One step high yield affinity purification of shiga-like toxin II variants and quantitation using enzyme linked immunosorbent assays

We have previously purified both shiga-like toxin (SLT) I and II using the toxins' affinity to P1 glycoprotein (P1gp) from hydatid cyst material (HCM). Binding of these toxins is based on their affinity for terminal Galα1 → 4Gal disaccharide residues present in HCM. Although the binding specificity of SLT-II variants (v) differs from that of STL-II they are reported to recognize Gb3 and should bind to P1 gp. Therefore we examined the usefulness of HCM to purify SLT-IIv of porcine (p) and human (h) origin. Toxins were purified from fermenter culture supernatants of Escherichia coli HB101(pDLW5) (SLT-IIvp), and E. coli DH5α(pJES210) (SLT-IIvh) utilizing HCM. SLT-IIvh and SLT-IIvp consisted of A and B subunits, as determined by SDS-PAGE. We obtained 0.16 mg SLT-IIvp and 0.12 mg SLT-IIvh/I of culture (yields >65%). Various capture systems to detect shiga toxin, SLT-II, SLT-IIvp and SLT-IIvh by ELISA were examined. All toxins bound to HCM, and all except SLT-IIvp bound to the monoclonal antibody 4D1. Only SLT-IIvp bound to the glycolipid Gb4, and only shiga toxin bound significantly to Gb3. Similarities in the level of Gb4 expression in HeLa 229 (ATCC) and Vero cells may explain the lack of differential cytoxicity between SLT-IIvp and SLT-IIvh on these cell lines.     

Hybridization of Escherichia coli producing Shiga-like toxin I, Shiga-like toxin II, and a variant of Shiga-like toxin II with synthetic oligonucleotide probes.

Synthetic oligonucleotides, constructed from the nucleotide sequences of genes coding for the A subunit of Shiga-like toxin (SLT) I and the B subunit of SLT-II, were used as probes at different degrees of stringency to identify Escherichia coli producing different types of SLTs. At 45 degrees C, the A-I oligonucleotide probe hybridized with E. coli producing SLT-I, SLT-II, and variant of SLT-II (SLT-IIv). At 53 degrees C, only SLT-I-producing E. coli hybridized with this probe. At 45 degrees C, the B-II oligonucleotide probe hybridized with SLT-II- and SLT-IIv-producing E. coli. At 53 degrees C, this probe hybridized with only SLT-II-producing E. coli. The A-I and B-II oligonucleotide probes were subsequently tested for hybridization with 73 SLT-producing E. coli and 49 non-SLT-producing E. coli isolated in Asia and Canada. At 45 degrees C, the A-I oligomer had a sensitivity of 97% and a specificity of 100% in identifying SLT-producing E. coli. At 53 degrees C, the A-I oligonucleotide probe had a sensitivity of 92% and a specificity of 91% in identifying E. coli containing genes encoding SLT-I. At 45 degrees C, the B-II oligonucleotide had a 100% sensitivity and 97% specificity in identifying E. coli that hybridized with the SLT-II probe. Of 17 E. coli that hybridized only with the SLT-II probe, 10 did not hybridize with the B-II oligonucleotide at 53 degrees C. All 10 isolates were cytotoxic to Vero cells but not to HeLa cells, confirming that the B-II oligonucleotide probe used at 53 degrees C will differentiate isolates producing SLT-II and SLT-IIv.     

In vivo formation of hybrid toxins comprising Shiga toxin and the Shiga-like toxins and role of the B subunit in localization and cytotoxic activity.

Shiga toxin, Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) are cell-associated cytotoxins that kill both Vero cells and HeLa cells, whereas Shiga-like toxin II variant (SLT-IIv) is an extracellular cytotoxin that is more cytotoxic for Vero cells than for HeLa cells. The basis for these differences in cytotoxin localization and host cell specificity were examined in this study. The A and B subunit genes of Shiga toxin and the SLTs were recombined by two methods so that hybrid toxins would be formed in vivo. Complementation of heterologous subunits was accomplished by cloning the individual A and B subunit genes of SLT-I, SLT-II, and SLT-IIv on plasmid vectors of different incompatibility groups so that they could be maintained in double transformants of Escherichia coli. In addition, six operon fusions were constructed so that the A and B subunit genes of Shiga toxin, SLT-II, and SLT-IIv could be expressed as a single operon. The activities of the hybrid cytotoxins were assessed in three ways: (i) level of cytotoxicity, (ii) ratio of HeLa to Vero cell cytotoxicity, and (iii) ratio of extracellular to cell-associated cytotoxicity. Neither the A subunit of Shiga toxin nor SLT-I associated with a heterologous B subunit to form an active cytotoxin. However, in all other cases the hybrid molecules formed by subunit complementation or operon fusion were cytotoxic. Furthermore, the cytotoxic specificity and localization of the hybrid cytotoxins always corresponded to the activities of the native toxin possessing the same B subunit.     

Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies.

Shiga toxin from Shigella dysenteriae 60R was purified to homogeneity by a novel one-step receptor analog affinity chromatography method. The method was based on the binding affinity of Shiga toxin for a specific disaccharide, Gal alpha 1----4Gal, which was also present in glycoproteins with P1 blood group seroreactivity produced in hydatid cysts from sheep infected with Echinococcus granulosus. Having shown that cyst fluid P1 glycoprotein bound Shiga toxin on a solid phase, a P1 glycoprotein affinity column was made by coupling P1-active substance to Sepharose 4B. Shiga toxin was purified by this method in large quantities (5 to 10 mg/20-liter batch) with a consistently good yield (greater than 80% of starting toxin). Shiga-like toxins I and II (SLT-I and -II, respectively) from Escherichia coli were also purified by the same method. A preparation containing SLT-II and SLT-I purified by receptor analog affinity chromatography was used to raise four monoclonal antibodies (MAbs) that were reactive with SLT-II by enzyme-linked immunosorbent assay. Three of these antibodies also reacted with Shiga toxin, which was the first clear demonstration of cross-reactivity between these toxins. One MAb, 4D1, which was specific for the B subunit of SLT-II and Shiga toxin, neutralized both toxins in a HeLa cell cytotoxicity assay. Two MAbs recognized the A subunit of both SLT-II and Shiga toxin by Western blot (immunoblot) analysis but were unable to neutralize either toxin. In addition, one B-subunit-specific MAb neutralized SLT-II alone, and a previously described Shiga toxin B-subunit-specific MAb was shown to be specific for Shiga toxin but not SLT-II.     

Blood group specific oligosaccharides from faeces of a blood group A breast-fed infant

Four different oligosaccharides were isolated from faeces collected from a blood group A, secretor, breast-fed infant. Three of these, GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-4Glc (A-tetrasaccharide), GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-4[Fuc alpha 1-3]Glc (A-pentasaccharide) and 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc (A-heptasaccharide) have previously found in urine, whereas GalNAc alpha 1-3[Fuc alpha 1-2]Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (A-hexasaccharide) is a new compound. Structures were deduced by mass spectrometry of permethylated and N-trifluoroacetylated oligosaccharide alditols. The latter gave more structural information than the corresponding N-acetyl derivatives. The four oligosaccharides were tested for blood group A activity and all were found to inhibit the binding of anti-A antibody to blood group A substance.     

Shiga toxin-glycosphingolipid interaction: Status quo of research with focus on primary human brain and kidney endothelial cells

Shiga toxin (Stx)-mediated injury of the kidneys and the brain represent the major extraintestinal complications in humans upon infection by enterohemorrhagic Escherichia coli (EHEC). Damage of renal and cerebral endothelial cells is the key event in the pathogenesis of the life-threatening hemolytic uremic syndrome (HUS). Stxs are AB₅ toxins and the B-pentamers of the two clinically important Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid globotriaosylceramide (Gb3Cer, Galα4Galβ4Glcβ1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1), which are expected to reside in lipid rafts in the plasma membrane of the human endothelium. This review summarizes the current knowledge on the Stx glycosphingolipid receptors and their lipid membrane ensemble in primary human brain microvascular endothelial cells (pHBMECs) and primary human renal glomerular endothelial cells (pHRGECs). Increasing knowledge on the precise initial molecular mechanisms by which Stxs interact with cellular targets will help to develop specific therapeutics and/or preventive measures to combat EHEC-caused diseases.     

Expression and purification of Shiga-like toxin II B subunits.

Shiga-like toxins (SLTs), which are produced by certain strains of Escherichia coli, are composed of enzymatically active A and B subunit multimers responsible for the toxin's binding. We have previously purified large amounts of the SLT-I B subunit by using a hyperexpression vector in Vibrio cholerae under the control of the trc promoter. In this study we examined various expression vectors to maximize yields of the SLT-II B subunit. The SLT-II B subunit has been expressed by using both the T7 promoter and the tac promoter in E. coli. When expressed from a plasmid containing the structural gene for SLT-II B deleted of the leader sequence, SLT-II B was able to form multimers when cross-linked, although SLT-II B production from this plasmid was unreproducible. SLT-II B expressed in all three systems appeared to form unstable multimers, which did not readily bind to a monoclonal antibody which preferentially recognizes B subunit multimers. SLT-II B expression was not increased by moving any of the plasmids into V. cholerae. Polyclonal antibodies raised to SLT-II B in rabbits recognized B subunit in SLT-II holotoxin yet were poorly neutralizing. SLT-II B was also expressed as a fusion protein with maltose-binding protein and could be cleaved from maltose-binding protein with factor Xa. Although the expression vectors were able to make large amounts of SLT-II B, as determined by Western blotting (immunoblotting), the levels of purified SLT-II B subunit were low compared with those obtained previously for SLT-I B subunit, probably because of instability of the multimeric SLT-II B subunit.     

Increased oral virulence of Escherichia coli expressing a variant Shiga-like toxin type II operon is associated with both A subunit residues Met4 and Gly102

We have previously demonstrated thatEscherichia coliDH5α clones expressing closely-related Shiga-like toxin type II operons (designated SLT-II/OX3b and SLT-II/O48) had similar cytotoxicity for Vero cells, but differed in oral virulence for streptomycin-treated mice. Studies with chimeric toxin operons indicated that increased virulence was associated with the A subunit of SLT-II/OX3b, which differs from that of SLT-II/O48 by two amino acids (at positions 4 and 102). In the present study, we have constructed a series of additional chimeric derivatives of the SLT-II/OX3b and SLT-II/O48 operons and assessed the effect of single A subunit amino acid substitutions on oral virulence. Maximal virulence, as judged by median survival time after oral challenge, was associated only with the combination of Met4 and Gly102, as found in the A subunit of SLT-II/OX3b.     

Purification and some properties of a Vero toxin from a human strain of Escherichia coli that is immunologically related to Shiga-like toxin II (VT2)

A cytotoxin to Vero cells (Vero toxin), which was immunologically related to Shiga-like toxin II (SLT-II) (or VT2), was purified from a stain of Escherichia coli isolated from a patient with hemolytic uremic syndrome. The toxin was active on Vero cells but much less active on HeLa cells, a property similar to that of the recently identified SLT-II variant from E. coli strains that caused edema disease of swine. Thus the toxin purified in this report was tentatively named Shiga-like toxin II variant (Vero toxin 2 variant). The purification procedures consisted of ammonium sulfate fractionation, DEAE-Sepharose CL-6B column chromatography, chromatofocusing column chromatography, and repeated high performance liquid chromatography (HPLC) on TSK-gel G-2000SW column and on TSK-gel DEAE-5PW columns. About 90 micrograms of purified toxin was obtained from 451 of the culture supernatant with a yield of about 16%. The purified toxin consisted of A and B subunits of molecular sizes similar to those of SLT-II (VT2). The isoelectric point of the purified toxin was 6.1, which was different from that of SLT-II (VT2) (pI = 4.1). In an Ouchterlony double gel diffusion test, purified toxin and SLT-II (VT2) formed precipitin lines with spur formation against anti-purified toxin and anti-SLT-II (anti-VT2), respectively. The purified toxin was cytotoxic to Vero cells, about 6 pg of the toxin killing 50% of the Vero cells, and showed lethal toxicity to mice when injected intraperitoneally, the LD50 being about 2.7 ng per mouse.     

Synthetic antigens as immunogens: Part II: Antibodies to synthetic T antigen

Antibody to the carbohydrate moiety of T antigen was developed. The synthetic antigen (Gal beta 1----3 GalNAc alpha 1----OC6H4N = N-BSA) was prepared by coupling the diazonium salt of the disaccharide derivative Gal beta 1----3 GalNAc alpha 1----OC6H4NH2 (o) with bovine serum albumin. Specificity of the antibody produced was examined with structurally related synthetic saccharides using the enzyme immunoassay technique. The presence of a glycosyl group at 0-6 of either the Gal or the GalNAc residue of the disaccharide Gal beta 1----3 GalNAc did not prevent binding of the antisera to the saccharide moiety. However, the antisera did not bind either the trisaccharide moiety NeuAc2----3 Gal beta 1----3 GalNAc alpha 1----OC6H4NO2 (o) or GlcNAc beta 1----3 Gal beta 1----3 GalNAc alpha OBn. These observations indicate that antibody approach to the antigen is to the 0-3 side of the terminal galactose in the disaccharide Gal beta 1----3 GalNAc. We have also observed that the antibody prefers Gal beta 1----3 GalNAc alpha 1----to Gal beta 1----3 GalNAc beta 1----disaccharide derivatives in its binding capacity. The antibody was found to bind natural T antigen present on neuraminidase-treated red blood cells and, by immunohistochemical analysis, it was found to bind to naturally occurring T antigen on breast tumor cells.     

Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II.

Shiga-like toxin type II (SLT-II) is one of two antigenically distinct cytotoxins produced by enterohemorrhagic Escherichia coli that are believed to play a central role in the pathogenesis of enterohemorrhagic E. coli-induced disease. SLT-II is a bipartite toxin with an enzymatically active A subunit that inhibits protein synthesis and an oligomeric B subunit that binds to the glycolipid globotriaosylceramide on eukaryotic cells. In this study, functional boundaries of the slt-II operon were mapped. Mutant proteins lacking the last four amino acids from the carboxy terminus of the 70-amino-acid mature SLT-II B polypeptide had no cytotoxic activity. However, when only two amino acids were removed from the carboxy terminus of the B subunit, the cytotoxic activity of the holotoxin was not altered drastically. Furthermore, a 21-amino-acid extension to the carboxy terminus of the SLT-II B polypeptide was tolerated with a minimum reduction in cytotoxic activity of the holotoxin. Deletion of the region coding for amino acids 3 through 18 of the 296-amino-acid mature SLT-II A polypeptide resulted in complete ablation of the cytotoxic activity of the holotoxin as well as abolition of the enzymatic activity of the A subunit. Thus, it appears that both 5'- and 3'-terminal coding sequences are essential for function of the slt-II operon.     

Pseudomonas aeruginosa exoenzyme S is an adhesion.

Exoenzyme S from Pseudomonas aeruginosa has been studied as an adhesion for glycosphingolipids and buccal cells. Binding of exoenzyme S to gangliotriosylceramide (GalNAc beta 1-4Gal beta 1-4Glc beta 1-1Cer), gangliotetraosylceramide (Gal beta 1-3 GalNAcT beta 1-4 Gal beta 1-4Glc beta 1-1Cer), and lactosylceramide (Gal beta 1-4Glc beta 1-1Cer) separated on thin-layer chromatograms was observed. Binding curves for exoenzyme S with dilutions of gangliotetraosylceramide immobilized on plastic plates were similar to previously reported results for the intact bacteria. Binding of exoenzyme S to sialylated counterparts of these glycosphingolipids was not seen, indicating that the addition of a sialic acid residue interferes with binding. Exoenzyme S and monoclonal antibody to exoenzyme S inhibit the binding of P. aeruginosa to buccal cells. The presence of exoenzyme S on the surface of P. aeruginosa was detected by immunogold labeling of bacteria with antibodies to exoenzyme S. Results of these studies led us to conclude that exoenzyme S is an important adhesion of P. aeruginosa.     

Frequent loss of Shiga-like toxin genes in clinical isolates of Escherichia coli upon subcultivation.

Forty-five consecutive patients with various gastrointestinal disorders were identified as having Shiga-like toxin (SLT)-producing Escherichia coli infections. This was shown by the cytotoxic effect of stool extracts in Vero cell cultures which was neutralizable by antibodies to SLTs and by isolation of E. coli that hybridized with DNA probes complementary to SLT-I and SLT-II sequences. When we tested the same strains for SLT genes after subcultivation, the isolates from 15 patients became negative by colony hybridization and polymerase chain reaction and failed to produce SLTs. The instability of SLT genes warrants direct screening methods for clinical material and the development of new culture methods to prevent the loss of SLT genes.     

Type 4 chain H expression by bile ductules and hepatocytes in cirrhosis

The monoclonal antibody MBr1 defines the blood group H determinant with beta 1----3N-acetylgalactosamine linkage (Fuc alpha 1----2Gal beta 1----3GalNAc----R) carried by type 3 or 4 backbone. The distribution of the antigen detected by this antibody was studied immunohistochemically in liver tissues. Although bile ducts with a diameter of more than about 100 microns normally expressed the MBr1-reactive antigen supranuclearly, smaller bile ducts and bile ductules did not express the antigen. In cirrhotic liver, proliferated bile ductules extensively expressed the MBr1-reactive antigen. In spite of the absence in normal liver cells, the antigen was expressed membranously in some cirrhotic liver cells. Under subcellular fractionation, MBr1 reactivity was almost exclusively recovered in the microsomal fraction. By HPTLC immunostaining, the major MBr1-reactive antigen was shown to be carried by type 4 chain H glycolipid (globo-H, Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer). MBr1 reactive glycoprotein was not found. In conclusion, although type 4 chain H glycolipid is not expressed by normal bile ductules and liver cells, it is actively synthesized and expressed by proliferated bile ductules and some of the liver cells in cirrhosis in the absence of any neoplastic change.     

Restricted accumulation of globotriaosylceramide in the hearts of atypical cases of Fabry's disease

Immunohistochemical and biochemical analyses of several tissues were performed in two unusual cases of Fabry's disease which showed accumulation of globotriaosylceramide (Gal alpha 1-4Gal beta 1-4 Glc-Cer, Gb3Cer) only in the hearts, but no clinical signs of the disease. Immunohistochemical study revealed that the hearts from our cases (cases no. 1 and 2) contained large amounts of anti-Gb3Cer antibody-positive granules in cytoplasms as in typical Fabry's disease. The contents of accumulated Gb3Cer in the hearts from case no. 1, case no. 2, and a typical Fabry's disease case were approximately 100, 340, and 100 times higher than those from normal controls, respectively. While typical Fabry's diseased kidney and liver contained approximately 40 and 50 times higher amounts of Gb3Cer than did controls, no accumulation of Gb3Cer was observed in kidney and liver of our cases. The only exception was a slight increase of Gb3Cer in kidney of case no. 2 (about two times higher than controls), in which epithelial cells of the glomeruli but not of other types of cells were positively stained by anti-Gb3Cer antibody. Case no. 1 kidney and liver were not stained by the antibody. The glomerular endothelium and epithelium, tubular epithelium, smooth muscle of renal arteries, and several hepatocytes were Gb3Cer-positive in the typical Fabry's disease case. The involvements of our cases differed distinctly from the typical Fabry's disease case.     

Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells

Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.     

Differences in the binding specificities of Pseudomonas aeruginosa M35 and Escherichia coli C600 for lipid-linked oligosaccharides with lactose-related core regions.

Membrane glycolipids contain the lactose sequence (galactose linked to glucose), and the oligosaccharide is variously extended such that there is a cell-type-specific repertoire. In this study, binding of Pseudomonas aeruginosa M35 to lipid-linked lactose (Gal beta 1-4Glc [structure 1]), lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc [structure 2]), lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc [structure 3]), and asialo GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc [structure 4]) was evaluated and compared with binding of Escherichia coli C600 to these compounds. Oligosaccharides were linked to the lipid phosphatidylethanolamine dipalmitoate, and the resulting neoglycolipids were resolved on thin-layer chromatograms or coated onto plastic microtiter wells. Lipid-linked structures 1 to 4 were bound by P. aeruginosa and E. coli in the chromatogram assay, but only structure 4 was bound in the microtiter well assay. As shown previously for E. coli binding to lipid-linked structures 1 to 3, binding to lipid-linked structure 4 was not inhibited with oligosaccharide, indicating a requirement for lipid and oligosaccharide. With few exceptions, sialylation and fucosylation of structures 1 to 4 resulted in impaired or abolished binding. Comparisons of binding intensities in the chromatogram assay indicated that recognition by P. aeruginosa and recognition by E. coli are not identical. Presence of the additional disaccharide unit, as in structure 2, resulted in enhanced binding of P. aeruginosa but diminished binding of E. coli relative to lactose binding; fucosylation at galactose of lactose resulted in markedly diminished binding of P. aeruginosa only. In the microtiter well assay, binding of E. coli to asialo GM1 was much weaker than P. aeruginosa binding. The saccharide-plus-lipid-dependent adhesion may be an important factor in increased susceptibility to infection of epithelia already damaged by microbial and chemical agents; the differing strengths of adhesion to the structural variants may relate to tissue tropism.     

Modification of the glycolipid-binding specificity of vero cytotoxin by polymyxin B and other cyclic amphipathic peptides.

Polymyxin B, an amphipathic cyclic decapeptide produced by Bacillus polymyxa, is routinely used in the extraction of the components from the periplasmic space of gram-negative bacteria. Vero cytotoxin 1 (VT1) is an Escherichia coli-elaborated subunit toxin which binds to the glycolipid globotriosylceramide (Gal-alpha 1-4-Gal beta 1-4-Glc-ceramide [Gb3]) and has been strongly implicated in the etiology of the hemolytic uremic syndrome and hemorrhagic colitis. We now show by in vitro glycolipid-binding assays that in the presence of low concentrations of polymyxin B, globotetraosylceramide (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-ceramide [Gb4]) is also recognized by both the VT1 B (binding) subunit and holotoxin. Melittin, a 26-amino-acid cyclic peptide of similar amphipathic nature, produced the same effect, whereas a hydrophobic blocking agent did not. Triton X-100 did not increase binding of VT1 to Gb4 but prevented glycolipid binding in toto at concentrations above 0.5%. Caution is therefore advised in the analysis of VT1 glycolipid binding in the presence of amphipathic peptides.     

Physicochemical and biological properties of purified Escherichia coli Shiga-like toxin II variant.

Purified Escherichia coli Shiga-like toxin II variant (SLT-IIv) was characterized with regard to selected physical, chemical, and biological properties. N-terminal amino acid sequencing confirmed the identities of 33,000-, 27,500-, and 7,500-molecular-weight (MW) bands seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified SLT-IIv as the A subunit, A1 fragment, and B subunit, respectively. The arginine-serine bond between amino acids 247 and 248 in the A subunit was determined to be the site for proteolytic cleavage into A1 and A2 fragments. As with other SLTs, gel filtration chromatography of SLT-IIv gave estimates of the MW of holotoxin that were variable and less than predicted for a 1-A-subunit-5-B-subunit configuration. The MWs were estimated to be 40,000 and 43,000 by Sephacryl S-100 and Sephadex G-100 and less than 2,000 by Bio-Sil Sec-250 gel filtration chromatography. The isoelectric point of SLT-IIv holotoxin was 9.0. Cytotoxicity of SLT-IIv was destroyed by heating at 65 degrees C for 30 min and by incubation with 2-mercaptoethanol and dithiothreitol, but it increased 30-fold by incubation with trypsin, chymotrypsin, or pepsin and 2-fold by incubation with thermolysin. SLT-IIv cytotoxic activity was stable at neutral and alkaline pH values but was lost at pHs 3, 4, and 5. SLT-IIv was stable in fluid from the anterior and posterior small intestines of pigs but was not enterotoxic in pig intestinal loops. The smallest doses of SLT-IIv that inhibited protein synthesis in porcine endothelial cells and Vero cells were 0.1 ng and 0.1 fg, respectively.     

Cryptococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipid lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), a possible adhesion receptor for yeasts.

The role of glycosphingolipids as adhesion receptors for yeasts was examined. Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae, as well as Histoplasma capsulatum and Sporotrichum schenckii (in their yeast phases), bound specifically to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), as measured by overlaying glycosphingolipid chromatograms with 125I-labeled organisms. An unsubstituted galactosyl residue was required for binding, because the yeasts did not bind to glucosylceramide (Glc beta 1-1Cer) derived from lactosylceramide by treatment with beta-galactosidase or to other neutral or acidic glycosphingolipids tested that contained internal lactosyl residues. Interestingly, the yeasts preferentially bound to the upper band of the lactosylceramide doublet in human lung and bovine erythrocytes, suggesting that the ceramide structure also affects binding. Active metabolism of the yeasts was required for binding to lactosylceramide, as binding was maximal in buffer containing glucose and was almost completely abolished in nutrient-deficient medium. C. neoformans also bound to human glioma brain cells grown in monolayers, and this binding was inhibited by liposomes containing lactosylceramide but not by liposomes containing glucosylceramide. Lactosylceramide is a major glycosphingolipid in these cells and the only one to which the yeasts bound. As lactosylceramide is widely distributed in epithelial tissues, this glycosphingolipid may be the receptor for yeast colonization and disseminated disease in humans.