Exposure to anthrax toxin alters human leucocyte expression of anthrax toxin receptor 1

Anthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-γ responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

GM-CSF receptor targeted treatment of primary AML in SCID mice using Diphtheria toxin fused to huGM-CSF

The severe combined immunodeficient (SCID) mouse model may be used to evaluate new approaches for the treatment of acute myeloid leukemia (AML). We have previously demonstrated the killing of SCID mouse leukemia initiating cells by in vitro incubation with human GM-CSF fused to Diphtheria toxin (DT-huGM-CSF). In this report, we show that in vivo treatment with DT-huGM-CSF eliminates AML growth in SCID mice. Seven cases of AML were studied. SCID mice were treated intraperitoneally with the maximally tolerated dose of 75 microg/kg/day for 7 days. Antileukemic efficacy was determined at days 40 and 80 after transplantation, by enumerating the percentages of human cells in SCID bone marrow using flow cytometry and short tandem repeat polymerase chain reaction (STR-PCR) analysis. Four out of seven AML cases were sensitive to in vivo treatment with DT-huGM-CSF at both evaluation time points. In three of these cases, elimination of human cells was demonstrated by flow cytometry and STR-PCR. One AML case showed moderate sensitivity for DT-huGM-CSF, and growth of the two remaining AML cases was not influenced by DT-huGM-CSF. Sensitivity was correlated with GM-CSFR expression. Our data show that DT-huGM-CSF can be used in vivo to reduce growth of AML and warrant further development of DT-huGM-CSF for the treatment of human AML.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009-2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Exploring the relationship between toxin and spore production in the human enteric pathogen Clostridium difficile

RESUMO: Clostridium difficile é presentemente a principal causa de doença gastrointestinal associada à utilização de antibióticos em adultos. C. difficile é uma bactéria Gram-positiva, obrigatoriamente anaeróbica, capaz de formar endósporos. Tem-se verificado um aumento dos casos de doença associada a C. difficile com sintomas mais severos, elevadas taxas de morbilidade, mortalidade e recorrência, em parte, devido à emergência de estirpes mais virulentas, mas também devido à má gestão do uso de antibióticos. C. difficile produz duas toxinas, TcdA e TcdB, que são os principais fatores de virulência e responsáveis pelos sintomas da doença. Estas são codificadas a partir do Locus de Patogenicidade (PaLoc) que codifica ainda para um regulador positivo, TcdR, uma holina, TcdE, e um regulador negativo, TcdC. Os esporos resistentes ao oxigénio são essenciais para a transmissão do organismo e recorrência da doença. A expressão dos genes do PaLoc ocorre em células vegetativas, no final da fase de crescimento exponencial, e em células em esporulação. Neste trabalho construímos dois mutantes de eliminação em fase dos genes tcdR e tcdE. Mostrámos que a auto-regulação do gene tcdR não é significativa. No entanto, tcdR é sempre necessário para a expressão dos genes presentes no PaLoc. Trabalho anterior mostrou que, com a exceção de tcdC, os demais genes do PaLoc são expressos no pré-esporo. Mostrámos aqui que TcdA é detectada à superfície do esporo maduro e que a eliminação do tcdE não influencia a acumulação de TcdA no meio de cultura ou em associação às células ou ao esporo. Estas observações têm consequências para o nosso entendimento do processo infecioso: sugeremque o esporo possa ser também um veículo para a entrega da toxina nos estágios iniciais da infecção, que TcdA possa ser libertada durante a germinação do esporo, e que o esporo possa utilizar o mesmo receptor reconhecido por TcdA para a ligação à mucosa do cólon.---------------------------ABSTRACT: Clostridium difficile is currently the major cause of antibiotic-associated gastrointestinal diseases in adults. This is a Gram-positive bacterium, endospore-forming and an obligate anaerobe that colonizes the gastrointestinal tract. Recent years have seen a rise in C. difficile associated disease (CDAD) cases, associated with more severe disease symptoms, higher rates of morbidity, mortality and recurrence, which were mostly caused due to the emergence of “hypervirulent” strains but also due to changing patterns of antibiotics use. C. difficile produces two potent toxins, TcdA and TcdB, which are the main virulence factors and the responsible for the disease symptoms. These are codified from a Pathogenicity Locus (PaLoc), composed also by the positive regulator, TcdR, the holin-like protein, TcdE, and a negative regulator, TcdC. Besides the toxins, the oxygen-resistant spores are also essential for transmission of the organism through diarrhea; moreover, spores can accumulate in the environment or in the host, which will cause disease recurrence. The expression of the PaLoc genes occurs in vegetative cells, at the end of the exponential growth phase, and in sporulating cells. In this work, we constructed two in-frame deletion mutants of tcdR and tcdE. We showed that the positive auto regulation of tcdR is not significant. However, tcdR is always necessary for the expression of the PaLoc genes. A previous work showed that, except tcdC, all the PaLoc genes are expressed in the forespore. Here, we detected TcdA at the spore surface. Furthermore, we showed that the in-frame deletion of tcdE does not affect the accumulation of TcdA in the culture medium or in association with cells or spores. This data was important for us to conclude about the infeccious process: it suggests that the spore may be the vehicle for the delivery of TcdA in early stages of infection, that TcdA may be released during spores germination and that this spore may use the same receptor recognized by TcdA to bind to the colonic mucosa.

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines

La toxine stable à la chaleur de type b (STb) est une des toxines produites par les souches Enterotoxigenic Escherichia coli (ETEC) impliquée dans le développement de la diarrhée. Une étude antérieure par Goncalves et al. (2009) a démontré que les cellules ayant internalisé la toxine STb démontraient une morphologie qui rappelle l’apoptose. Le changement du potentiel membranaire observé par Goncalves et al. (2009) nous a incité à vérifier la capacité de la toxine STb à induire l’apoptose des cellules HRT-18 et IEC-18 par la voie intrinsèque. Les cellules HRT-18 et IEC-18 ont été traitées avec de la toxine purifiée pour une durée de 24 heures puis ells ont été récoltées et examinées pour des caratéristiques de l’apoptose. L’activation des caspases-9 et -3, mais pas de la caspase-8, a été observée dans les deux lignées cellulaires à l’aide des substrats fluorescents spécifiques pour chaque caspase. L’ADN extrait des cellules HRT-18 et IEC-18 a révélé une fragmentation lorsque migré sur gel d’agarose. La condensation et la fragmentation des noyaux ont été observées en microscopie à fluorescence suite à une coloration de l’ADN au Hoechst 33342. Les indices apoptotiques des cellules HRT-18 et IEC-18 traitées avec des quantités croissantes de STb montrent une dose-réponse pour les deux lignées. L’activation de la caspase-9 est une indication que la voie intrinsèque de l’apoptose est activée dans les cellules HRT-18 et IEC-18. L’absence de l’activation de la caspase-8 démontre que la voie extrinsèque n’est pas impliquée dans la mort cellulaire médiée par STb.

Structure of the snake-venom toxin convulxin

Snake venoms contain a number of proteins that interact with components of the haemostatic system that promote or inhibit events leading to blood- clot formation. The snake- venom protein convulxin ( Cvx) binds glycoprotein ( GP) VI, the platelet receptor for collagen, and triggers signal transduction. Here, the 2.7 Angstrom resolution crystal structure of Cvx is presented. In common with other members of this snake-venom protein family, Cvx is an alphabeta- heterodimer and conforms to the C- type lectin- fold topology. Comparison with other family members allows a set of Cvx residues that form a concave surface to be putatively implicated in GPVI binding. Unlike other family members, with the exception of flavocetin- A ( FL- A), Cvx forms an (alphabeta)(4) tetramer. This oligomeric structure is consistent with Cvx clustering GPVI molecules on the surface of platelets and as a result promoting signal transduction activity. The Cvx structure and the location of the putative binding sites suggest a model for this multimeric signalling assembly.

Sialyloligosaccharides inhibit cholera toxin binding to the GM1 receptor

It is recognised that cholera toxin (Ctx) is a significant cause of gastrointestinal disease globally, particularly in developing countries where access to uncontaminated drinking water is at a premium. Ctx vaccines are prohibitively expensive and only give short-term protection. Consequently, there is scope for the development of alternative control strategies or prophylactics. This may include the use of oligosaccharides as functional mimics for the cell-surface toxin receptor (GM I). Furthermore, the sialic acid component of epithelial receptors has already been shown to contribute significantly to the adhesion and pathogenesis of Ctx. Here, we demonstrate the total inhibition of Ctx using GM1-competitive ELISA with 25 mg mL(-1) of a commercial preparation of sialyloligosaccharides (SOS). The IC50 value was calculated as 5.21 mg mL(-1). One-hundred percent inhibition was also observed at all concentrations of Ctx-HRP tested with 500 ng mL(-1) GM1-OS. Whilst SOS has much lower affinity for Ctx than GM1-OS, the commercial preparation is impure containing only 33.6% carbohydrate; however, the biantennary nature of SOS appears to give a significant increase in potency over constituent monosaccahride residues. It is proposed that SOS could be used as a conventional food additive, such as in emulsifiers, stabilisers or sweeteners, and are classified as nondigestible oligosaccharides that pass into the small intestine, which is the site of Ctx pathogenesis. (C) 2008 Elsevier Ltd. All rights reserved.

Carbohydrate-based anti-adhesive inhibition of Vibrio cholerae toxin binding to GM1-OS immobilized into artificial planar lipid membranes

We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10 mg/mL. The SOS EC50 value of 5.521 mg/mL is high compared with 0.2811 mu g/mL (182.5 pM or 1.825 x 10(-10) M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E (R) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%. 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease. (C) 2009 Elsevier Ltd. All rights reserved

Galactooligosaccharides (GOS) inhibit Vibrio cholerae toxin binding to its GM1 receptor

It is widely reported that cholera toxin (Ctx) remains a significant cause of gastrointestinal disease globally, particularly in developing countries where access to clean drinking water is at a premium. Vaccines are prohibitively expensive and have shown only short-term protection. Consequently, there is scope for continued development of novel treatment strategies. One example is the use of galactooligosaccharides (GOS) as functional mimics for the cell-surface toxin receptor (GM1). In this study, GOS fractions were fractionated using cation exchange chromatography followed by structural characterization using a combination of hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) such that their molecular weight profiles were known. Each profile was correlated against biological activity measured using a competitive inhibitory GM1-linked ELISA. GOS fractions containing > 5% hexasaccharides (DP6) exhibited > 90% binding, with EC50 values between 29.27 and 56.04 mg/mL. Inhibition by GOS DP6, was dose dependent, with an EC50 value of 5.10 mg/mL (5.15 mu M MW of 990 Da). In removing low molecular weight carbohydrates that do possess prebiotic, nutraceutical, and/or biological properties and concentrating GOS DP5 and/or DP6, Ctx antiadhesive activity per unit of (dry) weight was improved. This could be advantageous in the manufacture of pharmaceutical or nutraceutical formulations for the treatment or prevention of an acute or chronic disease associated with or caused by the adhesion and/or uptake of a Ctx or HLT.

Pectins and pectic-oligosaccharides inhibit Escherichia coli O157 : H7 Shiga toxin as directed towards the human colonic cell line HT29

Pectins and pectic-oligosaccharides, as derived by controlled enzymatic hydrolysis, were evaluated for their ability to interfere with the toxicity of Shiga-like toxins from Escherichia coli O157:H7. Both types of material resulted in some degree of protection but this was significantly higher (P > 0.01) with the oligosaccharide fractions (giving 90-100% cell survival, compared to 70-80% with the polymer). An effect of methylation on the protective effect was detected with lower degrees being more active. The pectic-oligosaccharides and galabiose, the minimum toxin receptor analogue, were shown to inhibit toxicity and were both protective at 10 mg ml(-1), but not at lower concentrations. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Development and validation of a qPCR-based method for quantifying Shiga toxin-encoding and other lambdoid bacteriophages

P>To address whether seasonal variability exists among Shiga toxin-encoding bacteriophage (Stx phage) numbers on a cattle farm, conventional plaque assay was performed on water samples collected over a 17 month period. Distinct seasonal variation in bacteriophage numbers was evident, peaking between June and August. Removal of cattle from the pasture precipitated a reduction in bacteriophage numbers, and during the winter months, no bacteriophage infecting Escherichia coli were detected, a surprising occurrence considering that 1031 tailed-bacteriophages are estimated to populate the globe. To address this discrepancy a culture-independent method based on quantitative PCR was developed. Primers targeting the Q gene and stx genes were designed that accurately and discriminately quantified artificial mixed lambdoid bacteriophage populations. Application of these primer sets to water samples possessing no detectable phages by plaque assay, demonstrated that the number of lambdoid bacteriophage ranged from 4.7 x 104 to 6.5 x 106 ml-1, with one in 103 free lambdoid bacteriophages carrying a Shiga toxin operon (stx). Specific molecular biological tools and discriminatory gene targets have enabled virus populations in the natural environment to be enumerated and similar strategies could replace existing propagation-dependent techniques, which grossly underestimate the abundance of viral entities.

The role of type 1 and curli fimbriae of Shiga toxin-producing Escherichia coli in adherence to abiotic surfaces

Biofilm formation on abiotic surfaces may provide a source of microbial contamination and may also enhance microbial environmental survival. The role of fimbrial expression by Shiga toxin-producing Escherichia coli (STEC) in biofilm formation is poorly understood. This study aimed to investigate the role of STEC type 1 and curli fimbriae in adhesion to and biofilm formation on abiotic surfaces. None of 13 O157:H7 isolates expressed either fimbrial type whereas 11 of 13 and 5 of 13 non-O157 STEC elaborated type 1 fimbriae and curli fimbriae, respectively. Mutants made by allelic exchange of a diarrhoeal non-O157 STEC isolate, O128:H2 (E41509), unable to elaborate type 1 and curli fimbriae were made for adherence and biofilm assays. Elaboration of type 1 fimbriae was necessary for the adhesion to abiotic surfaces whereas curliation was associated with both adherence and subsequent biofilm formation. STEC O157:H7 adhered to thermanox and glass but poorly to polystyrene. Additionally, STEC O157:H7 failed to form biofilms. These data indicate that certain STEC isolates are able to form biofilms and that the elaboration of curli fimbriae may enhance biofilm formation leading to possible long-term survival and a potential source of human infection.

Interaction with avian cells and colonisation of specific pathogen free chicks by Shiga-toxin negative Escherichia coli O157:H7 (NCTC 12900)

The prevalence of Escherichia coli O157:H7 infection in birds is low but several deliberate inoculation studies show that poultry are readily and persistently infected by this organism indicating a possible threat to public health. The mechanisms of colonisation of poultry are not understood and the aim is to establish models to study the interaction of E. coli O157:H7, at the cellular and whole animal levels. A non-toxigenic E. coli O157:H7 (NCTC 12900) was used in adherence assays with an avian epithelial cell line (Div-1) and used to inoculate 1-day-old SPF chicks. In vitro, NCTC 12900 induced micro-colonies associated with cytoskeletal arrangements and pedestal formation with intimate bacterial attachment. In the 1-day-old SPF chick, a dose of 1 x 10(5) cfu resulted in rapid and extensive colonisation of the gastrointestinal tract and transient colonisation of the liver and spleen. The number of E. coli O157:H7 organisms attained approximately 10(8) cfu/ml caecal homogenate 24 h after inoculation and approximately 10(7) cfu/ml caecal homogenate was still present at day 92. Faecal shedding persisted for 169 days, ceasing 9 days after the birds came into lay and 6% of eggs were contaminated on the eggshell. Histological analysis of tissue samples from birds dosed with 1 x 10(7) cfu gave evidence for E coli O157:H7 NCTC 12900 induced micro-colonies on the caecal mucosa, although evidence for attaching effacing lesions was equivocal. These models may be suitable to study those factors of E. coli O157:H7 that mediate persistent colonisation in avian species.

Role for flagella but not intimin in the persistent infection of the gastrointestinal tissues of specific-pathogen-free chicks by Shiga toxin-negative Escherichia coli O157:H7

Shiga toxin (Stx)-positive Escherichia coli O157:117 readily colonize and persist in specific-pathogen-free (SPF) chicks, and we have shown that an Stx-negative E. coli O157:117 isolate (NCTC12900) readily colonizes SPF chicks for up to 169 days after oral inoculation at 1 day of age. However, the role of intimin in the persistent colonization of poultry remains unclear. Thus, to investigate the role of intimin and flagella, which is a known factor in the persistence of non-O157 E. coli in poultry, isogenic single- and double-intimin and aflagellar mutants were constructed in E. coli O157:117 isolate NCTC12900. These mutants were used to inoculate (10(5) CFU) 1-day-old SPF chicks. In general, significant attenuation of the aflagellate and intiminaflagellate mutants, but not the intimin mutant, was noted at similar time points between 22 and 92 days after inoculation. The intimin-deficient mutant was still being shed at the end of the experiment, which was 211 days after inoculation, 84 days more than the wild type. Shedding of the aflagellar and intimin-aflagellar mutants ceased 99 and 113 days after inoculation, respectively. Histological analysis of gastrointestinal tissues from inoculated birds gave no evidence for true microcolony formation by NCTC12900 or intimin and aflagellar mutants to epithelial cells. However, NCTC12900 mutant derivatives associated with the mucosa were observed as individual cells and/or as large aggregates. Association with luminal contents was also noted. These data suggest that O157 organisms do not require intimin for the persistent colonization of chickens, whereas flagella do play a role in this process.