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Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are important enteric pathogens. In humans they cause diarrhea and hemorrhagic colitis, and these can progress to potentially fatal systemic sequelae, such as the hemolytic-uremic syndrome (HUS) (6, 10, 11, 16). HUS is characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure, and it is a leading cause of acute renal failure in children (6). During infections STEC strains colonize the gut and release Stx into the gut lumen; the STEC strains do not invade the gut mucosa, but toxin is absorbed into the circulation and targets tissues displaying the appropriate glycolipid receptor (particularly the microvasculature of the gut, kidneys, and brain). This accounts for both the severe gastrointestinal symptoms and the systemic manifestations of STEC disease. There are several distinct classes of Stx which differ in amino acid sequence. All Stx types associated with human disease (Stx1, Stx2, Stx2c, and Stx2d) recognize the same glycolipid receptor, globotriaosyl ceramide (Gb₃), which has the structure Gal(14)Gal(14)Glc-ceramide (7). In a recent study we exploited this specificity to develop a recombinant bacterium expressing a mimic of the Gb₃ oligosaccharide on its surface (13). This involved insertion of a plasmid (pJCP-Gb₃) carrying two Neisseria galactosyltransferase genes, lgtC and lgtE (2), into a derivative of E. coli R1 (CWG308), which has a waaO mutation in the outer core lipopolysaccharide (LPS) biosynthesis locus such that a truncated LPS core terminating in glucose (Glc) is produced (3). Expression of lgtC and lgtE resulted in the linkage of Gal(14)Gal(14) onto the terminal Glc. This bacterium adsorbed and neutralized Stx1, Stx2, Stx2c, and Stx2d with very high efficiency in vitro, and oral administration protected mice from an otherwise fatal challenge with highly virulent STEC strains (13). Oral administration of this novel agent to individuals diagnosed with, or at risk for, STEC infection has the potential to adsorb and neutralize free Stx in the gut lumen, thereby preventing absorption of toxin into the bloodstream and the concomitant life-threatening systemic sequelae associated with STEC disease in humans.

The above construct, however, was somewhat less effective at neutralizing the variant toxin Stx2e produced by STEC strains associated with piglet edema disease. This was not unexpected, since Stx2e has a different receptor specificity, recognizing globotetraosyl ceramide [Gb₄; GalNAc(13)Gal(14)Gal(14)-Glc-ceramide] preferentially over Gb₃ (1). Piglet edema disease is a serious, frequently fatal STEC-related illness characterized by neurological symptoms, including ataxia, convulsions, and paralysis; edema is typically present in the eyelids, brain, stomach, intestine, and mesentery of the colon. It is caused by particular STEC serotypes (most commonly O138:K81, O139:K82, and O141:K85) which are not associated with human disease (4, 9). The altered glycolipid receptor specificity affects the tissue tropism of the toxin, accounting for the distinctive clinical presentation of edema disease. Edema disease occurs principally at the time of weaning, and so incorporation of an effective Stx2e binding agent into the feed should be a means of preventing disease outbreaks and the associated economic losses. Accordingly, in the present study we have constructed a recombinant bacterium expressing globotetraose on its surface and examined its capacity to bind and neutralize Stx2e in vitro.

Construction of an E. coli CWG308 derivative expressing GalNAc(13)Gal(14)Gal(14)Glc. Globotetraose differs from globotriose only by the additional N-acetylgalactosamine (GalNAc) linked 13 to the terminal galactose (Gal). Thus, insertion of a gene encoding the appropriate GalNAc transferase into pJCP-Gb₃ would be expected to direct globotetraose expression when the plasmid is introduced into CWG308. The Neisseria lgt locus includes such a gene (lgtD), but this contains a poly(G) tract and so is unstable because of susceptibility to slipped-strand mispairing (2, 19). To overcome this we mutagenized the lgtD gene by overlap extension PCR, using N. gonorrhoeae chromosomal DNA as the template. The 5' portion of lgtD was amplified using the primers 5'-CAGACGGGATCCGACGTATCGGAAAAGGAGAAAC-3' (LGTDF), incorporating a BamHI site (underlined), and 5'-GCGCGCAATATATTCACCGCCACCCGACTTTGCC-3' (LGTDOLR). The 3' portion of lgtD was amplified using the primers 5'-GGCAAAGTCGGGTGGCGGTGAATATATTGCGCGC-3' (LGTDOLF) and 5'-CATGATGGATCCTGTTCGGTTTCAATAGC-3' (LGTDR), also incorporating a BamHI site. PCR was performed using the Expand high-fidelity PCR system (Roche Molecular Biochemicals) under conditions recommended by the supplier. The two PCR products were then purified, aliquots were mixed, and the complete lgtD coding sequence with the desired modifications was amplified using the primers LGTDF and LGTDR. This procedure mutates GGG codons in the poly(G) tract to GGT or GGC (all of which encode Gly), eliminating the risk of slipped-strand mispairing without changing the encoded amino acid sequence. The modified PCR product was digested with BamHI and cloned into similarly digested pJCP-Gb₃ between lgtC and lgtE, as shown in Fig. 1, and then it was transformed into E. coli JM109 (20). Insertion of lgtD with the correct mutations and the appropriate orientation was confirmed by sequence analysis of plasmid DNA using custom-made oligonucleotide primers and dye terminator chemistry on an ABI model 377 automated DNA sequencer. This plasmid (designated pJCP-lgtCDE) was then transformed into E. coli CWG308.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Construction of pJCP-Gb3 derivatives. pJCP-Gb3 is a derivative of pK184 (5) carrying Neisseria lgtC and lgtE, as described previously (13). The N. gonorrhoeae lgtD gene was amplified and mutagenized to stabilize its poly(G) tract by overlap extension PCR and cloned into the BamHI site between lgtC and lgtE to generate pJCP-lgtCDE. wbnF and gne genes were then amplified from E. coli O113 and cloned into the HindIII site downstream of lgtE to generate pJCP-lgtCDE/wbnF and pJCP-lgtCDE/gne, respectively. Restriction sites: E, EcoRI; B, BamHI; H, HindIII. All genes are in the same orientation and are located immediately downstream of the vector promoter (Plac).

View larger version (72K): [in this window] [in a new window]   FIG. 2.   Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LPS purified from E. coli CW308 derivatives. Lanes: 1, CWG308; 2, CWG:308-pJCP-Gb3; 3, CWG308:pJCP-lgtCDE; 4 and 5, CWG308:pJCP-lgtCDE/gne (two separate preparations); 6, CWG308:pJCP-lgtCDE/wbnF. Each lane contains approximately 3 µg of LPS.

Adsorption and neutralization of Stx. The capacity of the above CWG308 derivatives to adsorb and neutralize various Stx types was then assessed. Filter-sterilized French pressure cell (FPC) lysates of E. coli JM109:pJCP522 (12) and JM109:pJCP521 (15) were used as a source of Stx1 and Stx2c, respectively. For Stx2e, we first PCR-amplified the complete stx_2e operon from chromosomal DNA extracted from an O141 STEC strain isolated from a piglet with edema disease. The primers used were 5'-GCATCATGCGTTGTTAGCTC-3' and 5'-AAAGACGCGCATAAATAAACCG-3'. The PCR product was purified, blunt-cloned into SmaI-digested pBluescript SK (Stratagene, La Jolla, Calif.), and then transformed into E. coli JM109. The insert of this plasmid (designated pJCP543) was sequenced and found to be identical to the sequence previously published for stx_2e (18), except for a single nucleotide substitution in the A subunit coding region which did not affect the amino acid sequence. Accordingly, an FPC lysate of JM109:pJCP543 was used as a source of Stx2e. The crude Stx extracts were prepared by growing the various E. coli JM109 derivatives in 10 ml of Luria-Bertani broth supplemented with 50 µg of ampicillin/ml overnight at 37°C. Cells were harvested by centrifugation and resuspended in 10 ml of phosphate-buffered saline, pH 7.2 (PBS), and lysed in an FPC operated at 12,000 lb/in². Lysates were then sterilized by passage through a 0.45-µm-pore-size filter.

Conclusions. In the present study we have modified the globotriose-expressing bacterium CWG308:pJCP-Gb₃ so that it expresses globotetraose (the preferred receptor for Stx2e) by introducing additional genes encoding a GalNAc transferase (lgtD) and a UDP-GalNAc-4-epimerase (gne). The addition of an extra sugar residue to the outer LPS core required both genes and was demonstrated by electrophoretic analysis. Furthermore, the fact that the LPS migrated as a single species implied that this reaction proceeds to completion. The globotetraose-expressing bacterium had a reduced capacity to neutralize Stx1 and Stx2c in vitro, presumably because the terminal GalNAc residue sterically hinders the interaction between the Stx B subunit and the (now subterminal) globotriose moiety. However, its capacity to bind Stx2e was similar to that of the globotriose-expressing construct: both neutralized 98.4% of the cytotoxicity in lysates of E. coli JM109 expressing cloned stx_2e. It has long been held that the piglet edema disease-associated toxin Stx2e has a higher affinity for Gb₄ than for Gb₃ (1, 7, 17). Thus, the findings of this study were somewhat unexpected. Some of the early studies on Stx receptor specificity involved overlaying glycolipids separated by thin-layer chromatography with toxin. However, it has been suggested that the polyisobutylmethacrylate used in these studies (to stabilize the silica gel prior to reaction of the separated lipids with toxin) may have induced conformational changes in the carbohydrate moieties which affected toxin-receptor interactions (21). Receptor specificity was also examined on the basis of susceptibility of cell lines containing various amounts of Gb₃ and Gb₄ to the toxin. Interestingly, fatty acyl chain length is known to influence the interaction of Gb₃ with Stx1 and Stx2 to different extents, and so it is possible that factors other than the structure of the oligosaccharide component may have been compounding factors in the cell culture studies (7). In the present study the globotriose and globotetraose moieties were expressed on an otherwise identical platform comprising the inner core oligosaccharide and the lipid A components of E. coli LPS. Thus, differences in toxin-receptor interactions (or lack thereof) truly reflect the impact of oligosaccharide structure and conformation. Of course, the protective efficacy of oral administration of recombinant bacteria expressing mimics of Stx receptors needs to be examined in a piglet edema disease challenge model. On the basis of the results obtained in this study, it is clear that both the globotriose- and the globotetraose-expressing constructs should be tested.