Shiga toxin binding to glycolipids and glycans

Background

Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable.

Methodology

We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells.

Results

By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells.

Conclusions

Stx1 binds primarily to the glycan, but Stx2 binding is influenced by residues in the ceramide portion of Gb3 and the lipid environment. Nanomolar affinities were obtained for both toxins to immobilized glycolipids mixtures, while the effective dose for 50% inhibition (ED₅₀) of protein synthesis was about 10⁻¹¹ M. The failure of preincubation with Gb3 to protect cells from Stx2 suggests that in addition to glycolipid expression, other cellular components contribute to toxin potency.     

Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

Background

Stx toxin is a member of the AB₅ family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes.

Methods and Findings

In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA.

Conclusion

In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.     

Characterizing RecA-independent induction of Shiga toxin2-encoding phages by EDTA treatment

Background

The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA.

Methodology/Principal Findings

The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg²⁺. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction.

Conclusions/Significance

Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains.     

A Polyclonal Antibody Based Immunoassay Detects Seven Subtypes of Shiga Toxin 2 Produced by Escherichia coli in Human and Environmental Samples

Background

Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains.

Methods and Findings

Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity.

Conclusions

The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.     

Shiga Toxin and Lipopolysaccharide Induce Platelet-Leukocyte Aggregates and Tissue Factor Release,a Thrombotic Mechanism in Hemolytic Uremic Syndrome

Background

Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)–bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF.

Methodology/Principal Findings

The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF–expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery.

Conclusions/Significance

By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.     

Glycolipid Binding Preferences of Shiga Toxin Variants

The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx), an AB₅ toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h) significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA) to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) in the presence of cell membrane components such as phosphatidylcholine (PC), cholesterol (Ch) and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.     

Action of Shiga Toxin Type-2 and Subtilase Cytotoxin on Human Microvascular Endothelial Cells

The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 −a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.     

Shiga Toxin Receptor Gb3Cer/CD77: Tumor-Association and Promising Therapeutic Target in Pancreas and Colon Cancer

Background

Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas.

Methodology/Principal Findings

Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors.

Conclusions/Significance

Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.     

A New Immunoassay for Detecting All Subtypes of Shiga Toxins Produced by Shiga Toxin-Producing E. coli in Ground Beef

Background

Shiga toxin (Stx) is a common virulence factor of all Shiga toxin producing E. coli (STEC) that cause a wide spectrum of disease, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although several commercial kits are available for detection of Stx produced by STEC, none of them are capable of recognizing all subtypes of Stxs, which include three subtypes of Stx1 and seven subtypes of Stx2.

Methods and Findings

New monoclonal and polyclonal antibodies against Stx1 and Stx2 were developed. A universal sandwich ELISA capable of detecting all known subtypes of Stx1 and Stx2 was established using a pool of newly developed antibodies. To precisely monitor the sensitivity of the assay for each subtype of Stxs, recombinant toxoids were created and used as standards in ELISAs. Because of the high affinity of the antibodies incorporated, the ELISA assay is highly sensitive with a limit of detection for the different subtypes of Stx1a and Stx2a between 10 and 50 pg/mL in phosphate buffered saline (PBS). The assay was also able to identify STEC based on the production of Stxs using the supernatants of culture fluids or even single colonies on agar plates without lengthy enrichment in liquid medium. When applied to ground beef samples, this newly developed ELISA was capable of distinguishing beef samples spiked with a single bacterial cell.

Conclusions

A highly sensitive and universal assay for all subtypes of Stx1 and Stx2 was developed. It has significantly improved upon the current technologies by avoiding false negative results due to the narrow detection range of the assay. The assay developed in this study can be useful for prompt detection of new and emerging serotypes and screening ground beef samples for contamination of STEC at an early stage in the food supply chain, thus avoiding the need for possible recall.     

Novel Cell-Based Method To Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity

Escherichia coli O157:H7 is a leading cause of food-borne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stx2 and inhibitors of toxin activity. A Vero cell line harboring a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the toxin-induced inhibition of protein synthesis. This Vero-d2EGFP cell line produced a fluorescent signal which could be detected by microscopy or with a plate reader. However, a greatly attenuated fluorescent signal was detected in Vero-d2EGFP cells that had been incubated overnight with either purified Stx2 or a cell-free culture supernatant from Stx1- and Stx2-producing E. coli O157:H7. Dose-response curves demonstrated that the Stx2-induced inhibition of enhanced green fluorescent protein fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and identified a picogram-per-milliliter threshold for toxin detection. To establish our Vero-d2EGFP assay as a useful tool for the identification of toxin inhibitors, we screened a panel of plant compounds for antitoxin activities. Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1- and Stx2-containing medium in the presence of either grape seed or grape pomace extract. The antitoxin properties of the grape extracts were confirmed with an independent toxicity assay that monitored the overall level of protein synthesis in cells treated with purified Stx2. These results indicate that the Vero-d2EGFP fluorescence assay is an accurate and sensitive method to detect Stx2 activity and can be utilized to identify toxin inhibitors.Shiga toxin-producing Escherichia coli, with E. coli O157:H7 as the most common serotype, is an enteric pathogen known to cause human gastrointestinal illnesses ranging from bloody diarrhea and hemorrhagic colitis to life-threatening hemolytic-uremic syndrome (HUS) (1, 20). It has been estimated that E. coli O157 causes approximately 73,000 cases of illness per year in the United States from food- and waterborne sources. Shiga toxins (Stx1 and Stx2) are major virulence factors in E. coli O157 pathogenicity. These toxins inhibit protein synthesis by inactivating the ribosome and are thought to contribute to the development of HUS, a potentially fatal disease for which treatment is currently limited to supportive care (13, 14, 26). Toxin inactivation would prevent the development of HUS, but antitoxin therapeutics are not currently available (26). Detection methods to prevent the distribution of E. coli O157 in foods are thus an important component of food safety programs.The rise in food-related outbreaks of E. coli O157 infection has heightened the importance of developing better methods to rapidly detect and characterize Stxs from E. coli O157 strains (26). Several methods have been developed to examine Stx activity against mammalian cells. Current assays that measure the viability of intoxicated Vero cells require several days of incubation and often produce poor quantitative data (5, 9, 19). Other methods that are more quantitative and sensitive measure the incorporation of radioactive amino acids into newly synthesized proteins (6, 15). However, these radioactivity assays are complex and laborious and allow only a limited number of conditions to be examined. A quantitative luciferase-based assay was recently developed to measure Stx toxicity in a high-throughput format (31), but this system requires several preparatory and processing steps to detect luciferase expression.In the present study, we describe a simple cell-based assay for the detection of Stx2 and inhibitors of toxin activity by using a Vero cell line that expresses a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) to monitor the Stx2-induced inhibition of protein synthesis. This cell-based Vero-d2EGFP assay was used to screen a panel of natural compounds for anti-Stx activities, and we found that grape seed and grape pomace extracts both provided strong cellular protection against Stx2.     

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.     

Investigation of Encephalopathy Caused by Shiga Toxin 2c-Producing Escherichia coli Infection in Mice

A large outbreak of Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) O104:H4 occurred in northern Germany. From this outbreak, at least 900 patients developed hemolytic uremic syndrome (HUS), resulting in more than 50 deaths. Thirty percent of the HUS patients showed encephalopathy. We previously established a mouse model with encephalopathy associated with blood brain barrier (BBB) damage after oral infection with the Shiga toxin (Stx) 2c-producing Escherichia coli O157: H- strain {"type":"entrez-nucleotide","attrs":{"text":"E32511","term_id":"13026758","term_text":"E32511"}}E32511 ({"type":"entrez-nucleotide","attrs":{"text":"E32511","term_id":"13026758","term_text":"E32511"}}E32511). In this model, we detected high expression of the Stx receptor synthase enzyme, glycosphingolipid globotriaosylceramide (Gb3) synthase, in endothelial cells (ECs) and neurons in the reticular formation of the medulla oblongata by in situ hybridization. Caspase-3 was activated in neurons in the reticular formation of the medulla oblongata and the anterior horn of the spinal cord. Astrocytes (ASTs) were activated in the medulla oblongata and spinal cord, and a decrease in aquaporin 4 around the ECs suggested that BBB integrity was compromised directly by Stx2c or through the activation of ASTs. We also report the effectiveness of azithromycin (AZM) in our model. Moreover, AZM strongly inhibited the release of Stx2c from {"type":"entrez-nucleotide","attrs":{"text":"E32511","term_id":"13026758","term_text":"E32511"}}E32511 in vitro.     

Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

Background

Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts.

Principal Findings

We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility.

Significance

Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.     

Carriage of stx2a Differentiates Clinical and Bovine-Biased Strains of Escherichia coli O157

Background

Shiga toxin (Stx) are cardinal virulence factors of enterohemorrhagic E. coli O157:H7 (EHEC O157). The gene content and genomic insertion sites of Stx-associated bacteriophages differentiate clinical genotypes of EHEC O157 (CG, typical of clinical isolates) from bovine-biased genotypes (BBG, rarely identified among clinical isolates). This project was designed to identify bacteriophage-mediated differences that may affect the virulence of CG and BBG.

Methods

Stx-associated bacteriophage differences were identified by whole genome optical scans and characterized among >400 EHEC O157 clinical and cattle isolates by PCR.

Results

Optical restriction maps of BBG strains consistently differed from those of CG strains only in the chromosomal insertion sites of Stx2-associated bacteriophages. Multiplex PCRs (stx1, stx2a, and stx2c as well as Stx-associated bacteriophage - chromosomal insertion site junctions) revealed four CG and three BBG that accounted for >90% of isolates. All BBG contained stx2c and Stx2c-associated bacteriophage – sbcB junctions. All CG contained stx2a and Stx2a-associated bacteriophage junctions in wrbA or argW.

Conclusions

Presence or absence of stx2a (or another product encoded by the Stx2a-associated bacteriophage) is a parsimonious explanation for differential virulence of BBG and CG, as reflected in the distributions of these genotypes in humans and in the cattle reservoir.     

A Recombinant Fusion Toxin Based on Enzymatic Inactive C3bot1 Selectively Targets Macrophages

Background

The C3bot1 protein (∼23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes.

Methodology/Principal Findings

Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages.

Conclusions/Significance

In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.     

Shiga Toxin Increases Formation of Clathrin-Coated Pits through Syk Kinase

Clathrin-dependent endocytosis is a main entry mechanism for the glycolipid-binding Shiga toxin (Stx), although clathrin-independent pathways are also involved. Binding of Stx to its receptor Gb3 not only is essential for Stx retrograde transport to the endoplasmic reticulum and toxicity but also activates signaling through the tyrosine kinase Syk. We previously described that Syk activity is important for Stx entry, but it remained unclear how this kinase modulates endocytosis of Stx. Here we characterized the effects of Stx and Syk on clathrin-coated pit formation. We found that acute treatment with Stx results in an increase in the number of clathrin-coated profiles as determined by electron microscopy and on the number of structures containing the endocytic AP-2 adaptor at the plasma membrane determined by live-cell spinning disk confocal imaging. These responses to Stx require functional Syk activity. We propose that a signaling pathway mediated by Syk and modulated by Stx leads to an increased number of endocytic clathrin-coated structures, thus providing a possible mechanism by which Stx enhances its own endocytosis.     

Phylogenetic analysis of Shiga toxin 1 and Shiga toxin 2 genes associated with disease outbreaks

Background  

Shiga toxins 1 and 2 (Stx1 and Stx2) are bacteriophage-encoded proteins that have been associated with hemorrhagic colitis, hemolytic uremic syndrome and other severe disease conditions. Stx1 and Stx2 are genetically and immunologically distinct but share the same compound toxin structure, method of entry and enzymatic function.     

High temperature in combination with UV irradiation enhances horizontal transfer of stx2 gene from E. coli O157:H7 to non-pathogenic E. coli

Background

Shiga toxin (stx) genes have been transferred to numerous bacteria, one of which is E. coli O157:H7. It is a common belief that stx gene is transferred by bacteriophages, because stx genes are located on lambdoid prophages in the E. coli O157:H7 genome. Both E. coli O157:H7 and non-pathogenic E. coli are highly enriched in cattle feedlots. We hypothesized that strong UV radiation in combination with high temperature accelerates stx gene transfer into non-pathogenic E. coli in feedlots.

Methodology/Principal Findings

E. coli O157:H7 EDL933 strain were subjected to different UV irradiation (0 or 0.5 kJ/m²) combination with different temperature (22, 28, 30, 32, and 37°C) treatments, and the activation of lambdoid prophages was analyzed by plaque forming unit while induction of Stx2 prophages was quantified by quantitative real-time PCR. Data showed that lambdoid prophages in E. coli O157:H7, including phages carrying stx2, were activated under UV radiation, a process enhanced by elevated temperature. Consistently, western blotting analysis indicated that the production of Shiga toxin 2 was also dramatically increased by UV irradiation and high temperature. In situ colony hybridization screening indicated that these activated Stx2 prophages were capable of converting laboratory strain of E. coli K12 into new Shiga toxigenic E. coli, which were further confirmed by PCR and ELISA analysis.

Conclusions/Significance

These data implicate that high environmental temperature in combination with UV irradiation accelerates the spread of stx genes through enhancing Stx prophage induction and Stx phage mediated gene transfer. Cattle feedlot sludge are teemed with E. coli O157:H7 and non-pathogenic E. coli, and is frequently exposed to UV radiation via sunlight, which may contribute to the rapid spread of stx gene to non-pathogenic E. coli and diversity of shiga toxin producing E. coli.     

A Double-Blind,Randomised, Crossover Trial of Two Botulinum Toxin Type A in Patients with Spasticity

Background

Botulinum toxin type A (btxA) is one of the main treatment choices for patients with spasticity. Prosigne® a new released botulinum toxin serotype A may have the same effectiveness as Botox® in focal dystonia. However, there are no randomized clinical trials comparing these formulations in spasticity treatment. The aim of our study was to compare the efficacy and safety of Prosigne® with Botox® in the treatment of spasticity.

Methodology/Principal Findings

We performed a double-blind, randomized, crossover study consisting of 57 patients with clinically meaningful spasticity. The patients were assessed at baseline, 4 and 12 weeks after Prosigne® or Botox® administration. The main outcomes were changes in the patients’ Modified Ashworth Scale (MAS), Functional Independence Measure (FIM) and Pediatric Evaluation of Disability Inventory (PEDI) scores and adverse effects related to the botulinum toxin. Both of the toxins were significantly effective in relieving the level of spasticity in adults and children. There were no significant differences found between the Prosigne® and Botox® treatments regarding their MAS, FIM and PEDI scores. Likewise, the incidence of adverse effects was similar between the two groups.

Conclusion

Our results suggest that Prosigne® and Botox® are both efficient and comparable with respect to their efficacy and safety for the three month treatment of spasticity.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00819065","term_id":"NCT00819065"}}NCT00819065.     

Long-Term Effect of Antibodies against Infused Alpha-Galactosidase A in Fabry Disease on Plasma and Urinary (lyso)Gb3 Reduction and Treatment Outcome

Introduction

Enzyme replacement therapy (ERT) with alpha-Galactosidase A (aGal A) may cause antibody (AB) formation against aGal A in males with Fabry disease (FD). Anti agalsidase ABs negatively influence globotriaosylceramide (Gb3) reduction. We investigated the impact of agalsidase AB on Gb3 and lysoGb3 and clinical outcome in Fabry patients on ERT.

Methods

Adult male and female patients on ERT for at least one year were included. Urinary Gb3 was measured by HPLC, plasma lysoGb3 by LC-ESI-MS/MS and AB with a neutralization assay.

Results

Of the 59 patients evaluable patients, 0/30 females and 17/29 males developed anti-agalsidase antibodies (AB+). Only 3/17 males had transient (low) titers (tolerized). All AB+ patients developed antibodies during the first year of treatment. Change of agalsidase preparation (or dose) did not induce antibody formation. AB+ males had significant less decline in plasma lysoGb3 compared to AB− males (p = 0.04). Urinary Gb3 levels decreased markedly in AB− but remained comparable to baseline in AB+ males (p<0.01). (Lyso)Gb3 reduction in plasma and urine on ERT was correlated with LVmass reduction in females and development white matter lesions and stroke.

Conclusion

In male patients antibodies against aGal A remained present up to 10 years of ERT. The presence of these antibodies is associated with a less robust decrease in plasma lysoGb3 and a profound negative effect on urinary Gb3 reduction, which may reflect worse treatment outcome.