Infection with verotoxin producing Escherichia coli has been strongly implicated in the etiology of the hemolytic uremic syndrome (HUS). We have previously shown that this toxin specifically binds to a glycolipid receptor-globotriosyl ceramide (Gb₃). We have therefore quantitated the level of this glycolipid by HPLC in human renal cortex and medulla as a function of age. We have also measured the binding of verotoxin to Gb₃ isolated from each renal tissue sample. Gb₃ was a major component of the glycolipid fraction of all renal samples analyzed. The levels were found to be higher in the cortex than medulla, correlating with the clinical incidence of renal lesions in HUS, but reduced in the kidneys of infants as compared to adults. Verotoxin binding was directly proportional to the renal Gb₃ content. Thus, human renal tissue is a rich source of the verotoxin receptor glycolipid. However, changes in receptor concentration cannot explain the age-related incidence of HUS.

Verotoxin 1 (VT-1) and Shiga-like toxin II (SLT-H) bind to the glycosphingolipid (GSL), globotriaosylceramide (Gb3), whereas pig edema disease toxin (VTE) binds to globotetraosylceramide (Gb4) and to a lesser degree Gb3.Amino acids important in the GSL binding specificity of VT-1 and VTE have been identified by site-directed mutagenesis.One mutation, Asp-18 --Asn, in VT-1 resulted in binding to Gb4 in addition to Gb3 in a manner similar to VTE. Several mutations in VTE resulted in the complete loss of GSL binding; however, one mutation resulted in a change in the GSL binding specificity of the VTE B subunit. The double mutation Gln-64Glu and Lys-66 -* Gln (designated GT3) caused a selective loss of Gb4 binding, effectively changing the binding phenotype from VTE to VT-1. Both wild-type VTE and GT3 were purified to homogeneity and binding kinetics in vitro were determined with purified GSLs from human kidney. The cell cytotoxicity spectrum of the mutant toxin was also found to be altered in comparison with VTE. These changes were consistent with the GSL content of the target cells.

Very little is known about specific mechanisms for zinc accumulation and transport in bacteria. In this study a putative adhesin B in Hemophilus influenzae, the product of gene HI0119, has been identified as a periplasmic zinc-binding protein (PZP1). A pzp1-deficient mutant has been constructed which is defective for growth under aerobic conditions and grows poorly under anaerobic conditions. The growth defect is specifically rescued by supplementing the growth medium with high concentrations of zinc. Subcellular fractionation was used to localize PZP1 to the periplasmic region in a nontypeable H. influenzae strain and in a transfected recombinant Escherichia coli strain (TApzp1). Recombinant PZP1, purified from a periplasmic extract of E. coli strain TApzp1, contained ϳtwo zinc atoms/protein molecule as determined by neutron activation analysis and atomic absorption spectroscopy. The zinc atoms could be removed by incubation with EDTA, and, by further addition of zinc, a total of five zinc atoms/PZP1 could be bound. Direct binding of 65 Zn to the recombinant protein by Western blot was demonstrated. Taken together, these results provide direct evidence that PZP1 plays a key role in zinc uptake by H. influenzae.

All members of the verotoxin (VT) family specifically recognize globo-series glycolipids on the surface of susceptible cells. Those toxins that are associated with human disease, VT1, VT2, and VT2c, bind to globotriaosyl ceramide (Gb3) while VT2e, which is associated with edema disease of swine, binds preferentially to globotetraosyl ceramide (Gb4). We were recently able to identify, using site-directed mutagenesis, amino acids in the binding subunit of these toxins that are important in defining their glycosphingolipid (GSL) binding specificity (Tyrrell, G. J., K. Ramotar, B. Boyd, B. W. Toye, C. A. Lingwood, and J. L. Brunton. 1992. Proc. Natl. Acad. Sci. USA. 89:524). The concomitant mutation of Gln64 and Lys66 in the VT2e binding subunit to the corresponding residues (Glu and Gln, respectively) found in VT2 effectively converted the GSL binding specificity of the mutant toxin from Gb4 to Gb3 in vitro. We now report that the altered carbohydrate recognition of the mutant toxin (termed GT3) has biological significance, resulting in a unique disease after intravascular injection into pigs as compared with classical VT2e-induced edema disease. The tissue localization of radiolabeled GT3 after intravascular injection was elevated in neural tissues compared with VT2e accumulation, while localization of GT3 to the gastrointestinal tract was relatively reduced. Accordingly, the pathological lesions after challenge with GT3 involved gross edema of the cerebrum, cerebellum, and brain stem, while purified VT2e caused hemorrhage and edema of the cerebellum, and submucosa of the stomach and large intestine. In addition, both radiolabeled toxins bound extensively to tissues not directly involved in the pathology of disease. VT2e, unlike GT3 or VT1, bound extensively to red cells, which have high levels of Gb4. The overall tissue distribution of VT2e was thus found to be influenced by regional blood flow to each organ and not solely by the Gb4 levels of these tissues. Conversely, the distribution of GT3 (and VT1), which cleared more rapidly from the circulation, correlated with respective tissue Gb3 levels rather than blood flow. These studies indicate the primary role of carbohydrate binding specificity in determining systemic pathology, suggest that the red cells act as a toxin carrier in edema disease, and indicate that red cell binding does not protect against the pathology of systemic verotoxemia.