Functional significance of globotriaosyl ceramide in interferon-alpha(2)/type 1 interferon receptor-mediated antiviral activity

The N-terminus of the type 1 interferon receptor subunit, IFNAR1, has high amino acid sequence similarity to the receptor binding B subunit of the Escherichia coli-derived verotoxin 1, VT1. The glycolipid, globotriaosyl ceramide (Gb(3): Gal alpha(1) --> 4 Gal beta 1 --> 4 Glu beta 1 --> 1 Cer) is the specific cell receptor for VT1. Gb(3)-deficient variant cells selected for VT resistance are cross-resistant to interferon-alpha (IFN-alpha)-mediated antiproliferative activity. The association of eIFNAR1 with Gal alpha 1 --> 4 Gal containing glycolipids has been previously shown to be important for the receptor-mediated IFN-alpha signal transduction for growth inhibition. The crucial role of Gb(3) for the signal transduction of IFN-alpha-mediated antiviral activity is now reported. IFN-alpha-mediated antiviral activity, nuclear translocation of activated Stat1, and increased expression of PKR were defective in Gb(3)-deficient vero mutant cells, although the surface expression of IFNAR1 was unaltered. The VT1B subunit was found to inhibit IFN-alpha-mediated antiviral activity, Stat1 nuclear translocation and PKR upregulation. Unlike VT1 cytotoxicity, IFN-alpha-induced Stat1 nuclear translocation was not inhibited when RME was prevented, suggesting that the accessory function of Gb(3) occurs at the plasma membrane. IFN-alpha antiviral activity was also studied in Gb(3)-positive MRC-5 cells, which are resistant to IFN-alpha growth inhibition, partially resistant to VT1 but still remain fully sensitive to IFN-alpha antiviral activity, and two astrocytoma cell lines expressing different Gb(3) fatty acid isoforms. In both systems, long chain fatty acid-containing Gb(3) isoforms, which are less effective to mediate VT1 cytotoxicity, were found to correlate with higher IFN-alpha-mediated antiviral activity. Inhibition of Gb(3) synthesis in toto prevented IFN-alpha antiviral activity in all cells. We propose that the long chain Gb(3) fatty isoforms preferentially remain in the plasma membrane, and by associating with IFNAR1, mediate IFN-alpha antiviral signaling, whereas short chain Gb(3) fatty acid isoforms are preferentially internalized to mediate VT1 cytotoxicity and IFNAR1-dependent IFN-alpha growth inhibition.     

Globotriaosyl ceramide modulates interferon-α-induced growth inhibition and CD19 expression in Burkitt's lymphoma cells

Previous studies have indicated that globotriaosyl ceramide (Gb3 or CD77) plays a role in -interferon signal transduction and CD19-mediated homotypic adhesion in B cell lines derived from Burkitt's lymphoma. These roles for Gb3 may involve the proteins IFNAR-1 (subunit 1 of the interferon- receptor) and CD19, respectively, both of which have potential Gb3-binding sites in their extracellular domains which resemble those of the verotoxin (Shiga toxin and Shiga-like toxin) B subunit. The majority of this work was performed using wild-type Daudi cells and a single, Gb3-deficient mutant cell line, VT500. In the present investigations, these and additional Daudi-derived cells with varying degrees of sensitivity to interferon- were examined for Gb3 expression, interferon-induced growth inhibition and CD19 expression. The degree of interferon-induced growth inhibition and CD19 expression correlated with Gb3 expression in the various cell lines tested. In addition, reconstitution of the VT500 cell line with Gb3 but not other glycolipids partially restored the sensitivity of cells to IFN-induced growth inhibition. The degree to which reconstitution restored sensitivity to growth inhibition was similar to the results of previous studies in which Gb3 reconstitution restored sensitivity to verotoxin-induced cytotoxicity. These results demonstrate that Gb3 is specifically required for IFN-induced growth inhibition in Daudi cells and provide further evidence of a role for Gb3 in CD19 expression and function in these cells.     

Verotoxin 1 from Escherichia coli affects Gb3/CD77+ bovine lymphocytes independent of interleukin-2, tumor necrosis factor-alpha,and interferon-alpha

Verotoxin (VT)-induced immunomodulation has been implicated in the ability of VT-producing Escherichia coli (VTEC) to cause persistent infections in cattle. VT1, also referred to as Shiga toxin 1, is a potent cytotoxin that modulates cytokine secretions and functions. This prompted the current investigation to examine whether the inhibiting effect of VT1 on bovine lymphocytes correlates with the expression of the cellular VT1 receptor Gb3/CD77 or is mediated instead via perturbation of cytokine secretion. Using blood mononuclear cells stimulated by mitogens as a model, VT1 significantly blocked lymphoblast transformation and proliferation in the BoCD8+ T cell and BoCD21+ B cell population. In contrast, VT1 dramatically reduced the number of viable Gb3/CD77+ blast cells within all subpopulations identified (BoCD2+, BoCD4+, BoCD8+, WC1+ [i.e., gammadelta T cells] BoCD21+, and BoCD25+). Similar effects of VT1 were observed when the culture medium was supplemented with selected cytokines: tumor necrosis factor-alpha-sensitizing endothelial cells against VT1, interferon-alpha (IFN-alpha) as bovine IFN-alpha receptors are partially homologous to the B-subunit of VT1, and interleukin-2 that is critical for lymphocyte proliferation in vitro. The addition of these cytokines was neither able to mimic nor to overcome the effects of VT1. Therefore, it is concluded that VT1 directly acts on bovine lymphocytes rather than inducing a cytokine-mediated effect. VT1 considerably affects all main bovine lymphocyte subpopulations, implicating that the immune system is a predominant target for VT1 in cattle.     

The identification of three biologically relevant globotriaosyl ceramide receptor binding sites on the Verotoxin 1 B subunit.

The Verotoxin 1 (VT1) B subunit binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). Receptor-binding specificity is associated with the terminally linked Galalpha(1-4) Galbeta disaccharide sequence of the receptor. Recently, three globotriose (Galalpha[1-4] Galbeta [1-4] Glcbeta) binding sites per B-subunit monomer were identified by crystallography. Two of these sites (sites I and II) are located adjacent to phenylalanine-30. Site I was originally predicted as a potential Gb3 binding site on the basis of sequence conservation, and site II was additionally predicted based on computer modelling and receptor docking. The third (site III) was also identified by crystallography and is located at the N-terminal end of the alpha-helix. To determine the biological significance of sites II and III, and to support our previous findings of the significance of site I, we examined the binding properties and cytotoxicity of VT1 mutants designed to block Gb3 binding at each site selectively. The Scatchard analysis of saturation-binding data for each mutant revealed that only the amino acid substitutions predicted to affect site I (D-17E) or site II (G-62T) caused reductions in the binding affinity and capacity of VT1 for Gb3. Similarly, those mutations at sites I and II also caused significant reductions in both Vero and MRC-5 cell cytotoxicity (by seven and five logs, respectively, for G-62T and by four and two logs, respectively, for D-17E). In contrast, the substitution of alanine for W-34 at site III did not reduce the high-affinity binding of the B subunit, despite causing a fourfold reduction in the receptor-binding capacity. The corresponding mutant W-34A holotoxin had a two-log reduction in cytotoxicity on Vero cells and no statistically significant reduction on MRC-5 cells. We conclude that the high-affinity receptor binding most relevant for cell cytotoxicity occurs at sites I and II. In contrast, site III appears to mediate the recognition of additional Gb3 receptor epitopes but with lower affinity. Our results support the significance of the indole ring of W-34 for binding at this site.     

Verotoxin-resistant cell clones are deficient in the glycolipid globotriosylceramide: differential basis of phenotype

Escherichia coli-derived verotoxin is an extremely toxic protein and is highly selective toward certain primate cells. Two susceptible cell lines are the Daudi cell line (human Burkitt lymphoma) and the Vero cell line (Green African monkey kidney). Both of these cell lines contain significant levels of the verotoxin binding glycolipid globotriosylceramide (Gb3) (1 nmol/10(7) cells and 3 nmol/10(6) cells, respectively). A clone was selected from the Vero cell line for resistance to Verotoxin 2, while a mutant from the Daudi cell line was selected for resistance to Verotoxin 1. Both were found to be deficient in globotriosylceramide with a corresponding increase in the precursor glycolipid lactosylceramide. Cell free assay of alpha-galactosyltransferase activity revealed that the Vero cell clone (VRP) contained significantly reduced enzyme activity, whereas in the case of the Daudi mutant (VT20), no significant decrease in activity was noted in vitro. These observations suggest a complex regulation of Gb3 biosynthesis which is considered in relation to P blood group antigen expression.     

Globotetraosylceramide is recognized by the pig edema disease toxin

The pig edema disease toxin has been shown by a tlc glycolipid binding assay to bind specifically to globotetraosylceramide (Gb4, GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer.). Binding was reduced for globotriosylceramide (Gb3, Gal alpha 1-4Gal beta 1-4GlcCer) and more markedly for the Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer). Paragloboside, blood group A glycolipids, glycolipids terminating in Gal NAc beta 1-4Gal-, and glycolipids in which globoside was present as an internal sequence did not bind the toxin. Isogloboside (GalNAc beta 1-3Gal alpha 1-3Gal beta 1-4GlcCer) was efficiently recognized. This toxin is genetically related to the verotoxin (or Shiga-like) family of toxins for which Gb3 has been shown to be the receptor. The difference in susceptibility of cell lines to the cytotoxicity of the pig edema disease toxin and the Shiga and Shiga-like toxins is consistent with the difference in receptor glycolipid binding.     

Apparent cooperativity in multivalent verotoxin-globotriaosyl ceramide binding: kinetic and saturation binding studies with [(125)I]verotoxin

Verotoxin (VT) binding to the trisaccharide portion of globotriaosyl ceramide (Gb(3)) is believed to be a crucial step in the development of hemolytic uremic syndrome (HUS) commonly known as 'Hamburger disease'. This interaction is the initial step in the binding process and defines the specificity of verotoxin binding to cellular membranes. Although molecular modeling, co-crystallization and co-NMR studies with VT and the trisaccharide moiety of Gb(3) have indicated potential multiple sites for Gb(3) binding, little is known about their direct effects on kinetic and equilibrium binding. Here we describe how the binding of radiolabeled VT ([(125)I]VT1) to Gb(3) in a microtiter well format, is driven by two different association rate constants (k(+1a)=0.0075 and k(+1b)=0.275 min(-1) nM(-1)) with the high affinity site representing 15% of the total specific binding sites. Binding was reversible at room temperature, reached equilibrium after 2-3 h, and non-specific binding was less than 5%. Equilibrium binding studies defined by [(125)I]VT1 saturation binding to 15, 30, 60 and 120 ng Gb(3)/well, showed the presence of a single site with dissociation constants (K(d)s) ranging between 0.5 and 3 nM. However, the maximum density of specific [(125)I]VT1 binding sites (B(max)) did not directly correlate with the Gb(3) concentration per well: the most[(125)I]VT1 binding was observed for 60 ng Gb(3) (B(max)=1.28 nM; compared to 0. 23 nM for 30 ng Gb(3) and 0.65 nM for 120 ng Gb(3)). Furthermore, while Hill coefficients (n(H)) for 15, 30 and 120 ng Gb(3) were close to unity indicating single interactions, for the saturation isotherm for 60 ng Gb(3)/well n(H) was 1.4. Subsequent Scatchard analysis yielded a concave downward curve for [(125)I]VT1 binding to 60 ng Gb(3)/well, suggesting positive co-operativity. We present, for the first time, conclusive binding data confirming the presence of at least two discrete Gb(3) binding sites: these multivalent interactions between verotoxin VT-1 and Gb(3) were described by association reactions driven by two distinct rate constants, as well as by the positive co-operativity governing binding at a restricted receptor concentration. These results imply that the concentration of Gb(3) on the surface of target cells can have a complex, non-linear effect on verotoxin binding and thereby, on sensitivity to cytotoxicity.     

Rapid detection of diarrheagenic E. coli by real-time PCR

Enterovirulent Escherichia coli are among the most important causes of acute diarrhea in developing as well as in developed countries. We have adapted classical PCR to detect these organisms in stool specimens to real-time PCR using the LightCycler (LC) SYBR Green format followed by melting curve analysis. With only two different cycling protocols we could detect enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC) (duplex assay for both Verotoxin 1 (VT1) and Verotoxin 2 (VT2)) in one run and enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) (duplex assay detecting both heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT)) in another run. Using serial dilutions of control strains, the LC proved to be clearly more sensitive than conventional PCR for five out of seven investigated targets: VTEC (VT1 and VT2), ETEC (ST and LT) and EIEC. For EPEC and EAEC, LC and conventional PCR had identical sensitivities. With stool samples, we found an optimal agreement between LC-PCR and the conventional PCR when samples were tested in a 1:10 dilution. Only one specimen was discrepant, being repetitively positive for VT by LightCycler but not by conventional PCR. Given the significantly higher sensitivity of the LC-PCR for the VT target (up to a 10(-4) dilution factor by melting curve analysis and up to a 10(-6) dilution factor following gel electrophoresis), this is probably a false negative result by conventional PCR. We conclude that LightCycler PCR is more rapid, easier than and at least as sensitive as our conventional PCR for the detection of enterovirulent E. coli in stool specimens after culture on MacConkey.     

Differential intracellular transport and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies

Although verotoxin-1 (VT1) and verotoxin-2 (VT2) share a common receptor, globotriaosyl ceramide (Gb(3)), VT2 induces distinct animal pathology and is preferentially associated with human disease. Moreover VT2 cytotoxicity in vitro is less than VT1. We therefore investigated whether these toxins similarly traffic within cells via similar Gb(3) assemblies. At 4 degrees C, fluorescent-VT1 and VT2 bound both coincident and distinct punctate surface Gb(3) microdomains. After 10 min at 37 degrees C, similar distinct/coincident micropunctate intracellular localization was observed. Most internalized VT2, but not VT1, colocalized with transferrin. After 1 h, VT1 and VT2 coalesced during retrograde transport to the Golgi. During prolonged incubation (3-6 h), VT1, and VT2 (more slowly), exited the Golgi to reach the ER/nuclear envelope. At this time, VT2 induced a previously unreported, retrograde transport-dependent vacuolation. Cell surface and intracellular VT1 showed greater detergent resistance than VT2, suggesting differential 'raft' association. >90% (125)I-VT1 cell surface bound, or added to detergent-resistant cell membrane extracts (DRM), was in the Gb(3)-containing sucrose gradient 'insoluble' fraction, whereas only 30% (125)I-VT2 was similarly DRM-associated. VT1 bound more efficiently to Gb(3)/cholesterol DRMs generated in vitro. Only VT1 binding was inhibited by high cholesterol/Gb(3) ratios. VT2 competed less effectively for (125)I-VT1/Gb(3) DRM-binding but only VT2-Gb(3)/cholesterol DRM-binding was augmented by sphingomyelin. Differential VT1/VT2 Gb(3) raft-binding may mediate differential cell binding/intracellular trafficking and cytopathology.     

Structure-dependent pseudoreceptor intracellular traffic of adamantyl globotriaosyl ceramide mimics

The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb(3)), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb(3) is an amphipathic competitive inhibitor of VT1/VT2 Gb(3) binding. However, Gb(3)-negative VT-resistant CHO/Jurkat cells incorporate adaGb(3) to become VT1/VT2-sensitive. CarboxyadaGb(3), urea-adaGb(3), and hydroxyethyl adaGb(3), preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb(3) (two deacyl Gb(3)s linked to adamantane) protects against VT1/VT2 more effectively than adaGb(3) without incorporating into Gb(3)-negative cells. AdaGb(3) (but not hydroxyethyl adaGb(3)) incorporation into Gb(3)-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb(3)-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb(3)-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb(3) destabilized membrane cholesterol and reduced Gb(3) cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb(3). We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb(3) mimics and their structure-dependent modulation of endogenous intracellular Gb(3) vesicular traffic.     

Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity

In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to < 50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3.     

Treatment of neutral glycosphingolipid lysosomal storage diseases via inhibition of the ABC drug transporter, MDR1. Cyclosporin A can lower serum and liver globotriaosyl ceramide levels in the Fabry mouse model

We have shown that the ABC transporter, multiple drug resistance protein 1 (MDR1, P-glycoprotein) translocates glucosyl ceramide from the cytosolic to the luminal Golgi surface for neutral, but not acidic, glycosphingolipid (GSL) synthesis. Here we show that the MDR1 inhibitor, cyclosporin A (CsA) can deplete Gaucher lymphoid cell lines of accumulated glucosyl ceramide and Fabry cell lines of globotriaosyl ceramide (Gb3), by preventing de novo synthesis. In the Fabry mouse model, Gb3 is increased in the heart, liver, spleen, brain and kidney. The lack of renal glomerular Gb3 is retained, but the number of verotoxin 1 (VT1)-staining renal tubules, and VT1 tubular targeting in vivo, is markedly increased in Fabry mice. Adult Fabry mice were treated with alpha-galactosidase (enzyme-replacement therapy, ERT) to eliminate serum Gb3 and lower Gb3 levels in some tissues. Serum Gb3 was monitored using a VT1 ELISA during a post-ERT recovery phase +/- biweekly intra peritoneal CsA. After 9 weeks, tissue Gb3 content and localization were determined using VT1/TLC overlay and histochemistry. Serum Gb3 recovered to lower levels after CsA treatment. Gb3 was undetected in wild-type liver, and the levels of Gb3 (but not gangliosides) in Fabry mouse liver were significantly depleted by CsA treatment. VT1 liver histochemistry showed Gb3 accumulated in Kupffer cells, endothelial cell subsets within the central and portal vein and within the portal triad. Hepatic venule endothelial and Kupffer cell VT1 staining was considerably reduced by in vivo CsA treatment. We conclude that MDR1 inhibition warrants consideration as a novel adjunct treatment for neutral GSL storage diseases.     

Intracellular targeting of the endoplasmic reticulum/nuclear envelope by retrograde transport may determine cell hypersensitivity to verotoxin via globotriaosyl ceramide fatty acid isoform traffic

The pentameric B subunit of verotoxin (VT) mediates the attachment to cell surface globotriaosyl ceramide (Gb3) to facilitate receptor-mediated endocytosis of the toxin. In highly toxin-sensitive tumor cells, the holotoxin and VT1 B subunit is targeted intracellularly to elements of the endoplasmic reticulum (ER)/nuclear membrane. In less sensitive cells, the toxin is targeted to components of the Golgi apparatus. We have studied two cell systems: the induced VT hypersensitivity of human astrocytoma cell lines cultured in the presence of sodium butyrate (compared to sodium propionate and capronate) and the increased VT sensitivity of multiple drug-resistant mutants as compared to parental human ovarian carcinoma cells. In both cases, a difference in the intracellular retrograde transport of the receptor-bound internalized toxin to the ER/nuclear envelope, as opposed to the Golgi, correlated with a >1,000-fold increase in cell sensitivity to VT. This change in intracellular routing may be due to sorting of Gb3 fatty acid isoforms, since nuclear targeting was found in turn to correlate with the preferential synthesis of Gb3 containing shorter chain (primarily C16) fatty acid species. We propose that the isoform-dependent traffic of Gb3 from the cell surface to the ER/nuclear membrane provides a new signal transduction pathway for Gb3 binding proteins.     

Retroviral transfection of Madin-Darby canine kidney cells with human MDR1 results in a major increase in globotriaosylceramide and 10(5)- to 10(6)-fold increased cell sensitivity to verocytotoxin. Role of p-glycoprotein in glycolipid synthesis

Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell line with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resistance efflux pump, induces a major accumulation of the glycosphingolipid (GSL), globotriaosylceramide (Galalpha1-4Galbeta1-4glucosylceramide-Gb(3)), the receptor for the E. coli-derived verotoxin (VT), to effect a approximately million-fold increase in cell sensitivity to VT. The shorter chain fatty acid isoforms of Gb(3) (primarily C16 and C18) are elevated and VT is internalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines. P-gp (but not MRP) inhibitors, e.g. ketoconazole or cyclosporin A (CsA) prevented the increased Gb(3) and VT sensitivity, concomitant with increased vinblastine sensitivity. Gb(3) synthase was not significantly elevated in MDR1-MDCK cells and was not affected by CsA. In MDR1-MDCK cells, synthesis of fluorescent N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb(3) via NBD-glucosylceramide (GlcCer) from exogenous NBD-C(6)-ceramide, was prevented by CsA. We therefore propose that P-gp can mediate GlcCer translocation across the bilayer, from the cytosolic face of the Golgi to the lumen, to provide increased substrate for the lumenal synthesis of LacCer and subsequently Gb(3). These results provide a molecular mechanism for the observed increased sensitivity of multidrug-resistant tumors to VT and emphasize the potential of verotoxin as an antineoplastic. Two strains (I and II) of MDCK cells, which differ in their glycolipid profile, have been described. The original MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenotype and glycolipid synthase activities indicate MDCK-I cells. However, the partial drug resistance of MDCK-I cells precludes their being the parental cell. We speculate that the retroviral transfection per se, or the subsequent selection for drug resistance, selected a subpopulation of MDCK-I cells in the parental MDCK-II cell culture and that drug resistance in MDR1-MDCK cells is thus a result of both MDR1 expression and a second, previously unrecognized, component, likely the high level of GlcCer synthesis in these cells.     

Binding of verocytotoxin 1 to its receptor is influenced by differences in receptor fatty acid content.

Globotriaosylceramide [(Gal alpha 1-4Gal beta 1-4Glc-ceramide (Gb3)] was separated from human kidney, and the fatty acid composition was determined. Semisynthetic Gb3 molecular species of corresponding fatty acid chain length were prepared and compared for verotoxin (VT) binding affinity by TLC overlay, and a quantitative binding assay was performed in the presence of auxiliary lipids. Our results indicate that, within the natural range, fatty acid chain length has little effect on verotoxin binding but that Gb3 molecular species containing different fatty acids can interact to provide a higher affinity toxin receptor than any of the individual component receptor species. Receptor function as assayed by TLC overlay was not always found to correlate with binding in a lipid environment. Short-chain fatty acid Gb3 molecular species could not function as VT receptors under these conditions. Evidence is presented to suggest that fatty acid chain length can have a stereoselective effect on carbohydrate conformation.     

Inhibitory effect of intestinal anti-Gb3 IgA antibody on verotoxin-induced cytotoxicity

The effect of intestinal IgA antibody against the receptor for verotoxin (VT), globotriaosylceramide (Gb3), on VT-mediated cytotoxicity was examined. Intestinal IgA antibodies against Gb3 were prepared by oral immunization of mice with Gb3 and adjuvant monophosphoryl lipid A (MPL)-containing liposomes composed of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylserine and cholesterol (1 : 1 : 2, molar ratio) (PS-liposome). Oral administration with Gb3 and MPL-containing PS-liposome induced significant IgA responses to Gb3 in the intestinal lavage fluid in all mice tested. Furthermore, anti-Gb3 IgA antibodies in the lavage fluid effectively inhibited the cytotoxicity of VT2 to Vero cells in a dose-dependent manner. These results suggest that anti-Gb3 IgA antibodies produced in the intestinal tract, upon oral immunization with Gb3-containing liposome, function as inhibitors against VT and also indicate the potential usefulness of oral PS-liposome vaccines containing MPL for the induction of a protective mucosal immune response against intestinal diseases.     

Simultaneous detection of two verotoxin genes using dual‐label time‐resolved fluorescence immunoassay with duplex PCR

Verotoxin (VT) produced by several Escherichia coli serotypes causes haemorrhagic colitis and has been associated with haemolytic uraemic syndrome in humans. Two types of verotoxin are known. Conventional diagnosis of verotoxin‐producing Escherichia coli (VTEC) is conducted after isolation of bacteria from clinical specimens, followed by serological determination and identification of VTs. This method is complicated and time‐consuming. Recently, rapid, direct immunological methods for identification of VTEC, i.e. immunochromatography and latex agglutination, have been developed. However, these techniques continue to suffer from limited sensitivity and a lack of specificity. These difficulties arise from the fact that the antibody used in these procedures reacts exclusively with the O157 antigen; moreover, VTEC strains with non‐O157 antigens, such as O26, O103 and O111 antigens, exist. These VTEC groups did not react with anti‐O157 antibody. Consequently, it is necessary to diagnose the VT gene in these bacteria. Therefore, we have designed a sensitive and specific method for the detection of two VT genes simultaneously, utilizing duplex PCR with time‐resolved fluorescence immunoassay (TRFIA). Copyright © 2002 John Wiley & Sons, Ltd.     

Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysosomes and abrogation of toxicity

Verotoxins (VTs) are important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), a group of bacteria associated with severe disease sequelae in humans. The potent cytotoxic activity of VTs is important in pathogenicity, resulting in the death of cells expressing receptor Gb3 (globotriaosylceramide). EHEC, particularly serotype O157:H7, frequently colonize reservoir hosts (such as cattle) in the absence of disease, however, the basis to avirulence in this host has been unclear. The objective of this study was assessment of interaction between VT and intestinal epithelium, which represents the major interface between the host and enteric organisms. Bovine intestinal epithelial cells expressed Gb3 in vitro in primary cell cultures, localizing specifically to proliferating crypt cells in corroboration with in situ immunohistological observations on intestinal mucosa. Expression of receptor by these cells contrasts with the absence of Gb3 on human intestinal epithelium in vivo. Despite receptor expression, VT exhibited no cytotoxic activity against bovine epithelial cells. Sub-cellular localization of VT indicated that this toxin was excluded from endoplasmic reticulum but localized to lysosomes, corresponding with abrogation of cytotoxicity. VT intracellular trafficking was unaffected by treatment of primary cell cultures with methyl-beta-cyclodextrin, indicating that Gb3 in these cells is not associated with lipid rafts but is randomly distributed in the membrane. The combination of Gb3 isoform, membrane distribution and VT trafficking correlate with observations of other receptor-positive cells that resist verocytotoxicity. These studies demonstrate that intestinal epithelium is an important determinant in VT interaction with major implications for the differential consequences of EHEC infection in reservoir hosts and humans.     

Inhibition of Vero cell cytotoxic activity in Escherichia coli O157:H7 lysates by globotriaosylceramide, Gb3, from bovine milk

In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.