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Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) bacteria are not enteroinvasive but can cause hemorrhagic colitis. In some STEC-infected individuals, a life-threatening sequela of infection called the hemolytic uremic syndrome may develop that can lead to kidney failure. This syndrome is linked to the production of Stx by the infecting organism. For Stx to reach the kidney, the toxin must first penetrate the colonic epithelial barrier. However, the Stx receptor, globotriaosylceramide (Gb3), has been thought to be absent from human intestinal epithelial cells. Thus, the mechanisms by which the toxin associates with and traverses through the intestine en route to the kidneys have been puzzling aspects of STEC pathogenesis. In this study, we initially determined that both types of Stx made by STEC, Stx1 and Stx2, do in fact bind to colonic epithelia in fresh tissue sections and to a colonic epithelial cell line (HCT-8). We also discovered that globotetraosylceramide (Gb4), a lower-affinity toxin receptor derived from Gb3, is readily detectable on the surfaces of human colonic tissue sections and HCT-8 cells. Furthermore, we found that Gb3 is present on a fraction of HCT-8 cells, where it presumably functions to bind and internalize Stx1 and Stx2. In addition, we established by quantitative real-time PCR (qRT-PCR) that both fresh colonic epithelial sections and HCT-8 cells express Gb3 synthase mRNA. Taken together, our data suggest that Gb3 may be present in small quantities in human colonic epithelia, where it may compete for Stx binding with the more abundantly expressed glycosphingolipid Gb4.

Hemolytic-uremic syndrome is a clinical syndrome characterized by acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia that often follows infection by Shiga toxin- or verotoxin-producing strains of Escherichia coli . Because thrombocytopenia and platelet activation are hallmark features of hemolytic-uremic syndrome, we examined the ability of Shiga toxin to bind platelets by flow cytometry and high-performance thin-layer chromatography (HPTLC) of isolated platelet glycosphingolipids. By HPTLC, Shiga toxin was shown to bind globotriaosylceramide (Gb 3 ) and a minor platelet glycolipid with an R f of 0.03, band 0.03. In a survey of 20 human tissues, band 0.03 was identified only in platelets. In individuals, band 0.03 was expressed by 20% of donors and was specifically associated with increased platelet Gb 3 expression. Based on glycosidase digestion and epitope mapping, band 0.03 was hypothesized to represent a novel glycosphingolipid, IV 3 -β-Galα1-4galactosylglobotetraosylceramide. Based on incidence, structure, and association with increased Gb 3 expression, band 0.03 may represent the antithetical Luke blood group antigen. By flow cytometry, Shiga toxin bound human platelets, although the amount of Shiga toxin bound varied in donors. Differences in Shiga toxin binding to platelet membranes did not reflect differences in platelet Gb 3 expression. In contrast, there was a loose association between Shiga toxin binding and decreasing forward scatter, suggesting that Shiga toxin and verotoxins bind more efficiently to smaller, older platelets. In summary, Shiga and Shiga-like toxins may bind platelets via specific glycosphingolipid receptors. Such binding may contribute to the thrombocytopenia, platelet activation, and microthrombus formation observed in hemolytic-uremic syndrome.

Among 58 Escherichia coli urosepsis isolates, P1-antigen-containing dove and pigeon egg whites were significantly more effective inhibitors of P-adhesin-specific agglutination than were chicken egg whites or globoside. Globoside's inefficacy may have resulted from a proadherence effect of globoside's lipid tail. Adhesin phenotypes determined with dove and pigeon egg whites as as agglutination inhibitors corresponded closely with phenotypes defined by comparative hemagglutination of human P1 and p erythrocytes. These data suggest that avian P1-antigen-containing substances may provide a useful alternative method for P adhesin inhibition among uropathogenic E. coli strains.

A subtype of P fimbriae, encoded by the pap-2 gene cluster, has been analyzed for agglutination of erythrocytes and for binding to cryostat sections of the human kidney. We have demonstrated that pap-2-encoded fimbriae are capable of binding to erythrocytes from some animal species and to human erythrocytes which express globoside and the LKE (stage-specific embryonic antigen 4 [SSEA-4]) antigen. The pap-2 fimbriae bind to Bowman's capsule in the human kidney. Monoclonal antibodies directed against glycosphingolipids were used for the detection of specific P blood group-related antigens in the human kidney and on erythrocytes. Preincubation of kidney sections with monoclonal antibody MC813-70, which binds to the SSEA-4 antigen, inhibited adherence of purified pap-2-encoded fimbriae to Bowman's capsule. We suggest that one receptor for pap-2-encoded fimbriae is the antigen known as LKE (Luke) on human erythrocytes or SSEA-4 in the tissues.

Rotaviruses are the major etiologic agents of severe diarrhea in children. Many rotaviruses encode a hemagglutinin which binds to sialic acids. We report that rotaviruses specifically recognize the neutral glycosphingolipid gangliotetraosylceramide (asialo-GM1 or GA1). GA1 resolved by thin-layer chromatography is bound by rotavirus, and binding is blocked by neutralizing rotavirus antiserum. Similar glycosphingolipid structures, such as globoside, gangliotriaosylceramide, and GA1 oxidized with galactose oxidase are ineffective in binding rotavirus. Other enteric viruses also specifically bind GA1. GA1 adsorbed to polystyrene beads inhibits rotavirus replication in vitro (as do anti-GA1 antibodies). The use of orally administered immobilized GA1 or anti-GA1 antibodies may prove useful in preventing or attenuating rotaviral and other enteric viral infections.

35S-labeled Pseudomonas aeruginosa isolates were shown to bind to neutral glycosphingolipids (NGSLs) of rabbit corneal epithelia in culture by a thin-layer chromatogram overlay procedure. The lipids of the corneal epithelial cells grown in culture were extracted and partitioned into a chloroform-rich lower phase containing NGSLs and an aqueous upper phase containing gangliosides. By using a dot-blot assay, at least six times more radiolabeled P. aeruginosa isolates were shown to bind to the lipids in the lower phase compared with those in the upper phase. Thin-layer chromatography of the lower-phase lipids followed by staining with an orcinol spray revealed at least 10 NGSL components and several fast-migrating, nonglycosylated neutral lipid components (including cholesterol). 35S-labeled P. aeruginosa was shown to bind to NGSL components 1, 2, 5, 6, and 9. P. aeruginosa-reactive NGSL components 6 and 9 migrated with chromatographic mobilities similar to those of the standards ceramide trihexoside (CT) and ceramide monohexoside, respectively. Components 1 and 2 migrated slightly ahead of asialo GM1, and component 5 migrated faster than globoside but slower than CT. Among the various standards tested, P. aeruginosa bound to asialo GM1 and, to a lesser extent, to ceramide dihexoside and CT but not to GM1, GD1A, GM3, or ceramide monohexoside. It remains to be determined whether any of the five P. aeruginosa-reactive NGSL components of corneal epithelium identified in this study plays a role in the development of corneal infection. However, we have previously shown that component 9, one of the five P. aeruginosa-reactive NGSL components identified in this study, is present in significantly greater amounts in migrating epithelia than it is in nonmigrating epithelia (N. Panjwani, G. Michalopoulos, J. Song, G. Yogeeswaran, and J. Baum, Invest. Ophthalmol. Vis. Sci., in press). This may prove to be of biological significance because it is generally believed that traumatized (migrating) epithelia are more susceptible to infection than normal (nonmigrating) epithelia are.

A specific family of glycolipids, the globoseries, was shown to act as receptors on human uroepithelial cells and erythrocytes for the majority of uropathogenic Escherichia coli strains attaching to or hemagglutinating those cells. This was demonstrated in three different ways: (i) correlation between the natural presence of glycolipid in the target cell (erythrocytes of different species) and binding of bacteria; (ii) inhibition of attachment to human uroepithelial cells by preincubation of bacteria and glycolipid; and (iii) induction of binding to unreactive cells by coating of these cells with glycolipid. Strains reacting with the receptor agglutinated guinea pig erythrocytes in a mannose-resistant way after, but not before, coating of the cells with globotetraosylceramide. Unrelated glycolipids were not recognized. The reaction was made independent of simultaneous occurrence of mannose-sensitive adhesions on the strains by addition of D-mannose. The receptor-coated cells were used as a tool to screen for prevalence of receptor recognition in a collection of 453 E. coli strains isolated from patients with urinary tract infection or from the stools of healthy children. Of 150 strains attaching to human uroepithelial cells and agglutinating human erythrocytes, 121 bound to globotetraosylceramide (81%). Globoside recognition was especially frequent among pyelonephritis strains (74/81). The glycolipid composition of the urogenital epithelium and kidney tissue and the ability of uropathogenic E. coli to bind to these glycolipids may be a determinant in host-parasite interaction leading to urinary tract infection.