Structure-Activity Relationship Studies of Retro-1 Analogues against Shiga Toxin.

High-throughput screening has shown that Retro-1 inhibits ricin and Shiga toxins by diminishing their intracellular trafficking via the retrograde route, from early endosomes to the Golgi apparatus. To improve the activity of Retro-1, a structure-activity relationship (SAR) study was undertaken and yielded an analogue with a roughly 70-fold better half-maximal effective concentration (EC50) against Shiga toxin cytotoxicity measured in a cell protein synthesis assay.

Synthetic construction of sugar-amino acid hybrid polymers involving globotriaose or lactose and evaluation of their biological activities against Shiga toxins produced by Escherichia coli O157:H7.

Synthetic assembly of sugar moieties and amino acids in order to create "sugar-amino acid hybrid polymers" was accomplished by means of simple radical polymerization of carbohydrate monomers having an amino acid-modified polymerizable aglycon. Amines derived from globotriaoside and lactoside as glycoepitopes were condensed with known carbobenzyloxy derivatives, including Z-Gly, Z-l-Ala and Z-ß-Ala, which had appropriate spacer ability and a chiral center to afford fully protected sugar-amino acid hybrid compounds in good yields. After deprotection followed by acryloylation, the water-soluble glycomonomers were polymerized with or without acrylamide in the presence of a radical initiator in water to give corresponding copolymers and homopolymers, which were shown by SEC analysis to have high molecular weights. Evaluation of the biological activities of the glycopolymers against Shiga toxins (Stxs) was carried out, and the results suggested that glycopolymers having highly clustered globotriaosyl residues had high affinity against Stx2 (KD = 2.7∼4.0 µM) even though other glycopolymers did not show any affinity or showed very weak binding affinity. When Stx1 was used for the same assay, all of the glycopolymers having globotriaosyl residues showed high affinity (KD = 0.30∼1.74 µM). Interestingly, couple of glycopolymers having lactosyl moieties had weaker binding affinity against Stx1. In addition, when cytotoxicity assays were carried out for both Stxs, glycopolymers having highly clustered globotriaosyl residues showed higher affinity than that of the copolymers, and only highly clustered-type glycopolymers displayed neutralization potency against Stx2.

Protection against Shiga Toxins.

Shiga toxins consist of an A-moiety and five B-moieties able to bind the neutral glycosphingolipid globotriaosylceramide (Gb3) on the cell surface. To intoxicate cells efficiently, the toxin A-moiety has to be cleaved by furin and transported retrogradely to the Golgi apparatus and to the endoplasmic reticulum. The enzymatically active part of the A-moiety is then translocated to the cytosol, where it inhibits protein synthesis and in some cell types induces apoptosis. Protection of cells can be provided either by inhibiting binding of the toxin to cells or by interfering with any of the subsequent steps required for its toxic effect. In this article we provide a brief overview of the interaction of Shiga toxins with cells, describe some compounds and conditions found to protect cells against Shiga toxins, and discuss whether they might also provide protection in animals and humans.

Inhibitors of retrograde trafficking active against ricin and Shiga toxins also protect cells from several viruses, Leishmania and Chlamydiales.

Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.

Phage display and Shiga toxin neutralizers.

The current work presents an overview of the use of phage display technology for the identification and characterization of potential neutralizing agents for Shiga toxins. The last major Shiga toxin-associated disease outbreak, which took place in Germany in 2011, showed the international community that Shiga toxins remain a serious threat to public health. This is also demonstrated by the lack of specific therapies against Shiga toxin-induced Hemolytic Uremic Syndrome (HUS). Since its inception, phage display technology has played a key role in the development of antigen-specific (poly)-peptides or antibody fragments with specific biological properties. Herein, we review the current literature regarding the application of phage display to identify novel neutralizing agents against Shiga toxins. We also briefly highlight reported discoveries of peptides and heavy chain antibodies (VHH fragments or nanobodies) that can neutralize the cellular damage caused by these potent toxins.

A rapid and sensitive method to measure the functional activity of Shiga toxins in human serum.

Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins).

Novel enzymatic synthesis of spacer-linked P(k) trisaccharide targeting for neutralization of Shiga toxin.

A novel alkyl spacer-conjugated derivative of P(k) trisaccharide (P(k)), one of the active receptors of Shiga toxins (Stxs; Stx1 and Stx2) produced by pathogenic Escherichia coli (STEC), was designed and synthesized by a combination of cellulase-mediated condensation from Trichoderma reesei and α1,4-galactosyltransferase (LgtC) from Neisseria gonorrhoeae. The specific activity of N. gonorrhoeae LgtC was 66U/mg, which was 13-fold higher than that from N. meningitidis expressed in E. coli. 5-trifluoroacetamidopentyl-ß-P(k) (TFAP-P(k)) was synthesized (yield of 86%, based on the amount of TFAP-lactose added) and its binding to Stx1a-B and Stx2a-B was evaluated. The dissociation constants (KDs) of Stx1a-B and Stx2a-B to the spacer-linked P(k), immobilized on a CM5 sensor chip, were 6.8×10(-6) M (kon=4.1×10(1)M(-1)S(-1), koff=2.8×10(-4)S(-1)) and 2.2×10(-5)M (kon=3.9×10(2)M(-1)S(-1), koff=8.6×10(-3)S(-1)), respectively. This result suggests that the monovalent P(k)-derivative, conjugated to a pentylamino group, represents a promising Stx-neutralizing agent. This cellulase-mediated condensation using cellulase and glycosyltransferase is a valuable tool for the synthesis of spacer-linked oligosaccharide.

The inhibitory effect of colloidal bismuth hydroxide gel on Escherichia coli O157:H7 and on the activity of Shiga toxins.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is the causative agent of hemolytic uremic syndrome (HUS). Colloidal bismuth hydroxide gel (CBHG) is an anti-diarrheal and antisecretory compound, which does not inhibit gastrointestinal motility and reaches an in vivo gut concentration of 10.8 mg/ml of bismuth. Its action on bacteria has not been studied. We analyzed its inhibitory effects on STEC, as well as the deactivation of the Shiga toxin (Stx) and its ability to block the spread of genes encoding Stx. We determined a minimum inhibitory concentration and bactericidal concentration for the STEC O157:H7 strain (EDL933), with CBHG and Chobet® bismuth cream with pectin (CBCHP). We analyzed its effect on Stx by means of cytotoxicity assay and ELISA, as well as its effect on the free 933 W Stx phage. RESULTS: Effect on the EDL933 strain: CBHG: MIC 10 mg/ml of bismuth. CBCHP: MIC 6 mg/ml and MBC 15 mg/ml of bismuth. Effect on EDL933 virulence factors: significant decrease in active Stx and 933 W Stx phage titer. ELISA did not find significant differences with treatment. CONCLUSIONS: The results obtained may be useful in the development of new therapeutic strategies based on the use of CBHG to prevent or improve the prognosis of HUS, as it can be used to control STEC infections.

New Therapeutic Developments against Shiga Toxin-Producing Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is an etiologic agent of bloody diarrhea. A serious sequela of disease, the hemolytic uremic syndrome (HUS) may arise in up to 25% of patients. The development of HUS after STEC infection is linked to the presence of Stx. STEC strains may produce one or more Stxs, and the Stxs come in two major immunological groups, Stx1 and Stx2. A multitude of possible therapeutics designed to inhibit the actions of the Stxs have been developed over the past 30 years. Such therapeutics are important because antibiotic treatment of STEC infections is contraindicated due to an increased potential for development of HUS. The reason for the increased risk of HUS after antibiotic treatment is likely because certain antibiotics induce expression of the Stxs, which are generally associated with lysogenic bacteriophages. There are a few potential therapeutics that either try to kill STEC without inducing Stx expression or target gene expression within STEC. However, the vast majority of the treatments under development are designed to limit Stx receptor generation or to prevent toxin binding, trafficking, processing, or activity within the cell. The potential therapies described in this review include some that have only been tested in vitro and several that demonstrate efficacy in animals. The therapeutics that are currently the furthest along in development (completed phase I and II trials) are monoclonal antibodies directed against Stx1 and Stx2.

A retrograde trafficking inhibitor of ricin and Shiga-like toxins inhibits infection of cells by human and monkey polyomaviruses.

UNLABELLED: Polyomaviruses are ubiquitous pathogens that cause severe disease in immunocompromised individuals. JC polyomavirus (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), whereas BK polyomavirus (BKPyV) causes polyomavirus-induced nephropathy and hemorrhagic cystitis. Vaccines or antiviral therapies targeting these viruses do not exist, and treatments focus on reducing the underlying causes of immunosuppression. We demonstrate that retro-2(cycl), an inhibitor of ricin and Shiga-like toxins (SLTs), inhibits infection by JCPyV, BKPyV, and simian virus 40. Retro-2(cycl) inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum, a step necessary for productive infection. Retro-2(cycl) likely inhibits polyomaviruses in a way similar to its ricin and SLT inhibition, suggesting an overlap in the cellular host factors used by bacterial toxins and polyomaviruses. This work establishes retro-2(cycl) as a potential antiviral therapy that broadly inhibits polyomaviruses and possibly other pathogens that use retrograde trafficking. IMPORTANCE: The human polyomaviruses JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) cause rare but severe diseases in individuals with reduced immune function. During immunosuppression, JCPyV disseminates from the kidney to the central nervous system and destroys oligodendrocytes, resulting in the fatal disease progressive multifocal leukoencephalopathy. Kidney transplant recipients are at increased risk of BKPyV-induced nephropathy, which results in kidney necrosis and loss of the transplanted organ. There are currently no effective therapies for JCPyV and BKPyV. We show that a small molecule named retro-2(cycl) protects cells from infection with JCPyV and BKPyV by inhibiting intracellular viral transport. Retro-2(cycl) treatment reduces viral spreading in already established infections and may therefore be able to control infection in affected patients. Further optimization of retro-2(cycl) may result in the development of an effective antiviral therapy directed toward pathogens that use retrograde trafficking to infect their hosts.

[Studies on the mode of action of bacterial AB5 toxins].

Bacterial AB5 toxins are proteins, produced by pathogenic bacteria including of Vibrio cholerae, Shigella dysenteriae, and enterohaemorrhagic Escherichia coli, which are usually released into the extracellular medium and cause disease by killing or altering the metabolism of target eukaryotic cells. The toxins are usually composed of one A subunit (a toxic domain) and five B subunits (a receptor-binding domain). This article overviews the characteristics and mode of actions of AB5 toxins including cholera toxin, Shiga-like toxin, and subtilase cytotoxin, and highlights current topics related to the roles of the effectors in promoting bacterial infection.

N-methyldihydroquinazolinone derivatives of Retro-2 with enhanced efficacy against Shiga toxin.

The Retro-2 molecule protects cells against Shiga toxins by specifically blocking retrograde transport from early endosomes to the trans-Golgi network. A SAR study has been carried out to identify more potent compounds. Cyclization and modifications of Retro-2 led to a compound with roughly 100-fold improvement of the EC50 against Shiga toxin cytotoxicity measured in a cell protein synthesis assay. We also demonstrated that only one enantiomer of the dihydroquinazolinone reported herein is bioactive.

Synthesis and assessment of globotriose-chitosan conjugate, a novel inhibitor of shiga toxins produced by Escherichia coli.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes diarrhea and colitis in humans that can develop into a life-threatening hemolytic uremic syndrome (HUS). Developing efficient means of controlling STEC diseases, for which no drugs or vaccines are currently available, remains a high priority. We report here the construction and development of chitosan conjugates bearing the Stx ligand trisaccharide globotriose to demonstrate their potential as STEC disease treatment agents. The synthesis was accomplished by grafting a globotriose derivative containing an aldehyde-functionalized aglycone to chitosan amino groups. The obtained globotriose-chitosan conjugate bound with high affinity to Stx and efficiently neutralized its toxicity on Vero cells. Moreover, Stx levels in the gut of infected mice receiving oral doses of the conjugate were greatly diminished, enabling the mice to resist a fatal STEC challenge. The conjugate appears to function as a Stx adsorbent in the gut, preventing toxin entry into the bloodstream and consequent development of HUS. As such, the conjugate could act as a novel agent against STEC disease.

Small-molecule inhibitors of ricin and Shiga toxins.

This review summarizes the successes and continuing challenges associated with the identification of small-molecule inhibitors of ricin and Shiga toxins, members of the RNA N-glycosidase family of toxins that irreversibly inactivate eukaryotic ribosomes through the depurination of a conserved adenosine residue within the sarcin-ricin loop (SRL) of 28S rRNA. Virtual screening of chemical libraries has led to the identification of at least three broad classes of small molecules that bind in or near the toxin's active sites and thereby interfere with RNA N-glycosidase activity. Rational design is being used to improve the specific activity and solubility of a number of these compounds. High-throughput cell-based assays have also led to the identification of small molecules that partially, or in some cases, completely protect cells from ricin- and Shiga-toxin-induced death. A number of these recently identified compounds act on cellular proteins associated with intracellular trafficking or pro-inflammatory/cell death pathways, and one was reported to be sufficient to protect mice in a ricin challenge model.

Physicochemical, microbiological and sensory profiles of fermented milk containing probiotic strains isolated from kefir.

A two-strain starter culture containing Lactobacillus plantarum CIDCA 83114, a potential probiotic strain isolated from kefir grains, and Streptococcus thermophilus CIDCA 321 was tested for the preparation of a fermented milk product. Kluyveromyces marxianus CIDCA 8154, a yeast with immunomodulatory properties was included to formulate a three-strain starter culture. Supernatants of enterohaemorragic Escherichia coli, shiga-toxin-producing strain, along with a two-strain or a three-strain starter culture were included in the medium of Vero-cell surface cultures. The results demonstrated that these combinations of microorganisms antagonize the cytopathic action of shiga toxins. The cell concentration of Lb. plantarum did not decrease during fermentation, indicating that the viability of this strain was not affected by low pH, nor did the number of viable bacteria change during 21 days of storage in either fermented products. The number of viable yeasts increases during fermentation and storage. Trained assessors analyzed the general acceptability of fresh fermented milks and considered both acceptable. The milk fermented with the two-strain starter culture was considered acceptable after two week of storage, while the product fermented with the three-strain starter culture remained acceptable for less than one week. The main changes in sensory attributes detected by the trained panel were in sour taste, milky taste and also in fermented attributes. The correlation between different sensory attributes and acceptability indicated that the panel was positively influenced by milky attributes (taste, odour, and flavour) as well as the intensity of flavour. In conclusion, the two-strain starter culture would be the more promising alternative for inclusion of that potential probiotic lactobacillus in a fermented milk product.

Inhibition of retrograde transport protects mice from lethal ricin challenge.

Bacterial Shiga-like toxins are virulence factors that constitute a significant public health threat worldwide, and the plant toxin ricin is a potential bioterror weapon. To gain access to their cytosolic target, ribosomal RNA, these toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus. Here, we used high-throughput screening to identify small molecule inhibitors that protect cells from ricin and Shiga-like toxins. We identified two compounds that selectively block retrograde toxin trafficking at the early endosome-TGN interface, without affecting compartment morphology, endogenous retrograde cargos, or other trafficking steps, demonstrating an unexpected degree of selectivity and lack of toxicity. In mice, one compound clearly protects from lethal nasal exposure to ricin. Our work discovers the first small molecule that shows efficacy against ricin in animal experiments and identifies the retrograde route as a potential therapeutic target.

Screening and analysis of spices with ability to suppress verocytotoxin production by Escherichia coli O157.

To reduce the amounts of verocytotoxin (VT) produced by Escherichia coli O157:H7, various spices were screened for their ability to suppress VT production. Extracts of these spices were prepared with 70% ethyl alcohol. When E. coli O157:H7 cells were grown to the stationary phase at 37 degrees C in Luria-Bertani medium supplemented with 0.02% allspice extract, the production of both VT1 and VT2 was significantly reduced. Neither growth inhibition nor a delay in the lag phase was observed when the cells were cultured in the presence of 0.02% allspice extract. An active component of the allspice extract was purified by HPLC and was identified as eugenol. When we examined the suppressive effect of eugenol on VT production by E. coli O157:H7, the amounts of both intracellular and extracellular VTs were found to decrease with an increase in eugenol concentration. Our results suggest that eugenol is useful for reducing the virulence of E. coli O157:H7.

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

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

A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins.

A Gb3-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb3 antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb3 antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb3 antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb3 mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb3Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb3 mimic peptide potentially has great promise for use against Stxs.

[Role of the Shiga toxin in the hemolytic uremic syndrome]. / Papel de la toxina shiga en el síndrome urémico hemolítico.

In the last years, infection associated with Shiga toxin-producing Escherichia coli (STEC) and subsequent Hemolitic-Uremic Syndrome (HUS) became relevant as a public health since it was considered as one of the most important emergent patogen present in the food contaminated by cattle feces. STEC infection may be asymptomatic or begins with a watery diarrhea that may or may not progress to bloody diarrhea (hemorrhagic colitis) and HUS. In Argentina, HUS is the most common pediatric cause of acute renal insufficiency and the second cause of chronic renal failure. Up to now, STEC infection lacks of known effective treatment strategies that diminish risk of progression to HUS. The mechanisms by which Shiga toxin (Stx) induce HUS may help to find strategies to prevent or ameliorate HUS. In this article, recent progress that has contributed to understanding the disease pathogenesis of STEC is reviewed. New strategies to prevent further uptake of Shiga from the gut, either during the diarrheal phase or once HUS has developed are discussed.