Thin-Layer Chromatography in Structure and Recognition Studies of Shiga Toxin Glycosphingolipid Receptors.

Glycosphingolipids (GSLs) consist of a ceramide (Cer) lipid anchor, which is typically composed of the long-chain aminoalcohol sphingosine (d18:1) and a fatty acid (mostly C16-C24) and a sugar moiety harboring to a great extent one to five monosaccharides. GSLs of the globo-series are well-recognized receptors of Shiga toxins (Stxs) released by Stx-producing Escherichia coli (STEC). Receptors for the Stx subtypes Stx1a and Stx2a are globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), whereby Gb3Cer represents their high-affinity and Gb4Cer their low-affinity receptor. In addition to Gb3Cer and Gb4Cer, Gb5Cer and Forssman GSL are further receptors of the Stx2e subtype rendering Stx2e unique among the various Stx subtypes. Thin-layer chromatography (TLC) is a convenient and ubiquitously employed method for analyzing GSL mixtures of unknown composition. In particular, TLC immunochemical overlay detection allows for sensitive identification of Stx-binding GSLs in complex mixtures directly on the TLC plate. For this purpose, specific anti-GSL antibodies or Stxs themselves in conjunction with anti-Stx antibodies can be used. The described protocols of antibody-mediated detection of TLC-separated globo-series GSLs and corresponding identification of Stx-binding globo-series GSLs will provide detailed advice for successful GSL analysis and particularly highlight the power of the TLC overlay technique.

Multicenter evaluation of Verigene Enteric Pathogens Nucleic Acid Test for detection of gastrointestinal pathogens.

We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes. Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice.

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry.

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galß1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit.

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.

Baicalein Inhibits Stx1 and 2 of EHE: Effects of Baicalein on the Cytotoxicity, Production, and Secretion of Shiga Toxins of Enterohaemorrhagic Escherichia coli.

Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen. Baicalein (5,6,7-trihydroxylflavone), a flavone isolated from the roots of Scutellaria baicalensis, is considered as a potential antibacterial agent to control foodborne pathogens. Among seven compounds selected by in silico screening of the natural compound database, baicalein inhibited the cytotoxicity of both Shiga toxins 1 and 2 (Stx1 and Stx2) against Vero cells after pretreatment at 0.13 mmol/L. In addition, baicalein reduced the susceptibility of Vero cells to both Stx1 and Stx2. Real-time qPCR showed that baicalein increased transcription of stx1 but not of stx2. However, baicalein had no effects on production or secretion of Stx1 or Stx2. Docking models suggested that baicalein formed a stable structure with StxB pentamer with low intramolecular energy. The results demonstrate that inhibitory activity of baicalein against the cytotoxicity of both Stx1 and Stx2 might be due to of the formation of a binding structure inside the pocket of the Stx1B and Stx2B pentamers.

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, Galα4Galß4Glcß1Cer) and to less extent to globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1), 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.

Clustering of PK-trisaccharides on amphiphilic cyclodextrin reveals unprecedented affinity for the Shiga-like toxin Stx2.

Using amphiphilic cyclodextrin as a scaffold, the first class of PK-glycoconjugates capable of high avidity binding to both Stx1 and Stx2 toxins in solid-phase assay formats is reported. The generated glycomicroarray effectively mimics the plasma membrane surface while discriminating binding of the two Stx toxins, with unprecedented affinity to Stx2.

Shiga toxin glycosphingolipid receptors and their lipid membrane ensemble in primary human blood-brain barrier endothelial cells.

Shiga toxin (Stx)-mediated injury to microvascular endothelial cells in the brain significantly contributes to the pathogenesis of the hemolytic-uremic syndrome caused by enterohemorrhagic Escherichia coli (EHEC). Stxs are AB5 toxins and the B-pentamers of the two major Stx subtypes Stx1a and Stx2a preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) expressed by human endothelial cells. Here we report on comprehensive structural analysis of the different lipoforms of Gb3Cer (Galα4Galß4Glcß1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß3Galα4Galß4Glcß1Cer, the less effective Stx receptor) of primary human brain microvascular endothelial cells and their association with lipid rafts. Detergent-resistant membranes (DRMs), obtained by sucrose density gradient ultracentrifugation, were used as lipid raft-analogous microdomains of the liquid-ordered phase and nonDRM fractions were employed as equivalents for the liquid-disordered phase of cell membranes. Structures of the prevalent lipoforms of Gb3Cer and Gb4Cer were those with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0) determined by electrospray ionization mass spectrometry that was combined with thin-layer chromatography immunodetection using anti-Gb3Cer and anti-Gb4Cer antibodies as well as Stx1a and Stx2a subtypes. Association of Stx receptor GSLs was determined by co-localization with lipid raft-specific membrane protein flotillin-2 and canonical lipid raft marker sphingomyelin with Cer (d18:1, C16:0) and Cer (d18:1, C24:1/C24:0) in the liquid-ordered phase, whereas lyso-phosphatidylcholine was detectable exclusively in the liquid-disordered phase. Defining the precise microdomain structures of primary endothelial cells may help to unravel the initial mechanisms by which Stxs interact with their target cells and will help to develop novel preventive and therapeutic measures for EHEC-mediated diseases.

Adsorption of Shiga Toxin to Poly-γ-Glutamate Precipitated.

We screened foods containing indigestible ingredients in the ability to adsorb Shiga toxin (Stx). When 5 mg of foods and dietary fibers such as dry vegetables and inulin were mixed and incubated with 0.5 mL of Stx solution (100 ng/mL) containing 0.5% bovine serum albumin, both Stx1 and Stx2 seemed to be adsorbed by only a fermented food, natto (a traditional Japanese food prepared from steamed soybeans by the biological action of Bacillus subtilis). We purified the Stx-adsorbing substance from natto by extraction with H2 O, acid treatment, Proteinase K treatment, and an ion exchange chromatography. The purified substance showed an average molecular mass of about 600 kDa. We identified it as poly-γ-glutamate (PGA) by amino acid analysis of its hydrolysate and peptide analysis after its treatment with Proteinase K. Purified PGA (MW: molecular weight = about 600 kDa) was considered to adsorb both Stx1 and Stx2 when we separated adsorbed and unadsorbed Stxs (MW = about 72 kDa) by an ultrafiltration method with a centrifugal filter unit (MWCO: molecular weight cut-off = 100 K). However, PGA with the ability to adsorb Stx was an insoluble form precipitated in the filter unit during centrifugation. PGA precipitated beyond the saturated density was also confirmed to well adsorb both Stx1 and Stx2 by an equilibrated dialysis method. To the best of our knowledge, this is the 1st report on food-adsorbing Stx.

Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit.

Infections with shiga toxin-producing bacteria, like enterohemorrhagic Escherichia coli and Shigella dysenteriae, represent a serious medical problem. No specific and effective treatment is available for patients with these infections, creating a need for the development of new therapies. Recombinant lactic acid bacterium Lactococcus lactis was engineered to bind Shiga toxin by displaying novel designed albumin binding domains (ABD) against Shiga toxin 1 B subunit (Stx1B) on their surface. Functional recombinant Stx1B was produced in Escherichia coli and used as a target for selection of 17 different ABD variants (named S1B) from the ABD scaffold-derived high-complex combinatorial library in combination with a five-round ribosome display. Two most promising S1Bs (S1B22 and S1B26) were characterized into more details by ELISA, surface plasmon resonance and microscale thermophoresis. Addition of S1Bs changed the subcellular distribution of Stx1B, completely eliminating it from Golgi apparatus most likely by interfering with its retrograde transport. All ABD variants were successfully displayed on the surface of L. lactis by fusing to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA. Binding of Stx1B by engineered lactococcal cells was confirmed using flow cytometry and whole cell ELISA. Lactic acid bacteria prepared in this study are potentially useful for the removal of Shiga toxin from human intestine.

A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single-promoter vector and native signal peptide.

The entire stx1 region from Escherichia coli O157:H7, containing two open reading frames (stx1a and stx1b), was cloned into pET-32a with a single promoter. This region was transformed into E. coli TransB (DE3), which is a trxB and gor mutation strain. After expression in the E. coli periplasm in a completely soluble form, the rStx1 was purified and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ELISA, and Western blot analysis. Our rStx1 have Vero cell median cytotoxic dose (CD50 ) and median lethal dose (LD50 ) values of approximately 30 ng and 1.5 µg, respectively. The final yield of the purified rStx1 ranged from 2 to 3 mg/L by one-step nickel affinity gel column chromatography. This method is an easy approach to the large-scale preparation of Stx1 at a reasonable cost.

Microwave-accelerated bioassay technique for rapid and quantitative detection of biological and environmental samples.

Quantitative detection of molecules of interest from biological and environmental samples in a rapid manner, particularly with a relevant concentration range, is imperative to the timely assessment of human diseases and environmental issues. In this work, we employed the microwave-accelerated bioassay (MAB) technique, which is based on the combined use of circular bioassay platforms and microwave heating, for rapid and quantitative detection of Glial Fibrillary Acidic Protein (GFAP) and Shiga like toxin (STX 1). The proof-of-principle use of the MAB technique with the circular bioassay platforms for the rapid detection of GFAP in buffer based on colorimetric and fluorescence readouts was demonstrated with a 900W kitchen microwave. We also employed the MAB technique with a new microwave system (called the iCrystal system) for the detection of GFAP from mice with brain injuries and STX 1 from a city water stream. Control bioassays included the commercially available gold standard bioassay kits run at room temperature. Our results show that the lower limit of detection (LLOD) of the colorimetric and fluorescence based bioassays for GFAP was decreased by ~1000 times using the MAB technique and our circular bioassay platforms as compared to the commercially available bioassay kits. The overall bioassay time for GFAP and STX 1 was reduced from 4h using commercially available bioassay kits to 10min using the MAB technique.

A Conserved Structural Motif Mediates Retrograde Trafficking of Shiga Toxin Types 1 and 2.

Shiga toxin-producing Escherichia coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A-subunits block protein synthesis, while the B-subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B-subunit (STx1B) trafficking. Here, we show that, compared with STx1B, trafficking of the B-subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome-to-Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface-exposed loop in STx2B (ß4-ß5 loop) is required for its endosome-to-Golgi trafficking. We previously demonstrated that residues in the corresponding ß4-ß5 loop of STx1B are required for interaction with GPP130, the STx1B-specific endosomal receptor, and for endosome-to-Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved.

Recognition of human milk oligosaccharides by bacterial exotoxins.

The affinities of the most abundant oligosaccharides found in human milk for four bacterial exotoxins (from Vibrio cholerae and pathogenic Escherichia coli) were quantified for the first time. Association constants (Ka) for a library of 20 human milk oligosaccharides (HMOs) binding to Shiga toxin type 2 holotoxin (Stx2) and the B subunit homopentamers of cholera toxin, heat-labile toxin and Shiga toxin type 1 (CTB5, HLTB5 and Stx1B5) were measured at 25°C and pH 7 using the direct electrospray ionization mass spectrometry assay. Notably, all four bacterial toxins bind to a majority of the HMOs tested and five of the HMOs (2'-fucosyllactose, lacto-N-tetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II and lacto-N-fucopentaose III) are ligands for all four toxins. These five HMOs are also reported to bind to other bacterial toxins (e.g. toxin A and toxin B of Clostridium difficile). In all cases, the HMO affinities (apparent Ka) are relatively modest (≤15,000 M(-1)). However, at the high concentrations of HMOs typically ingested by infants, a significant fraction of these toxins, if present, is expected to be bound to HMOs. Binding measurements carried out with 2'-fucosyllactose or lacto-N-fucopentaose I, together with a high-affinity ligand based on the native carbohydrate receptor, revealed that all four toxins possess HMO-binding sites that are distinct from those of the native receptors, although evidence of competitive binding was found for lacto-N-fucopentaose I with Stx2 and 2'-fucosyllactose and lacto-N-fucopentaose I with HLTB5. Taken together, the results of this study suggest that, while HMOs are expected to bind extensively to these bacterial toxins, it is unlikely that HMO binding will effectively inhibit their interactions with their cellular receptors.

Do the A subunits contribute to the differences in the toxicity of Shiga toxin 1 and Shiga toxin 2?

Shiga toxin producing Escherichia coli O157:H7 (STEC) is one of the leading causes of food-poisoning around the world. Some STEC strains produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2) or variants of either toxin, which are critical for the development of hemorrhagic colitis (HC) or hemolytic uremic syndrome (HUS). Currently, there are no therapeutic treatments for HC or HUS. E. coli O157:H7 strains carrying Stx2 are more virulent and are more frequently associated with HUS, which is the most common cause of renal failure in children in the US. The basis for the increased potency of Stx2 is not fully understood. Shiga toxins belong to the AB5 family of protein toxins with an A subunit, which depurinates a universally conserved adenine residue in the α-sarcin/ricin loop (SRL) of the 28S rRNA and five copies of the B subunit responsible for binding to cellular receptors. Recent studies showed differences in the structure, receptor binding, dependence on ribosomal proteins and pathogenicity of Stx1 and Stx2 and supported a role for the B subunit in differential toxicity. However, the current data do not rule out a potential role for the A1 subunits in the differential toxicity of Stx1 and Stx2. This review highlights the recent progress in understanding the differences in the A1 subunits of Stx1 and Stx2 and their role in defining toxicity.

[Expression and identification of enterohemorrhagic Escherichia coli O157:H7 Shiga toxin1A subunit].

OBJECTIVE: To express and identify enterohemorrhagic Escherichia coli (EHEC) O157:H7 Shiga toxin 1 A subunit (Stx1A). METHODS: Stx1A encoded gene was amplified from EHEC O157:H7 genome by PCR, confirmed by sequencing and cloned into vector pET-22b(+). The recombinant plasmid pET-22b(+)-Stx1A was transformed into E.coli BL21(DE3) which was induced by IPTG to express the target protein. After purified by AKTA(TM);-His affinity chromatography, the recombinant protein was identified by mass spectrometry. With the recombinant protein, BALB/c mice were immunized to develop the anti-sera and evaluate its specific reaction with the natural Stx1A by Western blotting. RESULTS: The Stx1A gene with a size of 945 bp was amplified and cloned into prokaryotic expression vector pET22b(+) to form pET-22b(+)-Stx1A. The recombinant protein was effectively expressed in E.coli BL21(DE3) and purified by 6×His-based affinity chromatography. The mass spectrometry analysis showed that the target protein was Stx1A. Western blotting demonstrated that its immunized sera could react specifically with the natural Stx1A. CONCLUSION: The EHEC O157:H7 Stx1A gene was successfully cloned and expressed, which laid a solid foundation for the following researches.

Potentially pathogenic Escherichia coli in healthy, pasture-raised sheep on farms and at the abattoir in Brazil.

Sheep harbor pathogenic Escherichia coli, which may cause severe disease in humans. In this study, the prevalence of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) was examined in sheep feces and carcasses on three farms and at an abattoir in Brazil. The isolates were further characterized for the presence of markers recently associated with disease in humans, to investigate their possible origin and role as food-borne pathogens. At the abattoir, 99 carcass samples yielded two STEC and 10 EPEC isolates while 101 fecal samples yielded five EPEC and eight STEC isolates. On the other hand, on the farms, 202 samples yielded 44 STEC and eight EPEC isolates. The 77 isolates were typed by PFGE. Isolates with the same PFGE pattern and also those that were not restricted with XbaI were termed as "clones" (n=49). The isolates of any one clone mostly originated from the same sampling site. In addition, seven isolates encoded for novel Stx2 variants and five for Stx2e, the subtype related to porcine edema disease, which was for the first time isolated from sheep feces and carcasses. Also, three stx2-only isolates harbored genes of predicted Stx2 variants that were formed by A and B subunits of different types including Stx2a and Stx2d. The EPEC isolates were heterogeneous, 21 (91.3%) of them possessing efa1, ehxA, lpfAO113 or paa genes associated with diarrhea in humans. Thus, using markers recently associated with disease, we have demonstrated that E. coli similar to those pathogenic for humans are present in the sheep intestinal microflora, particularly at the abattoir, underlining the potential for food-borne transmission.

Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins.

The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG2b fusion protein, the pigeon α1,4-galactosyltransferase (α4Gal-T) and the core 2 ß1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG2b and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG2b. In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG2b and a P(k)-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG2b and P(k)-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon α4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG2b bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.

Recent progress of Shiga toxin neutralizer for treatment of infections by Shiga toxin-producing Escherichia coli.

Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC), including O157:H7, causes bloody diarrhea and hemorrhagic colitis in humans, occasionally resulting in fatal systemic complications, such as neurological damage and hemolytic-uremic syndrome. Because Stx is a major virulence factor of the infectious disease, a series of Shiga toxin neutralizers with various structural characteristics has been developed as promising therapeutic agents. Most of these agents function to bind to the toxin directly and inhibit the binding to its receptor present on the target cells. Other neutralizers do not inhibit receptor binding but induce aberrant intracellular transport of the toxin, resulting in effective detoxification. Such a novel type of Stx neutralizer provides a new therapeutic strategy against STEC infections. Here, recent progress of the development of Stx neutralizers is reviewed.

Identification of amino acids critical for the cytotoxicity of Shiga toxin 1 and 2 in Saccharomyces cerevisiae.

Shiga toxins (Stx1 and Stx2) are produced by E. coli O157:H7, which is a leading cause of foodborne illness. The A subunits of Stx1 (Stx1A) and Stx2 (Stx2A) are ribosome inactivating proteins (RIPs) that inhibit translation by removing an adenine from the highly conserved α-sarcin ricin loop (SRL) of the large rRNA. Here, we used mutagenesis in Saccharomyces cerevisiae to identify residues critical for cytotoxicity of Stx1A and Stx2A. The A subunits depurinated the SRL, inhibited translation and caused apoptotic-like cell death in yeast. Single mutations in Asn75, Tyr77, Glu167 and Arg176 reduced the cytotoxicity of both toxins around 10-fold. However, Asn75 and Tyr77 were more critical for the depurination activity of Stx2A, while Arg176 was more critical for the depurination activity of Stx1A. The crystal structures of the two proteins lack electron density for some surface loops, including one which is adjacent to the active site in both molecules. Modeling these loops changed neither the secondary nor the tertiary structures of the rest of the protein. Analysis of solvent accessible surface areas indicated that Asn75 and Tyr77 are more exposed in Stx2A, while Arg176 is more exposed in Stx1A, indicating that residues with higher surface exposure were more critical for enzymatic activity. Double mutations at Glu167 and Arg176 eliminated the depurination activity and cytotoxicity of both toxins. C-terminal deletions of A chains eliminated cytotoxicity of both toxins, but showed functional differences. Unlike Stx1A, cytotoxicity of Stx2A was lost before its ability to depurinate ribosomes. These results identify residues that affect enzymatic activity and cytotoxicity of Stx1A and Stx2A differently and demonstrate that the function of these residues can be differentiated in yeast. The extent of ribosome depurination and translation inhibition did not correlate with the extent of cell death, indicating that depurination of the SRL and inhibition of translation are not entirely responsible for cell death.