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Human major histocompatibility complex class II-negative colon carcinoma cells present staphylococcal superantigens to cytotoxic T lymphocytes: evidence for a novel enterotoxin receptor.

The staphylococcal enterotoxins (SE) bind to major histocompatibility complex (MHC) class II molecules on target cells and activate T cells expressing particular T cell receptor V beta sequences. In this report we demonstrate that SE bind to the MHC class II- SW620, Colo320DM and WiDr human colon carcinoma cell lines and direct cytotoxic T lymphocytes (CTL) to mediate strong target cell killing. Flow cytometry analysis, immunoprecipitation and Northern blotting experiments failed to demonstrate any surface expression of HLA-DR, HLA-DP and HLA-DQ isotypes on the SW620 colon carcinoma cell line, whereas abundant expression of these isotypes was seen on Raji cells, SEB and SEC1 were efficiently presented at picomolar concentration by the MHC class II- colon carcinoma cells and MHC class II+ Raji cells, whereas SEA and SED were preferentially presented on the MHC class II+ Raji cells. An anti-HLA-DR monoclonal antibody inhibited SEB-induced CTL targeting to Raji, but did not influence the killing of SW620 cells. Our data suggests the existence of functionally active SE-binding structures on human colon carcinoma cells which are distinct from the conventional MHC class II molecules. The possibility that these putative new SE receptors play a role in the enterotoxin action of SE must be considered.

Antigens, Bacterial↗

Glycosphingolipids: the putative receptor for Staphylococcus aureus enterotoxin-B in human kidney proximal tubular cells.

We have investigated the binding of 125I-staphylococcal enterotoxin-B (SEB) in cultured human proximal tubular cells. We found that the binding of 125I-SEB to PT cells was time and concentration dependent and competitively inhibited by antibody against SEB. Preincubation of cells with trypsin and neuraminidase or with fetuin did not significantly impair the binding of 125I-SEB to such cells. In contrast, treatment with endoglycoceramidase completely inhibited the binding of 125I-SEB to cells. Neutral glycosphingolipids exerted a concentration-dependent inhibition of 125I-SEB binding to such cells, maximum inhibition (96% compared to control) occurred upon incubation of PT cells with neutral glycosphingolipids. Taken together, our studies indicate that SEB specifically binds to a neutral glycosphingolipid in PT cells. In contrast, staphylococcal enterotoxin-A and toxic shock toxin (TST-1) are bound to a protein in such cells.

Bacterial Toxins↗

Comparison of receptors for Clostridium perfringens type A and cholera enterotoxins in isolated rabbit intestinal brush border membranes.

The rabbit intestinal brush border membrane (BBM) receptors for Clostridium perfringens type A (CPE) and cholera (CT) enterotoxins were compared. Initial studies characterized binding of 125I-CPE to isolated BBMs as specific, saturable, and irreversible. BBMs appear to contain a single type of CPE binding site. Protease pretreatment of BBMs strongly reduced subsequent specific binding of 125I-CPE but not 125I-CT, while neuraminidase pretreatment had little effect on binding of either enterotoxin. Proteases did not significantly release pre-bound 125I-CPE. Preincubation of CPE with an affinity-purified preparation containing a previously identified (Biochem. Biophys. Res. Commun. 112, 1099-105) CPE-binding protein resulted in reduced specific binding of 125I-CPE and an inhibition of CPE biologic activity. Similar experiments showed that CPE-binding protein had no effect on CT binding or biologic activity. Gangliosides had no significant effect on specific binding or biologic activity of CPE but reduced CT binding and biologic activity. Lipids, including gangliosides, separated by thin layer chromatography specifically bound CT but not CPE. Preincubation of BBMs with CT did not reduce subsequent binding of 125I-CPE; conversely, prebound CPE did not affect subsequent 125I-CT binding. These results strongly suggest that CPE does not share the CT BBM receptor ganglioside GM1, and support a role for the CPE-binding protein in CPE binding.

Animals↗

Biogenesis, cellular localization, and functional activation of the heat-stable enterotoxin receptor (guanylyl cyclase C).

Enterotoxigenic Escherichia coli elaborate a peptide called heat-stable enterotoxin (ST), which binds to and activates the intestinal ST receptor (STaR). STaR, also known as guanylyl cyclase C (GC-C), is a member of the transmembrane guanylyl cyclase receptor family. The mRNA for STaR encodes an approximately 120 kDa protein with the N-terminal ligand binding domain on the cell surface. Ligand affinity cross-linking studies have previously demonstrated several species of potential ST binding proteins, ranging in size from approximately 50 to 160 kDa. Although these smaller forms of STaR (50-80 kDa) have been proposed to act in vivo as toxin binding proteins, their biogenesis and localization have not previously been examined. Using pulse labeling in vivo and synchronized translation in vitro, we demonstrate that these smaller forms represent incomplete translational products and are not formed through limited proteolysis of the full-length receptor, as had previously been believed. We determined, using fluorescence confocal microscopy and surface labeling, that only approximately 25% of cellular receptors are expressed at the surface, while the remaining population is retained within the endoplasmic reticulum. Only full-length receptor is found at the surface of the cell, indicating this to be the biologically active form of STaR responsible for interacting with the heat-stable enterotoxin and other luminal intestinal peptides. The large intracellular receptor population, and potential for function before translocation to the cell surface, may impact on how pharmacologic modulators of this clinically important receptor are designed.

Amino Acid Sequence↗

Design of a photoreactive analogue of the Escherichia coli heat-stable enterotoxin STIb: use in identifying its receptor on rat brush border membranes.

The Escherichia coli heat-stable enterotoxin, STIb was prepared by solid-phase peptide synthesis and purified to homogeneity by high-pressure liquid chromatography. This analogue was iodinated and shown to bind specifically to rat intestinal membranes. The radiolabeled peptide was derivatized at the amino terminus with the photoreactive heterobifunctional crosslinking agent N-hydroxysuccinimidyl p-benzoylbenzoate. This photoreactive probe also exhibited binding specificity. It was mixed with rat intestinal brush border membranes and photolyzed in the presence or absence of excess unlabeled STIb. Polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate and 2-mercaptoethanol indicated that the peptide probe was crosslinked specifically to two molecular species of 57 and 75 kDa. One or both of these molecules appear to constitute the enterotoxin receptor or to be in close proximity to it.

Animals↗

Importance of disulfide bridges in the structure and activity of Escherichia coli enterotoxin ST1b.

A 13-amino acid sequence of the Escherichia coli heat-stable enterotoxin ST1b encodes its receptor-binding and diarrheal functions. This sequence includes six cysteines involved in three intramolecular disulfide bridges. To determine the importance of disulfide bridges to the biological activity of ST1b, we synthesized 15 analogues of the tridecapeptide representing all possible replacements of two of the six cysteines by alanines. Only 2 analogues--namely, A6,11ST1b-(6-18) and A10,18ST1b(6-18)--could inhibit the binding of a radiolabeled analogue of ST1b to rat intestinal cells. The purified peptides were, respectively, 4200 and 130 times less effective as inhibitors than ST1b(6-18), the sequence that includes all six cysteines. In addition, both peptides produce diarrhea when given orally to suckling mice. These analogues share in common only two cysteines (Cys-7 and Cys-15), suggesting that four cysteines, two of which are Cys-7 and Cys-15, are necessary for activity. A pattern of disulfide linkages is proposed where Cys-7 is paired to Cys-15, Cys-6 to Cys-11, and Cys-10 to Cys-18, the preceding disulfide bridges being ranked in descending order of importance in terms of their respective contribution to the activity of the enterotoxin. Using this disulfide bridge arrangement and constraints derived from NMR spectroscopy, we propose a folding pattern for the toxic domain of ST1b.

Animals↗

Determination of the binding site on the extracellular domain of guanylyl cyclase C to heat-stable enterotoxin.

Guanylyl cyclase C, one of the family of membrane-bound guanylyl cyclases, consists of an extracellular domain and an intracellular domain, which are connected by a single transmembrane polypeptide. The extracellular domain binds unique small polypeptides with high specificity, which include the endogenous peptide hormones, guanylin and uroguanylin, as well as an exogenous enterotoxigenic peptide, heat-stable enterotoxin, secreted by pathogenic Escherichia coli. Information on this specific binding is propagated into the intracellular domain, followed by the synthesis of cGMP, a second messenger that regulates a variety of intracellular physiological processes. This study reports the design of a photoaffinity labeled analog of heat-stable enterotoxin (biotinyl-(AC(5))(2)-[Gly(4), Pap(11)]STp(4-17)), which incorporates a Pap residue (p-azidophenylalanine) at position 11 and a biotin moiety at the N terminus, and the use of this analog to determine the ligand-binding region of the extracellular domain of guanylyl cyclase C. The endoproteinase Lys-C digestion of the extracellular domain, which was covalently labeled by this ligand, and mass spectrometric analyses of the digest revealed that the ligand specifically binds to the region (residue 387 to residue 393) of guanylyl cyclase C. This region is localized close to the transmembrane portion of guanylyl cyclase C on the external cellular surface. This result was further confirmed by characterization of site-directed mutants of guanylyl cyclase C in which each amino acid residue was substituted by an Ala residue instead of residues normally located in the region. This experiment provides the first direct demonstration of the ligand-binding site of guanylyl cyclase C and will contribute toward an understanding of the receptor recognition of a ligand and the modeling of the interaction of the receptor and its ligand at the molecular level.

Animals↗

A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor.

Secretory diarrhea is the leading cause of infectious diarrhea in humans. Secretory diarrhea may be caused by binding of heat-stable enterotoxins to the intestinal receptor guanylyl cyclase C (GCC). Activation of GCC catalyzes the formation of cGMP, initiating a signaling cascade that opens the cystic fibrosis transmembrane conductance regulator chloride channel at the apical cell surface. To identify proteins that regulate the trafficking or function of GCC, we used the unique COOH terminus of GCC as the "bait" to screen a human intestinal yeast two-hybrid library. We identified a novel protein, IKEPP (intestinal and kidney-enriched PDZ protein) that associates with the COOH terminus of GCC in biochemical assays and by co-immunoprecipitation. IKEPP is expressed in the intestinal epithelium, where it is preferentially accumulated at the apical surface. The GCC-IKEPP interaction is not required for the efficient targeting of GCC to the apical cell surface. Rather, the association with IKEPP significantly inhibits heat-stable enterotoxin-mediated activation of GCC. Our findings are the first to identify a regulatory protein that associates with GCC to modulate the catalytic activity of the enzyme and provides new insights in mechanisms that regulate GCC activity in response to bacterial toxin.

Adaptor Proteins, Signal Transducing↗

Modulation of enterotoxin binding and function in vitro and in vivo.

The use of the nontoxic B subunits of cholera and Escherichia coli enterotoxins in vitro and in vivo led to a decrease in toxin binding to target cells and a decrease in toxin-induced effects (i.e., morphological effects, adenylate cyclase activation, and fluid secretion). The reduction in toxin binding involves a process of down-regulation of cellular receptors for the toxin and not toxin occupancy of receptors. The extent of inhibition was dependent on the amount of B subunit used and on the duration of time after its use. Thus, in vivo exposure to a single bolus of B subunit was sufficient to block toxin binding and activity for up to 18 h. Because the B subunit binds extensively to the esophagus and the stomach, peroral administration will require a preparation that allows the subunit to reach the small bowel in a protected form. Our data provide a rationale for using B subunit therapy for short-term protection against the effects of enterotoxins, before the development of an immune response.

Animals↗

DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN IN FOOD.

Methods are described for the extraction and serological detection of trace amounts of enterotoxins A and B in foods incriminated in outbreaks of staphylococcal food poisoning. Evidence is presented for the probable applicability of the methods for the detection of unidentified enterotoxins.

Adsorption↗

The Escherichia coli heat-stable enterotoxin is a long-lived superagonist of guanylin.

The mechanism by which bacterial heat-stable enterotoxins (ST I STA) cause diarrhea in humans and animals has been linked to the activation of an intestinal membrane-bound guanylate cyclase. Guanylin, a recently discovered rat intestinal peptide, is homologous in structure to ST I and can activate guanylate cyclase present on the human colonic carcinoma cell line T84. To directly test the mechanistic association of guanylate cyclase activation with diarrhea, we synthesized guanylin and a guanylin analog termed N9P10 guanylin and compared their biological activities with those of a synthetic ST I analog, termed ST Ib(6-18). We report that guanylin is able to inhibit the binding of a radiolabeled ST I analog to rat intestinal cells but causes diarrhea in infant mice only at doses at least 4 orders of magnitude higher than that of ST Ib(6-18). In contrast, N9P10 guanylin was enterotoxic in mice at much lower doses than guanylin but proved to be a weaker inhibitor of radiolabeled ST I than guanylin in the receptor binding assay. The pattern of guanylate cyclase activation observed for ST Ib(6-18) and the two guanylin analogs parallels the results observed in the receptor binding assay rather than those observed in the diarrheal assay. Treatment of guanylin with chymotrypsin or lumenal fluid derived from newborn mouse intestines resulted in a rapid loss of binding activity. Together, these results suggest that ST I enterotoxins may represent a class of long-lived superagonists of guanylin.

Amino Acid Sequence↗

Binding of Escherichia coli heat-stable enterotoxin to receptors on rat intestinal cells.

This study was performed to determine whether receptors for Escherichia coli heat-stable enterotoxin (ST) exist on intestinal epithelial cells. Binding sites for 125I-ST were found on rat jejunal and ileal villus cells. Binding was rapid, reversible, linear with cell number, saturable, and temperature dependent. Significant degradation of 125I-ST occurred when incubated with cells at 37 degrees C but not at 25 degrees C. Binding was specific to ST since binding of 125I-ST was competitively inhibited by increasing concentrations of human or porcine ST but not by E. coli heat-labile, cholera, or staphylococcal enterotoxins. Addition of excess unlabeled ST to cells preincubated with 125I-ST resulted in dissociation of much but not all of the bound 125I-ST. Binding of 125I-ST to jejunal and ileal cells occurs with two affinities, and this is due to the phenomenon of negative cooperativity. The potency of ST for inhibiting the binding of 125I-ST was identical to the potency of ST in stimulating cGMP production. These data support the existence of receptors for ST on intestinal cells, and these receptors may be involved in the action of ST.

Animals↗

Staphylococcal enterotoxin B as a potent suppressant of T lymphocytes: trace levels suppress T lymphocyte proliferative responses.

Staphylococcal enterotoxins have long been known to be powerful stimulators of T lymphocytes in mouse and man. In a previous study we showed that high concentrations of staphylococcal enterotoxin serotype B (SEB) failed to stimulate strong proliferative responses by Lewis rat T lymphocytes. Moreover, concentrations of SEB (10-50 micrograms/ml) that stimulated optimal mouse T lymphocyte proliferative responses suppressed a mitogen- or antigen-induced rat T lymphocytes proliferative responses. The present study shows that SEB at low concentrations (as low as 10(-3)-10(-4) micrograms/ml) and often also trace levels (about 10(-6)-10(-7) micrograms/ml) suppresses both rat and mouse T lymphocytes proliferative responses to mitogen or antigen. Furthermore, under different circumstances, SEB may have conflicting effects on the same T cells. While high concentrations (1-50 micrograms/ml) of SEB stimulate certain mouse T cell clones, low concentrations or trace levels have a potent suppressive effect on the same clones. The results indicate that the in vitro conflicting effects of SEB on the same T cells are concentration dependent and may reflect its in vivo effects on SEB-reactive T lymphocytes. The suppression of the mitogen- or antigen-induced stimulation of T cell clones by SEB was direct and did not require the agency of suppressor cells. Furthermore, the suppression by low amounts of SEB was not major histocompatibility complex restricted and affected a large proportion of both rat and mouse T lymphocyte subpopulation, regardless of their antigenic specificity. The concomitant suppressogenic and stimulatory characteristics of SEB support the conclusion that, under different conditions, SEB can be considered a "super-suppressogen" as well as a "super-antigen". Overall, the results suggest that SEB, and possibly other bacterial toxins, could be useful in immunomodulation of specific T cell responses.

Animals↗

The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity.

This study addressed the question of whether the mucosal adjuvant property of cholera toxin (CT) and the structurally closely related Escherichia coli heat-labile toxin (LT) requires the enterotoxic and adenylate cyclase/cAMP activating property of these molecules. Therefore, we investigated the cytotoxic and adjuvant abilities of the enterotoxins and compared the results with those obtained with the non-toxic CT and LT derivatives; recombinant CTB (rCTB) and a mutated LT (mLT), which had a single amino acid substitution in position 112 (Glu----Lys) of the A subunit. Detailed functional studies revealed that, in contrast to the enterotoxins, both rCTB and mLT lacked ADP-ribosylating and cAMP-stimulating abilities. However, similar membrane ganglioside GM1-receptor binding ability of all the putative adjuvants was demonstrated. When the probe antigen, keyhole limpet hemocyanin (KLH), was given perorally together with CT or LT strong gut mucosal anti-KLH immune responses were stimulated, whereas no or very low anti-KLH responses were seen in the groups which received antigen admixed with rCTB or the mLT. Moreover, the specific serum antibody responses to the various immunization protocols closely paralleled the local anti-KLH response in the gut. From these results it appears that the adjuvant mechanism of LT, and probably also of CT, is linked to the ability to ADP-ribosylate and to stimulate cAMP formation. However, this study does not unequivocally rule out other possibilities such as interactions by the A1 fragment of CT or LT with other G-proteins than Gs alpha or events that parallel or precede the effects on the adenylate cyclase/cAMP system. Thus, the levels of ADP-ribosylation and cAMP-induction that are required and the key event or target cell that is responsible for the adjuvant effect of CT and LT remain to be elucidated. Studies are underway to address these issues.

Adjuvants, Immunologic↗

H-2 I-E molecules isolated from Mls1a stimulatory cells do not activate Mls1a-responsive T cells but do present exogenous staphylococcal enterotoxins.

The T cell response to allogeneic murine Mls determinants is not H-2 restricted but is dependent on H-2 class II molecules on the Mls-expressing stimulator cells. We have tested planar membranes containing H-2 class II I-E molecules alone or with I-A molecules for their ability to activate a panel of Mls1a-specific T hybrids. Despite the ability of the planar membranes to activate an alloreactive T hybrid and to present staphylococcal enterotoxins or an antigenic peptide to appropriately responsive T hybrids, they failed to stimulate the Mls1a-specific T hybrids. These findings, in the light of the various controls demonstrating sufficiency of the I-E molecules in the planar membranes, indicate that Mls1a determinants are not covalently bound to I-E molecules; the two molecular species are thus either not physically associated or are linked by a relatively weak interaction. In addition, our experiments show that isolated I-E molecules but not I-A molecules present staphylococcal enterotoxins A and B to two independently derived T hybrids expressing T cell receptor V beta 1, V beta 2 and V beta 6 elements.

Animals↗

Induction of clonal anergy by oral administration of staphylococcal enterotoxin B.

The staphylococcal enterotoxin is a major cause of food poisoning. The bacterial substance stimulates T cells expressing specific V beta T cell receptors (TcR) and is termed "the superantigen". We have previously demonstrated that intravenous injection of staphylococcal enterotoxin B (SEB) induces functional unresponsiveness (anergy) of reactive T cells as well as a partial deletion by activation-induced programmed cell death. In the present study, we examined the effect of oral administration of SEB in mice. Our results indicate that spleen T cells from SEB-primed mice are hyporesponsive to SEB stimulation in vitro, but the response to SEA was normal. V beta 8+ T cells purified from SEB-primed mice did not respond to stimulation of TcR. This SEB-specific unresponsiveness could not be reversed by exogenous interleukin-2, but was partially reversed by phorbol 12-myristate 13-acetate. Activation of mitogen-activated protein kinase during TcR-mediated stimulation was significantly inhibited in anergic T cells. Although the mechanisms of oral tolerance are not well understood, these results show that oral administration of SEB induce clonal anergy in peripheral T cells.

Administration, Oral↗

Evidence that induction of tolerance in vivo involves active signaling via a B7 ligand-dependent mechanism: CTLA4-Ig protects V beta 8+ T cells from tolerance induction by the superantigen staphylococcal enterotoxin B.

Co-stimulation through CD28 is thought to be necessary for the activation of unprimed CD4+ T cells, which are otherwise rendered tolerant. However, we previously found that CD4+ T cell priming was normal or augmented in mice which overexpressed a soluble form of CTLA4 where co-stimulation through CD28 was abrogated. To investigate this CD4+ T cell response, we exploited the capacity of the superantigen staphylococcal enterotoxin B to stimulate T lymphocytes bearing V beta 8+, which represent approximately 30% of all CD4+ T cells. In litter-mate controls of CTLA4-Ig transgenic mice, immunization with staphylococcal enterotoxin B leads to expansion, followed by deletion of V beta 8+ T cells, and the remaining cells are tolerant when stimulated in vitro. Comparable expansion and deletion of V beta 8 T cells occurs in CTLA4-Ig transgenic mice. However, in contrast to normal mice, the remaining V beta 8+ T cells from CTLA4-Ig transgenic mice are not anergic and remain responsive to superantigen in vitro.

Abatacept↗

Checkerboard immunoblotting recognizes twenty epitopes among the B subunit proteins of the cholera enterotoxin family.

Checkerboard immunoblotting (CBIB) was used to analyze the reactions of a series of monoclonal antibodies with proteins of the cholera enterotoxin (CT) family, including heat labile enterotoxins (LTs) produced by diarrheagenic strains of Escherichia coli and genetically engineered chimeric proteins in which single amino acids of the CT-B subunit protein or human (H) LT-B subunit protein were substituted for corresponding residues in porcine (P) LT-B. The result indicated that there were at least twenty different patterns of reactivity suggesting that there are at least twenty recognizable epitopes among the proteins studied. An epitope which includes Ala46 appears to be particularly important. This epitope is common to CT and H-LT but not P-LT, and the epitope is not blocked by the Gm1 ganglioside. Human convalescent sera react with this epitope. CBIB is a versatile technique for epitope analysis.

Amino Acid Sequence↗