Shiga Toxin Receptor Research Articles

Modeling Shiga toxin-induced human renal-specific microvascular injury.

Shiga toxin (Stx) causes significant renal microvascular injury and kidney failure in the pediatric population, and an effective targeted therapy has yet to be demonstrated. Here we established a human kidney microvascular endothelial cell line for the study of Stx mediated injuries with respect to their morphologic, phenotypic, and transcriptional changes, and modeled Stx induced thrombotic microangiopathy (TMA) in flow-mediated 3D microvessels. Distinct from other endothelial cell lines, both isolated primary and immortalized human kidney microvascular endothelial cells demonstrate robust cell-surface expression of the Stx receptor Gb3, and concomitant dose-dependent toxicity to Stx, with significant contributions from caspase-dependent cell death. Use of a glucosylceramide synthase inhibitor (GCSi) to target disruption of the synthetic pathway of Gb3 resulted in remarkable protection of kidney microvascular cells from Stx injury, shown in both cellular morphologies, caspase activation and transcriptional analysis from RNA sequencing. Importantly, these findings are recapitulated in 3D engineered kidney microvessels under flow. Moreover, whole blood perfusion through Stx-treated microvessels led to marked platelet binding on the vessel wall, which was significantly reduced with the treatment of GCSi. These results validate the feasibility and utility of a bioengineered ex vivo human microvascular model under flow to recapitulate relevant blood-endothelial interactions in STEC-HUS. The profound protection afforded by GCSi demonstrates a preclinical opportunity for investigation in human tissue approximating physiologic conditions. Moreover, this work provides a broad foundation for novel investigation into TMA injury pathogenesis and treatment. Insight Box: Shiga toxin (Stx) causes endothelial injury that results in significant morbidity and mortality in the pediatric population, with no effective targeted therapy. This paper utilizes human kidney microvascular cells to examine Stx mediated cell death in both 2D culture and flow-mediated 3D microvessels, with injured microvessels also developing marked platelet binding and thrombi formation when perfused with blood, consistent with the clinical picture of HUS. This injury is abrogated with a small molecule inhibitor targeting the synthetic pathway of the Shiga toxin receptor. Our findings shed light onto Stx-induced vascular injuries and pave a way for broad investigation into thrombotic microangiopathies.

The Shiga Toxin Receptor Globotriaosylceramide as Therapeutic Target in Shiga Toxin E. coli Mediated HUS.

In 90% of the cases, childhood hemolytic uremic syndrome (HUS) is caused by an infection with the Shiga toxin (Stx) producing E. coli bacteria (STEC-HUS). Stx preferentially binds to its receptor, the glycosphingolipid, globotriaosylceramide (Gb3), present on the surface of human kidney cells and various organs. In this study, the glycosphingolipid pathway in endothelial cells was explored as therapeutic target for STEC-HUS. Primary human glomerular microvascular endothelial cells (HGMVECs) and human blood outgrowth endothelial cells (BOECs) in quiescent and activated state were pre-incubated with Eliglustat (Cerdelga®; glucosylceramide synthase inhibitor) or Agalsidase alpha (Replagal®; human cell derived alpha-galactosidase) in combination with various concentrations of Stx2a. Preincubation of endothelial cells with Agalsidase resulted in an increase of α-galactosidase activity in the cell, but had no effect on the binding of Stx to the cell surface when compared to control cells. However, the incubation of both types of endothelial cells incubated with or without the pro-inflammatory cytokine TNFα in combination with Eliglustat resulted in significant decrease of Stx binding to the cell surface, a decrease in protein synthesis by Stx2a, and diminished cellular Gb3 levels as compared to control cells. In conclusion, inhibition of the synthesis of Gb3 may be a potential future therapeutic target to protect against (further) endothelial damage caused by Stx.

Primary Human Colon Epithelial Cells (pHCoEpiCs) Do Express the Shiga Toxin (Stx) Receptor Glycosphingolipids Gb3Cer and Gb4Cer and Are Largely Refractory but Not Resistant towards Stx.

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a.

Eliglustat prevents Shiga toxin 2 cytotoxic effects in human renal tubular epithelial cells.

Shiga toxin-producing Escherichia coli is responsible for post-diarrheal (D+) hemolytic uremic syndrome (HUS), which is a cause of acute renal failure in children. The glycolipid globotriaosylceramide (Gb3) is the main receptor for Shiga toxin (Stx) in kidney target cells. Eliglustat (EG) is a specific and potent inhibitor of glucosylceramide synthase, first step of glycosphingolipid biosynthesis, actually used for the treatment of Gaucher's disease. The aim of the present work was to evaluate the efficiency of EG in preventing the damage caused by Stx2 in human renal epithelial cells. Human renal tubular epithelial cell (HRTEC) primary cultures were pre-treated with different dilutions of EG followed by co-incubation with EG and Stx2 at different times, and cell viability, proliferation, apoptosis, tubulogenesis, and Gb3 expression were assessed. In HRTEC, pre-treatments with 50 nmol/L EG for 24 h, or 500 nmol/L EG for 6 h, reduced Gb3 expression and totally prevented the effects of Stx2 on cell viability, proliferation, and apoptosis. EG treatment also allowed the development of tubulogenesis in 3D-HRTEC exposed to Stx2. EG could be a potential therapeutic drug for the prevention of acute kidney injury caused by Stx2. For the first time, we have demonstrated that Eliglustat prevents Shiga toxin 2 cytotoxic effects on human renal epithelia, by reducing the expression of the toxin receptor globotriaosylceramide. The present work also shows that Eliglustat prevents Shiga toxin 2 effects on tubulogenesis of renal epithelial cells. Eliglustat, actually used for the treatment of patients with Gaucher's disease, could be a therapeutic strategy to prevent the renal damage caused by Shiga toxin.

Potent in vitro antitumor activity of B-subunit of Shiga toxin conjugated to the diphtheria toxin against breast cancer

Immunotoxins are protein-based drugs consist of a target-specific binding domain and a cytotoxic domain to eliminate target cells. Such compounds are potentially therapeutic to combat diseases such as cancer. Generally, the B-subunit of Shiga toxin (STXB) receptor, globotriaosylceramide (Gb3), is expressed in high amounts on a number of human tumors cancer cells. In this study, we evaluated a new antitumor candidate called DT389-STXB chimeric protein, which genetically fused the DT to B-subunit of Shiga-like toxin (STXB).First a chimeric protein, encoding DT389-STXB was synthesized. The optimized chimeric protein expressed in E.coli BL21 (DE3) and confirmed by anti-His Western blot analysis. T47D, SKBR3, 4T1 and MCF7 cell lines were treated separately with purified DT389-STXB recombinant protein and functional activity of DT389-STXB was analyzed by the cell enzyme-linked immunosorbentassay (ELISA), MTT, ICC, Western blot and apoptosis tests.The results indicated that the recombinant DT389-STXB fusion protein with a molecular weight of 53 kDa was successfully expressed in E.coli BL21 (DE3) and the anti-His western-blot was used to confirm the presence of the protein. The DT389-STXB fusion protein attached to T47D, SKBR3 and 4T1 cell lines with the proper affinity and induced dose-dependent cytotoxicity against GB3-expressing cancer cells in vitro.Our results showed that DT389-STXB fusion protein may be a promising candidate for antitumor therapy agent against breast cancer; however, further studies are required to explore its efficacy in vivo for therapeutic applications.

Blood group P1 antigen–bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.

Designing and Analyzing the Structure of DT-STXB Fusion Protein as an Anti-tumor Agent: An in Silico Approach.

Background & Objective:A main contest in chemotherapy is to obtain regulator above the biodistribution of cytotoxic drugs. The utmost promising strategy comprises of drugs coupled with a tumor-targeting bearer that results in wide cytotoxic activity and particular delivery. The B-subunit of Shiga toxin (STxB) is nontoxic and possesses low immunogenicity that exactly binds to the globotriaosylceramide (Gb3/CD77). Gb3/CD77 extremely expresses on a number of human tumors such as pancreatic, colon, and breast cancer and acts as a functional receptor for Shiga toxin (STx). Then, this toxin can be applied to target Gb3-positive human tumors. In this study, we evaluated DT390-STXB chimeric protein as a new anti-tumor candidate via genetically fusing the DT390 fragment of DT538 (Native diphtheria toxin) to STxB.Methods:This study intended to investigate the DT390- STxB fusion protein structure in silico. Considering the Escherichia coli codon usage, the genomic construct was designed. The properties and the structure of the protein were determined by an in silico technique. The mRNA structure and the physicochemical characteristics, construction, and the stability of the designed chimeric protein were analyzed using computational and bioinformatics tools and servers. Hence, the GOR4 and I-TASSER online web servers were used to predict the secondary and tertiary structures of the designed protein.Results:The results demonstrated that codon adaptation index (CAI) of dt390-stxB chimeric gene raised from 0.6 in the wild type to 0.9 in the chimeric optimized gene. The mfold data revealed that the dt390-stxB mRNA was completely stable to be translated effectively in the novel host. The normal activity of the fusion protein determined by considering the secondary and tertiary structure of each construct. Energy calculation data indicated that the thermodynamic ensemble for mRNA structure was -427.40 kJ/mol. The stability index (SI) of DT390-STxB was 36.95, which is quite appropriate to preserve the stability of the construct. Ultimately, the DT390-STxB was classified as a steady fusion protein according to the Ramachandran plot. Conclusion:Our results showed that DT390-STXB was a stable chimeric protein and it can be recruited as a candidate of novel anti-tumor agents for the development of breast cancer treatment.

Functional analyses of the UDP-galactose transporter SLC35A2 using the binding of bacterial Shiga toxins as a novel activity assay

SLC35A2 transports UDP-galactose from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum for glycosylation. Mutations in SLC35A2 induce a congenital disorder of glycosylation. Despite the biomedical relevance, mechanisms of transport via SLC35A2 and the impact of disease-associated mutations on activity are unclear. To address these issues, we generated a predicted structure of SLC35A2 and assayed for the effects of a set of structural and disease-associated mutations. Activity assays were performed using a rescue approach in ΔSLC35A2 cells and took advantage of the fact that SLC35A2 is required for expression of the glycosphingolipid globotriaosylceramide (Gb3), the cell surface receptor for Shiga toxin 1 (STx1) and 2 (STx2). The N- and C-terminal cytoplasmic loops of SLC35A2 were dispensable for activity, but two critical glycine (Gly-202 and Gly-214) and lysine (Lys-78 and Lys-297) residues in transmembrane segments were required. Residues corresponding to Gly-202 and Gly-214 in the related transporter SLC35A1 form a substrate-translocating channel, suggesting that a similar mechanism may be involved in SLC35A2. Among the eight disease-associated mutations tested, SLC35A2 function was completely inhibited by two (S213F and G282R) and partially inhibited by three (R55L, G266V, and S304P), providing a straight-forward mechanism of disease. Interestingly, the remaining three (V331I, V258M, and Y267C) did not impact SLC35A2 function, suggesting that complexities beyond loss of transporter activity may underlie disease due to these mutations. Overall, our results provide new insights into the mechanisms of transport of SLC35A2 and improve understanding of the relationship between SLC35A2 mutations and disease.

A CRISPR Screen Identifies LAPTM4A and TM9SF Proteins as Glycolipid-Regulating Factors

SummaryGlycosphingolipids (GSLs) are produced by various GSL-synthesizing enzymes, but post-translational regulation of these enzymes is incompletely understood. To address this knowledge disparity, we focused on biosynthesis of globotriaosylceramide (Gb3), the Shiga toxin (STx) receptor, and performed a genome-wide CRISPR/CAS9 knockout screen in HeLa cells using STx1-mediated cytotoxicity. We identified various genes including sphingolipid-related genes and membrane-trafficking genes. In addition, we found two proteins, LAPTM4A and TM9SF2, for which physiological roles remain elusive. Disruption of either LAPTM4A or TM9SF2 genes reduced Gb3 biosynthesis, resulting in accumulation of its precursor, lactosylceramide. Loss of LAPTM4A decreased endogenous Gb3 synthase activity in a post-transcriptional mechanism, whereas loss of TM9SF2 did not affect Gb3 synthase activity but instead disrupted localization of Gb3 synthase. Furthermore, the Gb3-regulating activity of TM9SF2 was conserved in the TM9SF family. These results provide mechanistic insight into the post-translational regulation of the activity and localization of Gb3 synthase.

Infrared MALDI mass spectrometry imaging of TLC-separated glycosphingolipids with emphasis on Shiga toxin receptors isolated from human colon epithelial cells

Thin-layer chromatography (TLC) combined with infrared MALDI mass spectrometry (IR-MALDI-MS) is a powerful analytical tool for detection and structural characterization of glyco- and phospholipids directly from the TLC plate. Here we coupled a pulsed IR-MALDI laser to a hybrid Synapt G2-S mass spectrometer (Waters) and obtained an effective focal spot size of ∼50×70μm2 in diameter. We used this new MALDI ion source configuration for TLC-IR-MALDI-MS imaging of neutral glycosphingolipids (GSLs), obtained from human colon epithelial HCT-8 cells, at 100μm step size. Our analytical focus was on the detection of globo-series GSLs globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the main receptors for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli (EHEC). The direct TLC-IR-MALDI-MSI analysis allowed precise visualization of the chromatographic separation of the various lipoforms of Gb3Cer and Gb4Cer, with ceramide moieties mainly ranging from Cer (d18:1, C16:0) to Cer (d18:1, C24:0/C24:1) as well as those of their precursor GSLs glucosylceramide (GlcCer) and lactosylceramide (Lc2Cer). Reference TLC overlay immunostaining assays were conducted on parallel developed TLC lanes for confirmation of anomeric sugar configuration of proposed structures. Together, the adopted protocol provided a rapid and near-comprehensive overview of the GSL composition of the investigated cell line of high medical relevance. This possibility could also be highly useful in glycolipidomics studies of complex biological matrices.

152 - The Shiga Toxin receptor Gb3/CD77, a promising therapeutical target in gastrointestinal cancer, is epigenetically regulated

152 - The Shiga Toxin receptor Gb3/CD77, a promising therapeutical target in gastrointestinal cancer, is epigenetically regulated

Gastric Adenocarcinomas Express the Glycosphingolipid Gb3/CD77: Targeting of Gastric Cancer Cells with Shiga Toxin B-Subunit.

The B-subunit of the bacterial Shiga toxin (STxB), which is nontoxic and has low immunogenicity, can be used for tumor targeting of breast, colon, and pancreatic cancer. Here, we tested whether human gastric cancers, which are among the most aggressive tumor entities, express the cellular receptor of Shiga toxin, the glycosphingolipid globotriaosylceramide (Gb3/CD77). The majority of cases showed an extensive staining for Gb3 (36/50 cases, 72%), as evidenced on tissue sections of surgically resected specimen. Gb3 expression was detected independent of type (diffuse/intestinal), and was negatively correlated to increasing tumor-node-metastasis stages (P = 0.0385), as well as with markers for senescence. Gb3 expression in nondiseased gastric mucosa was restricted to chief and parietal cells at the bottom of the gastric glands, and was not elevated in endoscopic samples of gastritis (n = 10). Gb3 expression in established cell lines of gastric carcinoma was heterogeneous, with 6 of 10 lines being positive, evidenced by flow cytometry. STxB was taken up rapidly by live Gb3-positive gastric cancer cells, following the intracellular retrograde transport route, avoiding lysosomes and rapidly reaching the Golgi apparatus and the endoplasmic reticulum. Treatment of the Gb3-expressing gastric carcinoma cell line St3051 with STxB coupled to SN38, the active metabolite of the topoisomerase type I inhibitor irinotecan, resulted in >100-fold increased cytotoxicity, as compared with irinotecan alone. No cytotoxicity was observed on gastric cancer cell lines lacking Gb3 expression, demonstrating receptor specificity of the STxB-SN38 compound. Thus, STxB is a highly specific transport vehicle for cytotoxic agents in gastric carcinoma. Mol Cancer Ther; 15(5); 1008-17. ©2016 AACR.

Upregulation of Shiga toxin receptor CD77/Gb3 and interleukin-1β expression in the brain of EHEC patients with hemolytic uremic syndrome and neurologic symptoms.

In 2011, a large outbreak of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) infections occurred in northern Germany, which mainly affected adults. Out of 3842 patients, 104 experienced a complicated course comprising hemolytic uremic syndrome and neurological complications, including cognitive impairment, aphasia, seizures and coma. T2 hyperintensities on magnet resonance imaging (MRI) bilateral in the thalami and in the dorsal pons were found suggestive of a metabolic toxic effect. Five of the 104 patients died because of toxic heart failure. In the present study, the post-mortem neuropathological findings of the five EHEC patients are described. Histological investigation of 13 brain regions (frontal, temporal, occipital cortex, corpora mammillaria, thalamus, frontal operculum, corona radiata, gyrus angularis, pons, medulla oblongata, cerebellar vermis and cerebellar hemisphere) showed no thrombosis, ischemic changes or fresh infarctions. Further, no changes were found in electron microscopy. In comparison with five age-matched controls, slightly increased activation of microglia and a higher neuronal expression of interleukin-1β and of Shiga toxin receptor CD77/globotriaosylceramide 3 was observed. The findings were confirmed by Western blot analyses. It is suggested that CD77/globotriaosylceramide upregulation may be a consequence to Shiga toxin exposure, whereas increased interleukin-1β expression may point to activation of inflammatory cascades.

Manganese induces oligomerization to promote down-regulation of the intracellular trafficking receptor used by Shiga toxin.

Manganese (Mn) protects cells against lethal doses of purified Shiga toxin by causing the degradation of the cycling transmembrane protein GPP130, which the toxin uses as a trafficking receptor. Mn-induced GPP130 down-regulation, in addition to being a potential therapeutic approach against Shiga toxicosis, is a model for the study of metal-regulated protein sorting. Significantly, however, the mechanism by which Mn regulates GPP130 trafficking is unknown. Here we show that a transferable trafficking determinant within GPP130 bound Mn and that Mn binding induced GPP130 oligomerization in the Golgi. Alanine substitutions blocking Mn binding abrogated both oligomerization of GPP130 and GPP130 sorting from the Golgi to lysosomes. Further, oligomerization was sufficient because forced aggregation, using a drug-controlled polymerization domain, redirected GPP130 to lysosomes in the absence of Mn. These experiments reveal metal-induced oligomerization as a Golgi sorting mechanism for a medically relevant receptor for Shiga toxin.

The ether lipid precursor hexadecylglycerol protects against Shiga toxins.

Shiga toxin-producing Escherichia coli bacteria cause hemorrhagic colitis and hemolytic uremic syndrome in humans. Currently, only supportive treatment is available for diagnosed patients. We show here that 24-h pretreatment with an ether lipid precursor, the alkylglycerol sn-1-O-hexadecylglycerol (HG), protects HEp-2 cells against Shiga toxin and Shiga toxin 2. Also the endothelial cell lines HMEC-1 and HBMEC are protected against Shiga toxins after HG pretreatment. In contrast, the corresponding acylglycerol, DL-α-palmitin, has no effect on Shiga toxicity. Although HG treatment provides a strong protection (~30 times higher IC₅₀) against Shiga toxin, only a moderate reduction in toxin binding was observed, suggesting that retrograde transport of the toxin from the plasma membrane to the cytosol is perturbed. Furthermore, endocytosis of Shiga toxin and retrograde sorting from endosomes to the Golgi apparatus remain intact, but transport from the Golgi to the endoplasmic reticulum is inhibited by HG treatment. As previously described, HG reduces the total level of all quantified glycosphingolipids to 50-70% of control, including the Shiga toxin receptor globotriaosylceramide (Gb3), in HEp-2 cells. In accordance with this, we find that interfering with Gb3 biosynthesis by siRNA-mediated knockdown of Gb3 synthase for 24 h causes a similar cytotoxic protection and only a moderate reduction in toxin binding (to 70% of control cells). Alkylglycerols, including HG, have been administered to humans for investigation of therapeutic roles in disorders where ether lipid biosynthesis is deficient, as well as in cancer therapy. Further studies may reveal if HG can also have a therapeutic potential in Shiga toxin-producing E. coli infections.

Thalamic involvement in patients with neurologic impairment due to Shiga toxin 2

The outbreak of hemolytic-uremic syndrome and diarrhea caused by Shiga toxin-producing Escherichia coli O104:H4 in Germany during May to July 2011 involved severe and characteristic neurologic manifestations with a strong female preponderance. Owing to these observations, we designed a series of experimental studies to evaluate the underlying mechanism of action of this clinical picture. A magnetic resonance imaging and electroencephalographic study of patients was performed to evaluate the clinical picture in detail. Thereafter, combinations of different experimental settings, including electrophysiological and histological analyses, as well as calcium imaging in brain slices of rats, were conducted. We report on 7 female patients with neurologic symptoms and signs including bilateral thalamic lesions and encephalopathic changes indicative of a predominant involvement of the thalamus. Experimental studies in rats revealed an enhanced expression of the Shiga toxin receptor globotriaosylceramide on thalamic neurons in female rats as compared to other brain regions in the same rats and to male animals. Incubation of brain slices with Shiga toxin 2 evoked a strong membrane depolarization and intracellular calcium accumulation in neurons, associated with neuronal apoptosis, predominantly in the thalamic area. These findings suggest that the direct cytotoxic effect of Shiga toxin 2 in the thalamus might contribute to the pathophysiology of neuronal complications in hemolytic-uremic syndrome.

Structure-dependent Pseudoreceptor Intracellular Traffic of Adamantyl Globotriaosyl Ceramide Mimics

The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb(3)), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb(3) is an amphipathic competitive inhibitor of VT1/VT2 Gb(3) binding. However, Gb(3)-negative VT-resistant CHO/Jurkat cells incorporate adaGb(3) to become VT1/VT2-sensitive. CarboxyadaGb(3), urea-adaGb(3), and hydroxyethyl adaGb(3), preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb(3) (two deacyl Gb(3)s linked to adamantane) protects against VT1/VT2 more effectively than adaGb(3) without incorporating into Gb(3)-negative cells. AdaGb(3) (but not hydroxyethyl adaGb(3)) incorporation into Gb(3)-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb(3)-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb(3)-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb(3) destabilized membrane cholesterol and reduced Gb(3) cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb(3). We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb(3) mimics and their structure-dependent modulation of endogenous intracellular Gb(3) vesicular traffic.

Shiga toxin 2-induced intestinal pathology in infant rabbits is A-subunit dependent and responsive to the tyrosine kinase and potential ZAK inhibitor imatinib

Shiga toxin producing Escherichia coli (STEC) are a major cause of food-borne illness worldwide. However, a consensus regarding the role Shiga toxins play in the onset of diarrhea and hemorrhagic colitis (HC) is lacking. One of the obstacles to understanding the role of Shiga toxins to STEC-mediated intestinal pathology is a deficit in small animal models that perfectly mimic human disease. Infant rabbits have been previously used to study STEC and/or Shiga toxin-mediated intestinal inflammation and diarrhea. We demonstrate using infant rabbits that Shiga toxin-mediated intestinal damage requires A-subunit activity, and like the human colon, that of the infant rabbit expresses the Shiga toxin receptor Gb3. We also demonstrate that Shiga toxin treatment of the infant rabbit results in apoptosis and activation of p38 within colonic tissues. Finally we demonstrate that the infant rabbit model may be used to test candidate therapeutics against Shiga toxin-mediated intestinal damage. While the p38 inhibitor SB203580 and the ZAK inhibitor DHP-2 were ineffective at preventing Shiga toxin-mediated damage to the colon, pretreatment of infant rabbits with the drug imatinib resulted in a decrease of Shiga toxin-mediated heterophil infiltration of the colon. Therefore, we propose that this model may be useful in elucidating mechanisms by which Shiga toxins could contribute to intestinal damage in the human.

Tumor-Specific Targeting of Pancreatic Cancer with Shiga Toxin B-Subunit

Pancreatic carcinoma is one of the most aggressive tumor entities, and standard chemotherapy provides only modest benefit. Therefore, specific targeting of pancreatic cancer for early diagnosis and therapeutic intervention is of great interest. We have previously shown that the cellular receptor for Shiga toxin B (STxB), the glycosphingolipid globotriaosylceramide (Gb(3) or CD77) is strongly increased in colorectal adenocarcinoma and their metastases. Here, we report an upregulation of Gb(3) in pancreatic adenocarcinoma (21 of 27 cases) as compared with matched normal tissue (n = 27). The mean expression was highly significantly increased from 30 ± 16 ng Gb(3)/mg tissue in normal pancreas to 61 ± 41 ng Gb(3)/mg tissue (mean ± SD, P = 0.0006), as evidenced by thin layer chromatography. Upregulation of Gb(3) levels did not depend on tumor stage or grading and showed no correlation with clinical outcome. Tumor cells and endothelial cells were identified as the source of increased Gb(3) expression by immunocytochemistry. Pancreatic cancer cell lines showed rapid intracellular uptake of STxB to the Golgi apparatus, following the retrograde pathway. The therapeutic application of STxB was tested by specific delivery of covalently coupled SN38, an active metabolite of the topoisomerase I inhibitor irinotecan. The cytotoxic effect of the STxB-SN38 compound in pancreatic cancer cell lines was increased more than 100-fold compared with irinotecan. Moreover, this effect was effectively blocked by competing incubation with nonlabeled STxB, showing the specificity of the targeting. Thus, STxB constitutes a promising new tool for specific targeting of pancreatic cancer.

Regulatory interplay between Escherichia coli O157:H7 factors governing cattle colonisation by manipulation of innate immunity

Event Abstract Back to Event Regulatory interplay between Escherichia coli O157:H7 factors governing cattle colonisation by manipulation of innate immunity David Gally1* 1 University of, Division of Immunity and Infection, United Kingdom Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is the most common EHEC serotype associated with human disease in the United Kingdom, with about 1000 cases a year. The bacterium originates from ruminant hosts, in particular cattle, where it colonises the terminal rectum. EHEC O157 colonisation of cattle is not associated with disease and this is mainly due to the different distribution of Gb3/CD77 Shiga toxin (Stx) receptors in cattle compared with humans. While the basis to the rectal tropism is still unclear a number of factors contribute to attachment at this site and therefore persistence in cattle. H7 flagella are involved in initial interactions followed by intimate binding conferred by a type III secretion system (T3SS). Vaccines combining antigens from these two systems have been shown to reduce both the number of cattle colonized and the level of bacterial excretion from animals that do become colonized following oral challenge with EHEC O157. Phage typing has been commonly applied to examine EHEC subpopulations; over the last decade EHEC O157 PT21/28 has been associated with the majority of human infections in the UK, although this now appears to be changing. A comprehensive study of cattle in Scotland, over the same period, showed that the same PT was common in cattle and was also more likely than other phage types to be sampled from cattle excreting high levels of the bacteria. Our previous work has also shown that T3S levels and regulation vary considerably between EHEC O157 strains. A key aim for our research is to understand the key genetic differences between strains underlying their capacity to be associated with disease. The simplest hypothesis being that higher or longer term shedding increases the environmental load and the likelihood of human infection. Our research is focused on the contribution of prophages, both functional and cryptic, to EHEC colonization of cattle. Genes encoding Stx and effector proteins secreted by the T3S system have been integrated into the EHEC genome as prophages and both contribute to innate response manipulation presumably to promote persistence in the bovine host. Our recent work now demonstrates that several of these prophages also control expression of T3S. Taken together, it is proposed that the prophage repertoire of an EHEC O157 strain is a critical determinant of colonization capacity and excretion level (Fig. 1). This talk will focus on this control and mathematical modeling of colonization that has provided novel insights into the timing and role of the innate response in restricting EHEC O157 colonisation in cattle and therefore why this response the key target for Stx and bacterial effector proteins. Keywords: EHEC O157:H7, Phage typing, Prophages, STX, type III secretion system Conference: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination, Ghent, Belgium, 2 Jul - 5 Jul, 2011. Presentation Type: Oral Presentation Topic: Infection and innate immunity, immunosupression and/or immunostimulation Citation: Gally D (2011). Regulatory interplay between Escherichia coli O157:H7 factors governing cattle colonisation by manipulation of innate immunity. Front. Immunol. Conference Abstract: ECMIS - E. coli and the Mucosal Immune System : Interaction, Modulation and Vaccination. doi: 10.3389/conf.fimmu.2011.01.00020 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 22 Sep 2011; Published Online: 13 Oct 2011. * Correspondence: Prof. David Gally, University of, Division of Immunity and Infection, Edinburgh, United Kingdom, d.gally@ed.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers David Gally Google David Gally Google Scholar David Gally PubMed David Gally Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.