Globotriaosyl ceramide receptor function - where membrane structure and pathology intersect.

The glycosphingolipid globotriaosyl ceramide, (Galalpha1-4Galss1-4 glucosyl ceramide-Gb(3)) also known as CD77 and the P(k) blood group antigen, is bound by both verotoxins and by the HIV adhesin, gp120. Gb(3) plays an important receptor role in VT induced hemolytic uremic syndrome (HUS) and HIV infection. The organization of glycolipids, including Gb(3), into lipid rafts is central to both pathologies. The fatty acid heterogeneity within the Gb(3) lipid moiety plays a central role in assembly within such ordered domains. Differential binding of verotoxins and gp120 to such Gb(3) isoforms in model and cell membranes indicates a significant role in the eventual pathogenic outcome. HUS may provide the first example whereby membrane Gb(3) organization provides a predictor for tissue selective in vivo pathology.

The medium is the message: glycosphingolipids and their soluble analogues.

We have made adamantylGSLs by substituting the fatty acids of primarily, globotriaosyl ceramide(Gb(3)) and sulfogalactosyl ceramide(SGC), with the rigid alpha-adamantane hydrocarbon frame. These analogues have proven to be remarkably water-soluble but retain the receptor function of the parent membrane GSL. AdaGb(3) prevents the binding of verotoxins to target cells but increased pathology in vivo, likely due to the partitioning into receptor negative target cells to provide pseudo-receptors. Preincubation of HIV with adaGb(3) prevents cellular infection in vitro and viral-host cell fusion. Cellular accumulation of Gb(3) reduces HIV susceptibility in vitro, whereas lack of Gb(3) promotes infection, suggesting that Gb(3) expression could be a novel risk factor for HIV susceptibility. AdaGb(3) has proven to be a new inhibitor for the MDR1 drug pump (P-glycoprotein) and can reverse drug resistance in cell culture. AdaSGC is bound by hsp70/hsc70 within the N-terminal ATPase domain and inhibits chaperone function. When added to cells transfected with the DeltaF508 CFTR mutant, adaSGC was able to decrease ER degradation of this mutant protein, an hsc70 dependent process. Our finding that DeltaF508 CFTR expressing cells show reduced SGC biosynthesis suggests that SGC could be an additional natural regulator of the hsp70 chaperone ATPase cycle.

3'Sulfogalactolipid binding specifically inhibits Hsp70 ATPase activity in vitro.

A method for the generation of soluble glycosphingolipid derivatives that retain the receptor activity of the parent (BBRC 257:391-394, Carb Res 335:91-100) was used to investigate the consequence of 3'sulfogalactolipid (SGL) specific binding within the N-terminal ATPase-containing domain of Hsc70. Sulfogalactosyl ceramide (SGC) was deacylated, and the resulting sulfogalactosylsphingosine coupled to an alpha-adamantane or a norbornane rigid hydrophobic frame. The resulting conjugate preferentially partitioned into water, as opposed to organic solvent. In the range of 100-300 microM, these conjugates inhibited the specific binding of bovine brain Hsc70 to immobilized SGLs. A similar dose-related inhibition of bovine brain Hsc70 ATPase activity was seen between 100 and 300 microM adamantylSGC (adaSGC). Adamantyl conjugates of glycolipids not bound by Hsp70s had no effect. Kinetic analysis indicated that adaSGC was a noncompetitive inhibitor of Hsc70 ATPase activity, a special case of mixed inhibition since the K(m) values were not statistically different, 0.89 +/- 0.024 microM to 0.93 +/- 0.038 microM, but the V(max) decreased from 0.20 +/- 0.012 pmol min(-1) microg(-1) to 0.15 +/- 0.016 pmol min(-1) microg(-1). A reproducible 5 min lag was observed prior to ATPase inhibition that could be eliminated by preincubation of adaSGC with Hsc70 or by adding the cochaperone Hdj-1. The dependence of ATPase inhibition on the rate of hydrolysis indicates that adaSGC binding occurs at a specific stage of the ATPase cycle. These studies identify a new mechanism for the regulation of Hsp70 ATPase activity.

Induction by sphingomyelinase of shiga toxin receptor and shiga toxin 2 sensitivity in human microvascular endothelial cells.

Shiga toxin-producing enterohemorrhagic Escherichia coli is the major cause of acute renal failure in young children. The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome. Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins. In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2. Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours. Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2. A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway. The product of CGT (glucosylceramide) was also increased. In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC. These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.

Synergistic effect of verotoxin and interferon-alpha on erythropoiesis.

Previous studies have shown that the verotoxin receptor globotriaosyl ceramide is involved in interferon-alpha (IFN-alpha) signaling pathways. The results of the present study indicate that verotoxin used in combination with IFN-alpha had a direct cytotoxic effect on erythrocyte development by targeting nucleated erythrocyte precursors. Toxin treatment alone had no significant effect on erythropoiesis. However, treatment with 100 ng/ml of verotoxin (VT) in combination with 100 U/ml of IFN-alpha was highly cytotoxic (>90%) to erythroid cells in human cord blood cultures by day 14 post-treatment. The lack of effect on other hematopoietic cells, and the relatively modest decrease in the number of erythroid colonies formed (28%) as a result of IFN-alpha/VT treatment, indicate that the cytotoxicity was targeted specifically toward cells committed to the erythrocyte lineage. IFN-alpha treatment alone did not result in cytotoxicity or a significant reduction in the number of erythroid colonies formed. However, IFN-alpha treatment did result in an increase in the surface expression of verotoxin receptors as determined by flow cytometry following labeling of cells with VT-FITC. Abnormalities in erythrocyte morphology and anemia are associated with infection by verotoxin-producing Escherichia coli such as serotype O157:H7. These symptoms are frequently attributed to passage of erythrocytes through partially occluded blood vessels following toxin-induced damage to endothelial cells. The present results document a synergistic cytotoxic effect of IFN-alpha and verotoxin on erythropoiesis, which could have relevance to clinical infection with verotoxin-producing bacteria.

Induction of epithelial cell death including apoptosis by enteropathogenic Escherichia coli expressing bundle-forming pili.

Infection with enteropathogenic Escherichia coli (EPEC) is a major cause of severe infantile diarrhea, particularly in parts of the developing world. The bundle-forming pilus (BFP) of EPEC is an established virulence factor encoded on the EPEC adherence factor plasmid (EAF) and has been implicated in both localized adherence to host cells and bacterial autoaggregation. We investigated the role of BFP in the ability of EPEC binding to kill host epithelial cells. BFP-expressing strains killed all three cell lines tested, comprising HEp-2 (laryngeal), HeLa (cervical), and Caco-2 (colonic) cells. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA, and morphological changes detected by electron microscopy indicated evidence of apoptosis. The extent of cell death was significantly greater for BFP-expressing strains, including E2348/69, a wild-type clinical isolate, as well as for a laboratory strain, HB101, transformed with a bfp-carrying plasmid. Strains which did not express BFP induced significantly less cell death, including a bfpA disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, HB101, and HB101 complemented with the locus of enterocyte effacement pathogenicity island. These results indicate a direct correlation between BFP expression and induction of cell death, including apoptosis, an event which may involve the targeting of host cell membrane phosphatidylethanolamine.

Enteropathogenic Escherichia coli virulence factor bundle-forming pilus has a binding specificity for phosphatidylethanolamine.

The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC), an established virulence factor encoded on the EPEC adherence factor (EAF) plasmid, has been implicated in the formation of bacterial autoaggregates and in the localized adherence of EPEC to cultured epithelial cells. While understanding of the pathogenic mechanism of this organism is rapidly improving, a receptor ligand for BFP has not yet been identified. We now report, using both solid-phase and liposome binding assays, that BFP expression correlates with phosphatidylethanolamine (PE) binding. In a thin-layer chromatogram overlay assay, specific recognition of PE was documented for BFP-expressing strains, including E2348/69, a wild-type EPEC clinical isolate, as well as a laboratory strain, HB101, transformed with a bfp-carrying plasmid. Strains which did not express BFP did not bind PE, including a bfpA disruptional mutant of E2348/69, EAF plasmid-cured E2348/69, and HB101. E2348/69 also aggregated PE-containing liposomes but not phosphatidylcholine- or phosphatidylserine-containing liposomes, while BFP-negative strains did not produce aggregates with any tested liposomes. Purified BFP preparations bound commercial PE standards as well as a PE-containing band within lipid extracts from human epithelial cells and from E2348/69. Our results therefore indicate a specific interaction between BFP and PE and suggest that PE may serve as a BFP receptor for bacterial autoaggregation and may promote localized adherence to host cells, both of which contribute to bacterial pathogenesis.

In vitro binding of Helicobacter pylori to monohexosylceramides.

H. pylori is the major cause of human gastritis, duodenal ulcer and thus gastric adenocarcinoma. Many glycosphingolipid species have been postulated as receptors for H. pylori and it is likely that H. pylori attachment requires multiple, perhaps sequential receptor/ligand interactions. In this study, the binding of a number of H. pylori clinical isolates, as well as stock strains, to acid and neutral glycosphingolipids separated on thin-layer chromatograms was characterized under microaerobic conditions. All H. pylori clinical isolates, laboratory strains and type culture collection strains recognized galactosylceramide (Galbeta1Cer) with ceramide containing sphingosine and hydroxylated fatty acid (type I), or non-hydroxylated fatty acid (type II), on thin-layer chromatograms and when incorporated into liposomes. The clinical isolates bound stronger to Galbeta1Cer (type II) than Galbeta1Cer (type I) on TLC, whereas lab and culture collection strains showed the opposite binding preference. A clear preference in binding to Galbeta1Cer (type I) incorporated into liposome was shown by most tested strains. Clinical isolates bound well to glucosylceramide (Glcbeta1Cer) with hydroxylated fatty acid, whereas weak binding to this glycolipid was detected with the lab and type collection strains. None of the tested strains bound Glcbeta1Cer with non-hydroxylated fatty acid on the solid surface, but some strains of both clinical or type collection origins showed weak or very weak binding in the liposome assay. A clear distinction between the binding specificity of living organisms (under microaerobic conditions) as opposed to dying organisms (under normoxic conditions) illustrates the importance of cellular physiology in this process. These studies illustrate lipid modulation of the potential receptor function of monohexosylceramides and the distinction between the receptor repertoire of H. pylori clinical isolates and cultured strains commonly used to study host-cell adhesion.

The aglycone of sulfogalactolipids can alter the sulfate ester substitution position required for hsc70 recognition.

3'-Sulfogalactolipids(SGLs), sulfogalactosyl ceramide (SGC), and sulfogalactoglycerolipid (SGG) bind to the N-terminal ATPase-containing domain of members of the heat shock protein 70 family. We have probed this binding specificity using a series of synthetic positional sulfated or phosphorylated glycolipid analogues, containing either a long-chain bisalkyl hydrocarbon-2-(tetradecyl)hexadecane (B30) or C(18) ceramide (SGC(18)) backbone. By TLC overlay and receptor ELISA, recombinant hsc70 bound ceramide-based glycoconjugates having 3'- or 4'-sulfogalactose glycone moieties and the 4'-sulfogalactose positional isomer conjugated to B30. Hsc70 binding was significantly decreased to the 3'-sulfogalactose conjugated to the long-chain branched alkane. 3'-Sulfoglucose conjugated to B30 was not bound, nor were similarly conjugated di-, tri-, and tetra-sulfated or phosphorylated galactolipids. These results highlight the importance of the position, rather than the number of sulfate esters within the galactose ring. This binding selectivity was shared by the sea urchin hsp70-related sperm receptor. A 3'-SGC-based soluble inhibitor, in which the acyl chain was replaced with an adamantyl group, inhibited binding of hsc70 to both 3'- and 4'-SGC species with an IC(50) of 50 and 75 microM, respectively, indicating a shared sulfogalactose binding site. These studies demonstrate the highly specific nature of hsc70/SGL binding and show, for the first time, that the lipid aglycone can alter the substitution position requirement for glycolipid recognition.

A verotoxin 1 B subunit-lambda CRO chimeric protein specifically binds both DNA and globotriaosylceramide (Gb(3)) to effect nuclear targeting of exogenous DNA in Gb(3) positive cells.

Inefficient nuclear incorporation of foreign DNA remains a critical roadblock in the development of effective nonviral gene delivery systems. DNA delivered by traditional protocols remains within endosomal/lysosomal vesicles, or is rapidly degraded in the cytoplasm. Verotoxin I (VT), an AB(5) subunit toxin produced by enterohaemorrhagic Escherichia coli, binds to the cell surface glycolipid, globotriaosylceramide (Gb(3)) and is internalized into preendosomes. VT is then retrograde transported to the Golgi, endoplasmic reticulum (ER), and nucleus of highly VT-sensitive cells. We have utilized this nuclear targeting of VT to design a unique delivery system which transports exogenous DNA via vesicular traffic to the nucleus. The nontoxic VT binding subunit (VTB) was fused to the lambda Cro DNA-binding repressor, generating a 14-kDa VTB-Cro chimera. VTB-Cro binds specifically via the Cro domain to a 25-bp DNA fragment containing the consensus Cro operator. VTB-Cro demonstrates simultaneous specific binding to Gb(3). Treatment of Vero cells with fluorescent-labeled Cro operator DNA in the presence of VTB-Cro, results in DNA internalization to the Golgi, ER, and nucleus, whereas fluorescent DNA alone is incorporated poorly and randomly within the cytoplasm. VTB-Cro mediated nuclear DNA transport is prevented by brefeldin A, consistent with Golgi/ER intracellular routing. Pretreatment with filipin had no effect, indicating that caveoli are not involved. This novel VTB-Cro shuttle protein may find practical applications in the fields of intracellular targeting, gene delivery, and gene therapy.

Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes.

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

Hsp70s contain a specific sulfogalactolipid binding site. Differential aglycone influence on sulfogalactosyl ceramide binding by recombinant prokaryotic and eukaryotic hsp70 family members.

Specific 3'-sulfogalactolipid [SGL-sulfogalactosyl ceramide (SGCer) and sulfogalactosylglycerolipid (SGG)] binding is compared for hsp70s cloned from Helicobacter pylori, Haemophilus influenzae, Chlamydia trachomatis serovar E, Escherichia coli, murine male germ cells, and the hsp70-like extracellular domain within the sperm receptor from Strongylocentrotus purpuratus. This lectin activity, conserved among the different hsp70 family members, is modulated by the SGL aglycone. This is shown by differential binding to both SGC fatty acid homologues and 3'-sulfogalactolipid neoglycoproteins generated by coupling bovine serum albumin (BSA) and glycosyl ceramide acids synthesized by oxidation of the double bond of sphingosine. Eukaryotic hsp70s preferentially bound the SGCer fatty acid homologues SG(24)Cer, SG(18)Cer, and SG(20:OH)Cer, while prokaryotic hsp70s bound SG(18:1)Cer and SG(20:OH)Cer. Eukaryotic hsp70s bound SGCer-BSA and SG(24)Cer-BSA conjugates where the latter is the main constituent in SGCer-BSA, while prokaryotic hsp70s bound SG(20:OH)Cer-BSA. None of the hsp70s bound sulfogalactosyl sphingosine (SGSph) or SGSph-BSA, further demonstrating the important role of the aglycone. Although the primary SGL recognition domain of all hsp70s is conserved, we propose that aglycone organization differentially influences the interaction with the sub-site. Heterogeneous SGCer aglycone isoforms in cells and the differential in vitro binding of eukaryotic and prokaryotic hsp70s may relate to their different adhesin roles in vivo as mediators of germ cell and bacterial/host interactions, respectively.

Heat-inducible surface stress protein (Hsp70) mediates sulfatide recognition of the respiratory pathogen Haemophilus influenzae.

The in vitro glycolipid binding specificity of clinical strains of nontypeable Haemophilus influenzae is altered to include sulfated glycolipids following a brief heat shock. We have constructed, expressed, and purified a recombinant protein of H. influenzae Hsp70, which showed significant specific binding to sulfated galactolipids in vitro. Furthermore, indirect immunofluorescence demonstrates that Hsp70 proteins are surface exposed in H. influenzae only after heat shock and are contained in the outer membrane protein fractions.

The ATPase domain of hsp70 possesses a unique binding specificity for 3'-sulfogalactolipids.

The region(s) of hsp70 critical for sulfogalactolipid (SGL) recognition has been defined through deletion analysis and site-directed mutagenesis. Truncated polymerase chain reaction products of hsp70 generated N-terminal fragments of 43, 35, 29, and 22 kDa. The C terminus substrate-binding domain (28 kDa) was also expressed. The N-terminal ATPase domain (rP43) shared the binding specificity of hsp70, because only sulfogalactosyl ceramide and sulfogalactosyl glycerolipid were recognized by both TLC overlay and RELISA. The C-terminal domain showed no binding. SGL binding of rP29 and rP22 was severely reduced. The loss of SGL binding for rP35 by RELISA but not TLC overlay was considered as a function of receptor presentation. The truncation of rP43 to rP35 demonstrates that residues 318-387 (the base of the ATP binding cleft) are critical for high affinity SGL binding. Mutagenesis showed that Arg(342) and Phe(198) are crucial for this process. SGL binding, mediated by these conserved residues within the ATPase domain of hsp70, implies that this binding specificity is evolutionarily conserved.

MHC class II proteins contain a potential binding site for the verotoxin receptor glycolipid CD77.

Globotriaosyl ceramide or CD77 functions as a cell surface receptor for toxins of the Shiga toxin/verotoxin family and as a marker for germinal center stage B-cells. The B-cell protein CD19 and the interferon-alpha receptor possess verotoxin-like amino acid sequences in their extracellular domains, and CD77 has been shown to function in CD19-mediated adhesion and interferon-induced growth inhibition. The Burkitt's lymphoma cell line, Daudi, is similar to germinal center B-cells in their expression of CD77, CD19 and MHC class II molecules. Using the multiple sequence alignment program, ClustalW, we have identified a verotoxin-like amino acid sequence on the beta-chain of human and murine MHC class II molecules. Binding of CD77 at this site could modulate the peptide-binding properties of these MHC class II molecules. Using Western blot analysis of whole cell extracts, we found that CD77-positive Daudi cells have higher levels of HLA-D proteins than VT500 cells, a Daudi-derived CD77-deficient mutant cell line. In contrast, MHC class II-mediated adhesion and surface expression are similar in the two cell lines. Therefore, CD77 could play a functional or regulatory role in MHC class II-mediated functions specifically relating to antigen presentation by B-cells to T helper cells.

Oxidation of aglycone of glycosphingolipids: serine and ceramide acid precursors for soluble glycoconjugates.

A new oxidation protocol for the cleavage of sphingosine double bonds is described. The procedure is applicable to both natural and deacyl glycolipids and can be applied to microgram quantities of precursors. Under neutral conditions, glycosyl ceramide acids are obtained and under basic conditions glycosyl serine acids are obtained. The glycosyl ceramide acid-based glycoconjugates--BSA-neoglycoprotein and adamantyl-neohydrocarbon--demonstrate the importance that an aglycone can play in carbohydrate-protein interaction. Studies with HIV coat protein gp120 and BSA-neoglycoprotein conjugates derived from galactosylceramide (GalC) showed that binding affinities of the conjugates depend on the manner in which the glycosyl unit is coupled to the protein. Deacyl-GalC conjugates, in which the glycosyl unit is coupled via the amine of the sphingosine, showed significantly lower affinity as compared to glycosylceramide acid conjugates. In the case of Gb3-VT1 binding, it was found that ceramide acid conjugates bound to VT1 better than the serine acid conjugates. These studies show that the aglycone organization, particularly the region adjacent to the carbohydrate region (or in a membrane environment, the aglycone-glycone interface) modulate carbohydrate presentation. It is possible that in each of the conjugates described above, the interface region could have different hydrogen-bonding networks (see Scheme 4.) This, in turn, could influence the solvation and/or conformation of this region and thereby influence ligand binding.

Verotoxin targets lymphoma infiltrates of patients with post-transplant lymphoproliferative disease.

Post-transplant lymphoproliferative disease (PTLD) is an invasive, EBV expressing B lymphoma and a major cause of morbidity and mortality following organ transplantation. Presently there is limited therapy available; rather the patient often loses the allograft or succumbs to the malignancy. CD77 (or globotriaosyl ceramide -Gb(3)) is a germinal center B cell marker [Gregory et al. Int J Cancer 1998;42:213-20; Gregory et al., J Immunol 1987;139:313-8; Mangeney et al. Eur J Immunol 1991;21:1131-40], expressed on most EBV infected B cells and is the receptor for the E. coli derived verotoxin (VT) [Lingwood CA. Advances in Lipid Research 1993;25:189-212]. We present the basis of a possible novel approach to PTLD therapy utilizing the specific targeting of VT to the infiltrating lymphoma cells. Biopsies of adenoid, kidney or liver tissue of four PTLD patients were stained with verotoxin to determine expression of CD77. VT is a potent inducer of necrosis/apoptosis of receptor positive cells. In each PTLD case, the infiltrating EBV positive B lymphoma cells were strongly and selectively stained with VT, identifying CD77 as a new marker for these cells. For such individuals, VT might provide the basis of an approach to control their malignancy.

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.

Enterohemorrhagic Escherichia coli induces apoptosis which augments bacterial binding and phosphatidylethanolamine exposure on the plasma membrane outer leaflet.

Enterohemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes watery diarrhea and hemorrhagic colitis and can lead to serious and even fatal complications such as hemolytic uremic syndrome. We investigated the ability of EHEC to kill host cells using three human epithelial cell lines. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA and morphological changes detected by electron microscopy changes revealed evidence of apoptotic cell death. The rates and extents of cell death were similar for both verotoxin-producing and nonproducing strains of EHEC as well as for a related gastrointestinal pathogen, enteropathogenic E. coli (EPEC). The induction of apoptosis by bacterial attachment was independent of verotoxin production and greater than that produced by a similar treatment with verotoxin alone. Expression of phosphatidylethanolamine, previously reported to bind EHEC and EPEC, was also increased on apoptotic cells but with little correlation to phosphatidylserine expression. Phosphatidylethanolamine levels but not phosphatidylserine levels on dying cells correlated with EHEC binding. Cells treated with phosphatidylethanolamine-containing liposomes also showed increased EHEC binding. These results suggest that bacterial induction of apoptosis offers an advantage for bacterial attachment by augmenting outer leaflet levels of the phosphatidylethanolamine receptor.