nonreactive Vbeta6+ T cells infiltrated the pulmonary lesions of SEB-primed MRL-+/+ mice, although this may be a secondary reaction for the Vbeta6+ T cells. The results of in vitro restimulation of spleen cells from SEB-primed BALB/c and SEB-primed MRL-+/+ mice with SEB suggest that incomplete induction of tolerance after the intratracheal administration of SEB may be involved in the pathogenesis of interstitial pneumonia induced in autoimmune mice. These results suggest participation of superantigens in the development of interstitial pneumonia in patients with autoimmune disease and other lung diseases such as idiopathic pulmonary fibrosis.

Animals↗

PubMed1999

Requirement of I-E molecule for thymocyte apoptosis induced by staphylococcal enterotoxin B in vivo.

In vivo administration of bacterial superantigen staphylococcal enterotoxin B (SEB) to BALB/c mice led to thymus atrophy resulting from thymocyte apoptosis. In this study, we demonstrated that SEB induced a substantial reduction in thymocyte numbers in BALB/c, B10. D2 (H-2(d) haplotype), B10.BR, C3H/HeJ, C3H/HeN (H-2(k)), and (BALB/c x B6)F1 (H-2(dxb)), but caused little or no effect in I-E- strains such as B6, B10, A.BY (H-2(b)), and A.SW (H-2(s)) mice. Elimination of CD4(+)CD8(+) cells predominantly accounted for the thymocyte loss, although the numbers of other subpopulations may also be reduced. Thymocyte apoptosis was shown by an increase in the level of DNA fragmentation in BALB/c but not in B6 mice after SEB administration. Treatment with anti-I-Ed monoclonal antibody to BALB/c mice blocked SEB-induced thymocyte apoptosis when anti-I-Ad exerted less effect. In contrast to SEB, staphylococcal enterotoxin A led to comparable levels of thymus atrophy in BALB/c and B6 mice. Studies on the surface marker expression indicated that CD25 expression was upregulated on BALB/c mouse thymocytes but with only a moderate increase in B6 mice. The CD4(+)CD8(+) cells were the major (>90%) population that expressed elevated levels of CD25 in BALB/c mice. An increase in the expression of TCRalphabeta, CD3, and CD69 surface markers was also observed on thymocytes from BALB/c mice, but not from I-E- strains. The differential response of I-E+ and I-E- mice to SEB may be exploited as a model for the study of apoptosis in the thymus.

Animals↗

PubMed2001

Roles of I-E molecule and CD28 costimulation in induction of suppression by staphylococcal enterotoxin B in vivo.

Exposure to bacterial superantigens leads to the induction of a subsequent state of immune hyporesponsiveness. Using a transwell coculture system, a previous report demonstrated that splenocytes from staphylococcal enterotoxin B (SEB)-injected BALB/c mice secreted soluble mediators to suppress the proliferative response of naive syngeneic splenocytes to SEB stimulation. We show in the present study that, in contrast to the suppressive effect induced by SEB in BALB/c (H-2(d) haplotype), MRL(+/+), and MRL-lpr/lpr (H-2(k)) mice, SEB-primed splenocytes from I-E(-) strains such as B6, B10, A. BY (H-2(b)), and A.SW (H-2(s)) mice failed to inhibit the CD25 expression and the proliferative activity of their syngeneic naive responder splenocytes. Further results revealed that the SEB-primed cells from BALB/c, but not B6, mice inhibited the CD25 expression and proliferation of naive responder cells from either BALB/c or B6 mice, indicating the critical regulatory role of the effector cells. Unlike SEB, staphylococcal enterotoxin A induced profound suppression in both BALB/c and B6 mice. Moreover, the suppressive competence of SEB-primed splenocytes was diminished in CD28-deficient BALB/c mice. Taken together, our results indicate that when SEB is used as a stimulator in vivo, both the I-E molecule and CD28 costimulation are required for the induction of regulatory cells bearing suppressive activity.

Animals↗

PubMed1993

High yield of active STb enterotoxin from a fusion protein (MBP-STb) expressed in Escherichia coli.

Maltose binding protein (MBP) fused to STb, a heatstable enterotoxin of Escherichia coli, was secreted into the periplasm. A factor Xa cleavage site is present between MBP and STb allowing MBP to be cleaved from STb. The gene fusion is under the control of the strong and inducible Ptac promoter. Three hours after induction with IPTG, cells were harvested. Following osmotic shock treatment of the cells, the MBP-STb fusion protein was released and affinity-purified using an amylose resin. The fusion protein purified in this way was biologically active in ligated intestinal segments of rats. Digestion of MBP-STb with factor Xa released native STb which was purified to homogeneity by reverse-phase chromatography using a PepRPC column. The toxin was eluted at approximately 38% acetonitrile. The 5000-Da toxin was shown to be pure by SDS-PAGE and immunoblotting. The recovered enterotoxin was active in the rat loop assay. Amino acid sequence analysis showed that the first eight residues were identical to those of native STb, confirming the identity of STb. The ultraviolet absorption spectra of purified STb revealed low absorption at 254 and 280 nm compared to 210-230 nm. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.6. Typically, 8 liters of bacterial culture resulted in 2.2 mg of pure STb. This genetic construction provides a readily obtainable source of biologically active STb toxin.

ATP-Binding Cassette Transporters↗

PubMed1996

Use of Vibrio spp. for expression of Escherichia coli enterotoxin B subunit fusion proteins: purification and characterization of a chimera containing a C-terminal fragment of DNA polymerase from herpes simplex virus type 1.

The nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a convenient carrier molecule for the attachment and delivery of heterologous peptides into eukaryotic cells. To evaluate the properties of such EtxB-based fusion proteins an efficient method for their production and purification is required. High-level production and purification of native EtxB has been achieved using heterologous expression and secretion in a marine Vibrio (Amin, T., and Hirst, T. R., 1994, Protein Expression Purif. 5, 198-204). However, the use of this method to isolate EtxB fusion proteins has been precluded because of their susceptibility to degradation by extracellular proteases secreted by members of the Vibrionaceae. In this paper a method is described for production of EtxB-pol, comprising the enterotoxin B subunit linked to a 27-residue C-terminal fragment of Pol, the catalytic subunit of DNA polymerase of herpes simplex virus type 1 (HSV-1). Following assessment of the relative efficacy of different Vibrio strains as hosts for EtxB-pol expression, the chimera was produced at the highest level of 3.5 mg/liter by cultures of Vibrio sp.60. Addition of 0.3 mM EDTA to the growth medium blocked proteolysis of the secreted EtxB-pol fusion protein, which was then purified to homogeneity using ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 57%. Purified EtxB-pol reacted with both anti-EtxB and anti-Pol peptide antibodies, and was able to specifically bind UL42, a processivity factor which normally binds to the C-terminal region of HSV-1 Pol. This modified method for expression and purification of EtxB-pol should be of general utility for the preparation of other EtxB-based fusion proteins.

Bacterial Toxins↗

PubMed2000

The envelope glycoprotein of simian immunodeficiency virus contains an enterotoxin domain.

By the use of a mouse model, the enteropathic effects of the simian immunodeficiency virus (SIV) surface unit (SU) envelope glycoprotein were explored. Purified SU (0.01-0.45 nmol) was administered intraperitoneally to 6- to 8-day-old mouse pups and induced a dose-dependent diarrheal response. Surgical introduction of SU into adult mouse intestinal loops revealed fluid accumulation without histological alterations and SU-treated unstripped intestinal mucosa induced chloride (Cl(-)) secretory currents in Ussing chambers. Similarly to rotavirus NSP4, the first described viral enterotoxin, SU induced a transient increase in intracellular calcium levels and increased inositol 1,4,5-triphosphate (IP(3)) levels in HT-29 cells. These data indicate the calcium response is mediated by IP(3). The presence of diarrhea and fluid accumulation within intestinal loops in the absence of histological alterations and induction of Cl(-) secretory currents demonstrate that SIV contains an enterotoxic domain localized within SU and is the second viral enterotoxin described.

Animals↗

PubMed1985

Purification by high performance liquid chromatography of Clostridium perfringens type A enterotoxin prepared from high toxin producers selected by a toxin-antitoxin halo.

High enterotoxin-producing substrains of Clostridium perfringens type A were selected reproducibly as colonies having toxin-antitoxin haloes on agar plates of Duncan-Strong medium containing antitoxin serum. Enterotoxin from these substrains was subjected to rapid purification by high performance liquid chromatography (HPLC). For this, the toxin was extracted by sonication from sporulating bacteria grown in Duncan-Strong sporulation medium, fractionated by ammonium sulfate (40% saturation) precipitation and differential solubilization and then purified by HPLC: gel permeation chromatography through a G2000SW column and ion-exchange chromatography on a Mono Q column. Purified toxin preparations had a similar specific activity (4.2 X 10(2) mouse MLD/mg protein) and homogeneity on polyacrylamide gel-electrophoresis to preparations obtained by conventional gel permeation through a Sephadex-G200 column. By further HPLC on a Mono Q column, minor nontoxin proteins were separated from the toxin without loss of the toxicity on a protein basis. The final yield of the purified toxin was about 15% of that in the bacterial extract. The two HPLC procedures each took only one hour.

Animals↗

PubMed1987

Nucleotide sequence of the staphylococcal enterotoxin C1 gene and relatedness to other pyrogenic toxins.

The nucleotide sequence for the structural gene entC1 encoding staphylococcal enterotoxin C1 was determined. The gene contained 801 bp and coded for a protein of 266 amino acids. Of these, 27 comprised the signal peptide. Cleavage of the signal peptide resulted in a mature protein with 239 amino acids and a calculated molecular weight of 27,496. The nucleotide sequence of entC1 shared considerable homology (74% and 59%, respectively) with genes encoding enterotoxin B and streptococcal pyrogenic exotoxin A. A similar degree of amino acid homology was observed after alignment of the respective proteins. Thus, certain regions of these three toxin molecules possess structural similarities that may be responsible for shared biological properties.

Amino Acid Sequence↗

PubMed1986

Enterotoxin A production in Staphylococcus aureus: inhibition by glucose.

In this study, we investigated the relationship between carbohydrate metabolism and repression of staphylococcus enterotoxin A (SEA) in Staphylococcus aureus 196E and a pleiotrophic mutant derived from strain 196E. The mutant, designated at strain 196E-MA, lacked a functional phosphoenolpyruvate phosphotransferase system (PTS). The mutant produced acid, under aerobic conditions, from only glucose and glycerol. The parent strain contained an active PTS, and aerobically produced acid from a large number of carbohydrates. Prior growth in glucose led to repression of SEA synthesis in the parent strain; addition to the casamino acids enterotoxin production medium (CAS) led to more severe repression of toxin synthesis. The repression was not related to pH decreases produced by glucose metabolism. When S. aureus 196E was grown in the absence of glucose, there was inhibition of toxin production as glucose level was increased in CAS. The inhibition was related to pH decrease and was unlike the repression observed with glucose-grown strain 196E. The inhibition of SEA synthesis in mutant strain 196E-MA was approximately the same in cells grown with or without glucose and was pH related. Repression of SEA synthesis similar to that seen with glucose-grown S. aureus 196E could not be demonstrated in the mutant. In addition, glucose-grown S. aureus 196E neither synthesized beta-galactosidase nor showed respiratory activity with certain tricarboxylic acid (TCA) cycle compounds. Glucose-grown strain 196E-MA, however, did not show suppressed respiration of TCA cycle compounds; beta-galactosidase was not synthesized because the mutant lacked a functional PTS.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Metabolism↗

PubMed1982

A simple purification method for enterotoxin F produced by Staphylococcus aureus and some properties of the toxin.

Staphyloccoccus aureus enterotoxin F (SEF), which is associated with S. aureus strains isolated from toxic-shock-syndrome patients, was purified by successive chromatography on CM sephadex C-25 and gelfiltration on sephadex G-75. When tested by disc-polyacrylamide gel-electrophoresis the toxin migrated as a homogeneous protein. In SDS-polyacrylamide gel-electrophoresis three protein bands were observed. The main component had a mol wt of 23 000 and the two minor components had a mol wt less than 13 000. By iso-electric focussing a main protein band with an iso-electric point of 7.2 was obtained. The LD50 for rabbits (3-3.5 kg) by subcutaneous and intravenous application of SEF was 6 micrograms and 180 micrograms, respectively. Antibodies to SEF prepared in a sheep did not react with other staphylococcal enterotoxins (A to E).

Antibodies↗

PubMed1994

Combined activation of murine lymphocytes with staphylococcal enterotoxin and interleukin-2 results in additive cytotoxic activity.

This report demonstrates that in vitro activation of murine spleen cells with interleukin-2 (IL-2) or the bacterial superantigen staphylococcal enterotoxin A (SEA) results in different patterns of activation and function of cytotoxic cells. Lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity (ADCC) are mainly mediated by IL-2 activated natural killer (NK) cells. SEA is the most powerful T cell mitogen known so far and retargets cytotoxic T lymphocytes (CTL) to tumors expressing major histocompatibility complex (MHC) class II in staphylococcal-enterotoxin-dependent cellular cytotoxicity (SDCC). Culture of mouse spleen cells with SEA led to expansion and activation of T cells, which demonstrated strong SDCC activity and some NK-like cytotoxicity after 5 days in culture. Cell sorting revealed that both CD8+ and CD4+ T cells mediated SDCC but the former were more effective. Phenotypic analysis showed that SEA preferentially stimulated and expanded T cells expressing T cell receptor V beta 11, in particular CD8+ T cells. Combined activation with SEA and IL-2 resulted in simultaneous induction of T and NK cell cytotoxicity. Moreover, IL-2 had additive effects on SEA-induced SDCC. Combined treatment with SEA and IL-2 might therefore be an approach to induce maximal cytotoxicity against tumors and to recruit both T and NK cells in tumor therapy.

Animals↗

PubMed1990

Indomethacin induced hepatic alterations in mono-oxygenase system and faecal Clostridium perfringens enterotoxin in the rat.

The administration of indomethacin to rats, at the dose of 10 mg/kg once daily for three days, caused a loss of microsomal cytochrome P-450 and cytochrome b5 in the liver, and a fall in drug-metabolizing enzyme activities (i.e. aminopyrine N-demethylase, NADP cyt. c. reductase). Indomethacin also induced intestinal lesions and a significant increase in Clostridium perfringens enterotoxin levels in the feces at 24 hours after both the second and third day of treatment. The above findings suggest that the development of intestinal lesion and the accompanying release of Clostridium perfringens enterotoxin, as well as hepatic enzyme alterations in the rat, result from indomethacin administration. Some of the data in this paper were presented at the Meeting of British Pharmacological Society in Ireland, July 6th-8th, 1988.

Aminopyrine N-Demethylase↗