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Sample records for heat-labile enterotoxin promotes

  1. Heat-labile enterotoxin of Escherichia coli promotes intestinal colonization of Salmonella enterica.

    PubMed

    Verbrugghe, Elin; Van Parys, Alexander; Leyman, Bregje; Boyen, Filip; Arnouts, Sven; Lundberg, Urban; Ducatelle, Richard; Van den Broeck, Wim; Yekta, Maryam Atef; Cox, Eric; Haesebrouck, Freddy; Pasmans, Frank

    2015-12-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of infantile and travellers' diarrhoea, which poses a serious health burden, especially in developing countries. In addition, ETEC bacteria are a major cause of illness and death in neonatal and recently weaned pigs. The production of a heat-labile enterotoxin (LT) promotes the colonization and pathogenicity of ETEC and may exacerbate co-infections with other enteric pathogens such as Salmonella enterica. We showed that the intraintestinal presence of LT dramatically increased the intestinal Salmonella Typhimurium load in experimentally inoculated pigs. This could not be explained by direct alteration of the invasion or survival capacity of Salmonella in enterocytes, in vitro. However, we demonstrated that LT affects the enteric mucus layer composition in a mucus-secreting goblet cell line by significantly decreasing the expression of mucin 4. The current results show that LT alters the intestinal mucus composition and aggravates a Salmonella Typhimurium infection, which may result in the exacerbation of the diarrhoeal illness. PMID:26616654

  2. Escherichia coli heat-labile enterotoxin promotes protective Th17 responses against infection by driving innate IL-1 and IL-23 production.

    PubMed

    Brereton, Corinna F; Sutton, Caroline E; Ross, Pádraig J; Iwakura, Yoichiro; Pizza, Mariagrazia; Rappuoli, Rino; Lavelle, Ed C; Mills, Kingston H G

    2011-05-15

    Escherichia coli heat-labile enterotoxin (LT) is a powerful mucosal adjuvant; however, it is associated with toxic effects when delivered intranasally, and its mechanism of action is poorly understood. In this article, we demonstrate that LT acts as a highly effective adjuvant when administered parenterally, promoting Ag-specific IL-17, as well as IFN-γ, IL-4, and IL-10 production in response to coadministered Ags. We found that the adjuvant activity of LT was mediated in part by inducing dendritic cell (DC) activation; LT promoted CD80 and CD86 expression by DCs and enhanced IL-1α, IL-1β, and IL-23 production. An LT mutant, LTK63, that lacks enzyme activity was less effective than the wild-type toxin in promoting DC maturation and the development of Ag-specific Th17 cells. LT enhanced IL-23 and IL-1α production from DCs via activation of ERK MAPK and IL-1β secretion through activation of caspase-1 and the NLRP3 inflammasome. These cytokines played a major role in promoting Th17 responses by LT and LTK63. The induction of Th17 cells in vivo in response to LT and LTK63 as adjuvants was significantly reduced in IL-1RI-deficient mice. Finally, using a murine respiratory infection model, we demonstrated that LT can act as a highly effective adjuvant for a pertussis vaccine, promoting Ag-specific Th17 cells and protection against Bordetella pertussis challenge, which was significantly reduced in IL-17-defective mice. Our findings provide clear evidence that LT can promote protective immune responses in part through induction of innate IL-1 and, consequently, Th17 cells. PMID:21490151

  3. Inhibition of heat-labile cholera and Escherichia coli enterotoxins by brefeldin A.

    PubMed

    Donta, S T; Beristain, S; Tomicic, T K

    1993-08-01

    Cholera enterotoxin and the related heat-labile enterotoxins of Escherichia coli enter their target cells through noncoated vesicles, but how the toxins are processed intracellularly and how they get to their targeted enzyme, adenylate cyclase, remain to be defined. Brefeldin A, an inhibitor of the trans-Golgi network, is shown herein to transiently block the morphologic and enzymatic effects of the toxin at a step distal to the initial binding process but prior to activation of adenylate cyclase by the toxin. It is likely, therefore, that these toxins are processed by the Golgi apparatus before trafficking to the membrane adenylate cyclase. PMID:8392970

  4. Inhibition of heat-labile cholera and Escherichia coli enterotoxins by brefeldin A.

    PubMed Central

    Donta, S T; Beristain, S; Tomicic, T K

    1993-01-01

    Cholera enterotoxin and the related heat-labile enterotoxins of Escherichia coli enter their target cells through noncoated vesicles, but how the toxins are processed intracellularly and how they get to their targeted enzyme, adenylate cyclase, remain to be defined. Brefeldin A, an inhibitor of the trans-Golgi network, is shown herein to transiently block the morphologic and enzymatic effects of the toxin at a step distal to the initial binding process but prior to activation of adenylate cyclase by the toxin. It is likely, therefore, that these toxins are processed by the Golgi apparatus before trafficking to the membrane adenylate cyclase. Images PMID:8392970

  5. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Spangler, B D

    1992-01-01

    Cholera and the related Escherichia coli-associated diarrheal disease are important problems confronting Third World nations and any area where water supplies can become contaminated. The disease is extremely debilitating and may be fatal in the absence of treatment. Symptoms are caused by the action of cholera toxin, secreted by the bacterium Vibrio cholerae, or by a closely related heat-labile enterotoxin, produced by Escherichia coli, that causes a milder, more common traveler's diarrhea. Both toxins bind receptors in intestinal epithelial cells and insert an enzymatic subunit that modifies a G protein associated with the adenylate cyclase complex. The consequent stimulated production of cyclic AMP, or other factors such as increased synthesis of prostaglandins by intoxicated cells, initiates a metabolic cascade that results in the excessive secretion of fluid and electrolytes characteristic of the disease. The toxins have a very high degree of structural and functional homology and may be evolutionarily related. Several effective new vaccine formulations have been developed and tested, and a growing family of endogenous cofactors is being discovered in eukaryotic cells. The recent elucidation of the three-dimensional structure of the heat-labile enterotoxin has provided an opportunity to examine and compare the correlations between structure and function of the two toxins. This information may improve our understanding of the disease process itself, as well as illuminate the role of the toxin in studies of signal transduction and G-protein function. Images PMID:1480112

  6. Heat-Labile Enterotoxin IIa, a Platform To Deliver Heterologous Proteins into Neurons

    PubMed Central

    Chen, Chen; Przedpelski, Amanda; Tepp, William H.; Pellett, Sabine; Johnson, Eric A.

    2015-01-01

    ABSTRACT Cholera toxin (CT) and the related heat-labile enterotoxins (LT) of Escherichia coli have been implicated as adjuvants in human therapies, but reactivity upon intranasal delivery dampened efforts to develop other clinical applications. However, each CT family member variant has unique biological properties that may warrant development as therapeutic platforms. In the current study, a nontoxic variant of the heat-labile enterotoxin IIa (LTIIa) was engineered to deliver heterologous, functional proteins into the cytosol of neurons. As proof of principle, the LTIIa variant delivered two cargos into neurons. LTIIa delivered β-lactamase efficiently into cells containing complex gangliosides, such as GD1b, as host receptors. LTIIa delivery of β-lactamase was sensitive to brefeldin A, an inhibitor that collapses the Golgi compartment into the endoplasmic reticulum, but not sensitive to treatment with botulinum neurotoxin D (BoNT/D), an inhibitor of synaptic vesicle cycling. LTIIa delivered a single-chain, anti-BoNT/A camelid antibody that inhibited SNAP25 cleavage during post-BoNT/A exposure of neurons. Delivery of functional, heterologous protein cargos into neurons demonstrates the potential of LTII variants as platforms to deliver therapies to inactivate toxins and microbial infections and to reverse the pathology of human neurodegenerative diseases. PMID:26265718

  7. GM1 erythroimmunoassay for detection and titration of Escherichia coli heat-labile enterotoxin.

    PubMed

    Germani, Y; Bégaud, E; Guesdon, J L; Moreau, J P

    1986-11-01

    A GM1 ganglioside erythroimmunoassay for the detection of heat-labile Escherichia coli enterotoxin (LT) was developed for use in poorly equipped laboratories in developing countries. This assay is based on the immunological similarity between Vibrio cholerae toxin and LT and uses cholera toxin antiserum and sheep anti-rabbit immunoglobulin covalently coupled to sheep erythrocytes as conjugate. This assay has the following advantages over other currently available techniques: the reagents it uses are stable, in particular, tanned and sensitized sheep erythrocytes; GM1 ganglioside is commercially available; erythro-adsorption can be read with the naked eye; the test can be completed in 1 day; and as little as 4 ng of V. cholerae toxin or LT per ml can be detected accurately. The GM1 ganglioside erythroimmunoassay showed good quantitative and qualitative correlation with the Vero cell assay and the conventional GM1 enzyme-linked immunosorbent assay. The GM1 ganglioside erythroimmunoassay was somewhat less sensitive than the GM1 enzyme-linked immunosorbent assay but more sensitive than the Vero cell assay. Results obtained for 12 LT-positive and 138 LT-negative E. coli strains correlated with results obtained with GM1 enzyme-linked immunosorbent and Vero cell assays. PMID:3533985

  8. Adjuvant effect of non-toxic mutants of E. coli heat-labile enterotoxin following intranasal, oral and intravaginal immunization.

    PubMed

    De Magistris, M T; Pizza, M; Douce, G; Ghiara, P; Dougan, G; Rappuoli, R

    1998-01-01

    Cholera toxin and Escherichia coli heat-labile enterotoxin (LT) are known to be very effective mucosal adjuvants, but their toxicity limits their use in humans. We genetically detoxified LT by substituting single residues in the active site of the enzymatic A subunit and obtained mutant molecules that retain mucosal adjuvant activity but are devoid of toxicity. These mutant LT molecules induce mucosal and systemic responses to antigens delivered intranasally, orally and intravaginally in mice. Furthermore, mucosal immunization with these molecules confers protection against systemic challenge with tetanus toxin (TT) and mucosal challenge with Helicobacter pylori. PMID:9554265

  9. Mutants of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines.

    PubMed

    Peppoloni, Samuele; Ruggiero, Paolo; Contorni, Mario; Morandi, Maurizio; Pizza, Mariagrazia; Rappuoli, Rino; Podda, Audino; Del Giudice, Giuseppe

    2003-04-01

    Cholera toxin and Escherichia coli heat-labile enterotoxin are powerful mucosal adjuvants but their high toxicity hampers their use in humans. Site-directed mutagenesis has allowed the generation of several cholera toxin and E. coli heat-labile enterotoxin mutants with abolished or strongly reduced toxicity that still retain strong mucosal adjuvanticity. Among them, LTK63 (Ser to Lys substitution at position 63 in the A subunit) is completely nontoxic and LTR72 (Ala to Arg at position 72) retains a very low residual enzymatic activity. Both of them have been shown to be safe and effective in enhancing the immunogenicity of intranasally coadministered vaccines, also resulting in protective responses in several animal models. Clinical grade preparations of these mutants have now been produced, tested in animals and proven to be totally safe. Indeed, they did not induce any inflammatory event in the respiratory tract nor, more importantly, in the olfactory bulbs and in the meninges. The fully nontoxic LTK63 mutant has now been successfully tested in human volunteers with a trivalent subunit influenza vaccine. PMID:12899578

  10. Inhibition of T-cell Response by Escherichia coli Heat-Labile Enterotoxin-Treated Epithelial Cells

    PubMed Central

    Lopes, Luciene M.; Maroof, Asher; Dougan, Gordon; Chain, Benjamin M.

    2000-01-01

    Escherichia coli heat-labile enterotoxin (LT) is an extensively studied adjuvant of mucosal responses. Nevertheless, its mode of action as an adjuvant remains incompletely understood. In this study, we describe a simplified in vitro model with which to look at some aspects of immunoregulation by LT. The interaction of LT with the apical surface of a monolayer of CaCo-2 epithelial cells induces the release of a soluble factor which inhibits the antigen-induced release of interleukin-2 by T cells cultured at the basolateral side of the cells. The release of this factor requires the ADP-ribosylating activity of LT since the isolated B subunit, as well as an enzymatically silent LT mutant, loses biological activity in this model. The inhibitory activity is likely to be due to prostaglandin release, since it is blocked by indomethacin. The contribution of LT-induced prostaglandin release to the complex immunoregulatory activity of LT is discussed. PMID:11083810

  11. Evaluation of heat-labile enterotoxins type IIa and type IIb in the pathogenicity of enterotoxigenic Escherichia coli for neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Type II heat-labile enterotoxins (LT-II) have been reported in Escherichia coli isolates from humans, animals, food and water samples. The roles of the antigenically distinguishable LT-IIa and LT-IIb subtypes in pathogenesis and virulence of enterotoxigenic E. coli (ETEC) have not been previously re...

  12. Comparison of two GM1-erythrocyte assays to detect heat-labile Escherichia coli enterotoxin in stool specimens.

    PubMed

    Germani, Y; Guesdon, J L; Phalente, L; Begaud, E; Moreau, J P

    1988-05-01

    Two erythrocyte immunoassay techniques to detect the presence of Escherichia coli heat-labile enterotoxin (LTh) in stool supernatants and cell-free culture supernatants were compared. In the competitive assay, GM1 ganglioside was coated onto V-shaped-well microdilution plates and enterotoxin was coupled to sheep erythrocytes. As little as 0.8 ng of LTh per ml was detected by this method, which was based on the competition between the LTh of the test sample and the sensitized erythrocytes. The second assay made use of chimera antibody prepared by coupling polyclonal anti-LTh antibody to a monoclonal antibody specific for sheep erythrocytes. In this case, LTh, which was specifically bound to a GM1 ganglioside-coated plate, was detected by successively adding the chimera antibody and sheep erythrocytes. The limit of detection of the chimera antibody erythrocyte immunoassay was 0.2 ng/ml. Stool samples were collected from 167 infants hospitalized for diarrhea in the hospital of Noumea, New Caledonia. False-negative reactions due to proteases present in the stool samples were avoided by the addition of phenylmethylsulfonyl fluoride. PMID:3290242

  13. Levels of Expression and Immunogenicity of Attenuated Salmonella enterica Serovar Typhimurium Strains Expressing Escherichia coli Mutant Heat-Labile Enterotoxin

    PubMed Central

    Covone, M. Giuseppina; Brocchi, Marcelo; Palla, Emanuela; da Silveira, W. Dias; Rappuoli, Rino; Galeotti, Cesira L.

    1998-01-01

    The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium Δcya Δcrp Δasd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated Δcya Δcrp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence. PMID:9423862

  14. Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Grant, C C; Messer, R J; Cieplak, W

    1994-01-01

    Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects. Images PMID:7927684

  15. Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Lobet, Y; Cluff, C W; Cieplak, W

    1991-01-01

    Previous studies of the S1 subunit of pertussis toxin, an NAD(+)-dependent ADP-ribosyltransferase, suggested that a small amino-terminal region of amino acid sequence similarity to the active fragments of both cholera toxin and Escherichia coli heat-labile enterotoxin represents a region containing critical active-site residues that might be involved in the binding of the substrate NAD+. Other studies of two other bacterial toxins possessing ADP-ribosyltransferase activity, diphtheria toxin and Pseudomonas exotoxin A, have revealed the presence of essential glutamic acid residues vicinal to the active site. To help determine the relevance of these observations to activities of the enterotoxins, the A-subunit gene of the E. coli heat-labile enterotoxin was subjected to site-specific mutagenesis in the region encoding the amino-terminal region of similarity to the S1 subunit of pertussis toxin delineated by residues 6 through 17 and at two glutamic acid residues, 110 and 112, that are conserved in the active domains of all of the heat-labile enterotoxin variants and in cholera toxin. Mutant proteins in which arginine 7 was either deleted or replaced with lysine exhibited undetectable levels of ADP-ribosyltransferase activity. However, limited trypsinolysis of the arginine 7 mutants yielded fragmentation kinetics that were different from that yielded by the wild-type recombinant subunit or the authentic A subunit. In contrast, mutant proteins in which glutamic acid residues at either position 110 or 112 were replaced with aspartic acid responded like the wild-type subunit upon limited trypsinolysis, while exhibiting severely depressed, but detectable, ADP-ribosyltransferase activity. The latter results may indicate that either glutamic acid 110 or glutamic acid 112 of the A subunit of heat-labile enterotoxin is analogous to those active-site glutamic acids identified in several other ADP-ribosylating toxins. Images PMID:1908825

  16. Preparation of biocompatible heat-labile enterotoxin subunit B-bovine serum albumin nanoparticles for improving tumor-targeted drug delivery via heat-labile enterotoxin subunit B mediation

    PubMed Central

    Zhao, Liang; Su, Rongjian; Cui, Wenyu; Shi, Yijie; Liu, Liwei; Su, Chang

    2014-01-01

    Heat-labile enterotoxin subunit B (LTB) is a non-catalytic protein from a pentameric subunit of Escherichia coli. Based on its function of binding specifically to ganglioside GM1 on the surface of cells, a novel nanoparticle (NP) composed of a mixture of bovine serum albumin (BSA) and LTB was designed for targeted delivery of 5-fluorouracil to tumor cells. BSA-LTB NPs were characterized by determination of their particle size, polydispersity, morphology, drug encapsulation efficiency, and drug release behavior in vitro. The internalization of fluorescein isothiocyanate-labeled BSA-LTB NPs into cells was observed using fluorescent imaging. Results showed that BSA-LTB NPs presented a narrow size distribution with an average hydrodynamic diameter of approximately 254±19 nm and a mean zeta potential of approximately −19.95±0.94 mV. In addition, approximately 80.1% of drug was encapsulated in NPs and released in the biphasic pattern. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that BSA-LTB NPs exhibited higher cytotoxic activity than non-targeted NPs (BSA NPs) in SMMC-7721 cells. Fluorescent imaging results proved that, compared with BSA NPs, BSA-LTB NPs could greatly enhance cellular uptake. Hence, the results indicate that BSA-LTB NPs could be a potential nanocarrier to improve targeted delivery of 5-fluorouracil to tumor cells via mediation of LTB. PMID:24851048

  17. Modulation of dendritic cell endocytosis and antigen processing pathways by Escherichia coli heat-labile enterotoxin and mutant derivatives.

    PubMed

    Petrovska, Liljana; Lopes, Luciene; Simmons, Cameron P; Pizza, Mariagrazia; Dougan, Gordon; Chain, Benjamin M

    2003-03-28

    Escherichia coli heat-labile enterotoxin (LT) is known to be a potent adjuvant of both the mucosal and systemic immune systems but the mechanism of action leading to adjuvant activity remains incompletely understood. This study investigates the action of LT and LT mutants with impaired enzymatic activity, on the function of dendritic cells. Wild-type LT and LTR72, which retains some ADP ribosyltransferase activity, induced a selective increase in cell surface expression of B7.1, and a selective decrease of CD40 expression on mouse bone marrow derived dendritic cells. LTK63 and LT-B had no obvious effect on the expression of these antigens on similar dendritic cells. LT-treated dendritic cells also showed a profoundly impaired ability to present protein antigen (ovalbumin) to cognate T cells, although this effect was not observed with non-toxic LT mutants. LT and LTR72-treated cells showed a slower rate of receptor-mediated endocytosis as measured by flow cytometric analysis of uptake of fluorescently labelled dextran. Furthermore, confocal microscopy showed changes in the intracellular distribution of endocytosed molecules, and of the class II containing acidic antigen processing compartments. This response of dendritic cells to toxin is likely to play an important role in determining the adjuvant activity of these molecules. PMID:12615441

  18. Inhibition of Class II Major Histocompatibility Complex Antigen Processing by Escherichia coli Heat-Labile Enterotoxin Requires an Enzymatically Active A Subunit

    PubMed Central

    Matousek, Milita P.; Nedrud, John G.; Cieplak, Witold; Harding, Clifford V.

    1998-01-01

    Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) were found to inhibit intracellular antigen processing. Processing was not inhibited by mutant LT with attenuated ADP-ribosyltransferase activity, CT B or LT B subunit, which enhanced presentation of preexisting cell surface peptide-class II major histocompatibility complex complexes. Inhibition of antigen processing correlated with A subunit ADP-ribosyltransferase activity. PMID:9632629

  19. Enhancement of humoral immunity by the type II heat-labile enterotoxin LT-IIb is dependent upon IL-6 and neutrophils.

    PubMed

    Greene, Christopher J; Hu, John C; Vance, David J; Rong, Yinghui; Mandell, Lorrie; King-Lyons, Natalie; Masso-Welch, Patricia; Mantis, Nicholas J; Connell, Terry D

    2016-08-01

    LT-IIb, a type II heat-labile enterotoxin produced by Escherichia coli, is a potent intradermal adjuvant that enhances immune responses to coadministered antigens. Although the immune mechanisms that promote this augmented immune response have not been well defined, prior intradermal immunization experiments suggested that early cellular and immunomodulatory events at the site of immunization modulated the augmentation of antigen-specific immune responses by LT-IIb. To investigate that hypothesis, mice were intradermally immunized with a recombinant ricin vaccine, a prospective toxin subunit antigen, in the presence and absence of LT-IIb. Analysis of tissue-fluid collection, coupled with histologic sections from the site of intradermal immunization, revealed that a single dose of LT-IIb induced local production of interleukin 6 and promoted a regional infiltration of neutrophils. The adjuvant effects of LT-IIb were abrogated in interleukin 6-deficient mice and when mice were depleted of neutrophils by pretreatment with anti-Ly6G. Overall, these data firmly demonstrated that LT-IIb, when used as an intradermal adjuvant, recruits neutrophils and is a potent rapid inducer of interleukin 6. PMID:27059843

  20. Intranasal Immunization with SAG1 and Nontoxic Mutant Heat-Labile Enterotoxins Protects Mice against Toxoplasma gondii

    PubMed Central

    Bonenfant, C.; Dimier-Poisson, I.; Velge-Roussel, F.; Buzoni-Gatel, D.; Del Giudice, G.; Rappuoli, R.; Bout, D.

    2001-01-01

    Effective protection against intestinal pathogens requires both mucosal and systemic immune responses. Intranasal administration of antigens induces these responses but generally fails to trigger a strong protective immunity. Mucosal adjuvants can significantly enhance the immunogenicities of intranasally administered antigens. Cholera toxin (CT) and heat-labile enterotoxin (LT) are strong mucosal adjuvants with a variety of antigens. Moreover, the toxicities of CT and LT do not permit their use in humans. Two nontoxic mutant LTs, LTR72 and LTK63, were tested with Toxoplasma gondii SAG1 protein in intranasal vaccination of CBA/J mice. Vaccination with SAG1 plus LTR72 or LTK63 induced strong systemic (immunoglobulin G [IgG]) and mucosal (IgA) humoral responses. Splenocytes and mesenteric lymph node cells from mice immunized with LTR72 plus SAG1, but not those from mice immunized with LTK63 plus SAG1, responded to restimulation with a T. gondii lysate antigen in vitro. Gamma interferon and interleukin 2 (IL-2) production by splenocytes and IL-2 production by mesenteric lymph node cells were observed in vitro after antigen restimulation, underlying a Th1-like response. High-level protection as assessed by the decreased load of cerebral cysts after a challenge with the 76K strain of T. gondii was obtained in the group immunized with LTR72 plus SAG1 and LTK63 plus SAG1. They were as well protected as the mice immunized with the antigen plus native toxins. This is the first report showing protection against a parasite by using combinations of nontoxic mutant LTs and SAG1 antigen. These nontoxic mutant LTs are now attractive candidates for the development of mucosally delivered vaccines. PMID:11179334

  1. Comparative Adjuvant Effects of Type II Heat-Labile Enterotoxins in Combination with Two Different Candidate Ricin Toxin Vaccine Antigens

    PubMed Central

    Vance, David J.; Greene, Christopher J.; Rong, Yinghui; Mandell, Lorrie M.; Connell, Terry D.

    2015-01-01

    Type II heat-labile enterotoxins (HLTs) constitute a promising set of adjuvants that have been shown to enhance humoral and cellular immune responses when coadministered with an array of different proteins, including several pathogen-associated antigens. However, the adjuvant activities of the four best-studied HLTs, LT-IIa, LT-IIb, LT-IIbT13I, and LT-IIc, have never been compared side by side. We therefore conducted immunization studies in which LT-IIa, LT-IIb, LT-IIbT13I, and LT-IIc were coadministered by the intradermal route to mice with two clinically relevant protein subunit vaccine antigens derived from the enzymatic A subunit (RTA) of ricin toxin, RiVax and RVEc. The HLTs were tested with low and high doses of antigen and were assessed for their abilities to stimulate antigen-specific serum IgG titers, ricin toxin-neutralizing activity (TNA), and protective immunity. We found that all four HLTs tested were effective adjuvants when coadministered with RiVax or RVEc. LT-IIa was of particular interest because as little as 0.03 μg when coadministered with RiVax or RVEc proved effective at augmenting ricin toxin-specific serum antibody titers with nominal evidence of local inflammation. Collectively, these results justify the need for further studies into the mechanism(s) underlying LT-IIa adjuvant activity, with the long-term goal of evaluating LT-IIa's activity in humans. PMID:26491037

  2. Crystal structure of a non-toxic mutant of heat-labile enterotoxin, which is a potent mucosal adjuvant.

    PubMed

    van den Akker, F; Pizza, M; Rappuoli, R; Hol, W G

    1997-12-01

    Two closely related bacterial toxins, heat-labile enterotoxin (LT-I) and cholera toxin (CT), not only invoke a toxic activity that affects many victims worldwide but also contain a beneficial mucosal adjuvant activity that significantly enhances the potency of vaccines in general. For the purpose of vaccine design it is most interesting that the undesirable toxic activity of these toxins can be eliminated by the single-site mutation Ser63Lys in the A subunit while the mucosal adjuvant activity is still present. The crystal structure of the Ser63Lys mutant of LT-I is determined at 2.0 A resolution. Its structure appears to be essentially the same as the wild-type LT-I structure. The substitution Ser63Lys was designed, based on the wild-type LT-I crystal structure, to decrease toxicity by interfering with NAD binding and/or catalysis. In the mutant crystal structure, the newly introduced lysine side chain is indeed positioned such that it could potentially obstruct the productive binding mode of the substrate NAD while at the same time its positive charge could possibly interfere with the critical function of nearby charged groups in the active site of LT-I. The fact that the Ser63Lys mutant of LT-I does not disrupt the wild-type LT-I structure makes the non-toxic mutant potentially suitable, from a structural point of view, to be used as a vaccine to prevent enterotoxigenic E. coli infections. The structural similarity of mutant and wild-type toxin might also be the reason why the inactive Ser63Lys variant retains its adjuvant activity. PMID:9416617

  3. Heat-labile enterotoxin-induced activation of NF-κB and MAPK pathways in intestinal epithelial cells impacts enterotoxigenic Escherichia coli (ETEC) adherence.

    PubMed

    Wang, Xiaogang; Gao, Xiaofei; Hardwidge, Philip R

    2012-08-01

    Enterotoxigenic Escherichia coli (ETEC) causes human morbidity and mortality in developing nations and is an emerging threat to food safety in developed nations. The ETEC heat-labile enterotoxin (LT) not only causes diarrheal disease by deregulating host adenylate cyclase, but also enhances ETEC adherence to intestinal epithelial cells. The mechanism governing this LT pro-adherence phenotype is unclear. Here we investigated intestinal epithelial cell signal transduction pathways activated by ETEC and quantified the relative importance of these host pathways to LT-induced ETEC adherence. We show that ETEC activates both NF-κB and mitogen-activated protein kinase signalling pathways through mechanisms that are primarily dependent upon LT. LT-induced NF-κB activation depends upon the cAMP-dependent activation of the Ras-like GTPase Rap1 but is independent of protein kinase A (PKA). By using inhibitors of these pathways, we demonstrate that inhibiting the p38 mitogen-activated protein kinase prevents LT from increasing ETEC adherence. By contrast, the LT pro-adherence phenotype appears unrelated to both LT-induced Rap1 activity and to subsequent NF-κB activation. We speculate that LT may alter host signal transduction to induce the presentation of ligands for ETEC adhesins in such a way that promotes ETEC adherence. Our findings provide insight into previously unexplored functions of LT and their relative importance to ETEC virulence. PMID:22452361

  4. Relationship between Heat-Labile Enterotoxin Secretion Capacity and Virulence in Wild Type Porcine-Origin Enterotoxigenic Escherichia coli Strains

    PubMed Central

    Wijemanne, Prageeth; Xing, Jun; Berberov, Emil M.; Marx, David B.; Francis, David H.; Moxley, Rodney A.

    2015-01-01

    Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and

  5. Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

    PubMed

    Wijemanne, Prageeth; Xing, Jun; Berberov, Emil M; Marx, David B; Francis, David H; Moxley, Rodney A

    2015-01-01

    Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and

  6. Antibodies to heat-labile Escherichia coli enterotoxins in human milk and sera. A study of Ethiopian and Swedish mothers and their children.

    PubMed

    Aust-Kettis, A; Gebre-Medhin, M; Habte, D; Khosla, N; Wadström, T

    1981-09-01

    Maternal serum and cord blood from 50 Ethiopian, 10 Costa Rican and 20 Swedish newly delivered mothers and their babies was examined for the presence of antibodies against heat labile (LT) enterotoxin from a human strain of E. coli. 96% of the Ethiopian, 80% of the Costa Rican and 30% of the Swedish mothers and infants had detectable antibody levels. The titres were significantly higher in the Ethiopian material. Furthermore, antibody titres to E. coli enterotoxin were determined in breast milk collected from Ethiopian mothers at 48 h and at 1 month after delivery. One third of these mothers had detectable levels of antibodies in samples from early lactation. Experiments performed with LT enterotoxin from another human and with LT from a porcine E. coli strain confirmed the results. Neutralization tests with cholera enterotoxin as antigen were all negative in sera and milk samples from all these groups. The material has been collected in three different geographical areas which are nonendemic for cholera. PMID:7032015

  7. Cholera toxin, LT-I, LT-IIa, and LT-IIb: the critical role of ganglioside-binding in immunomodulation by Type I and Type II heat-labile enterotoxins

    PubMed Central

    Connell, Terry D.

    2010-01-01

    The heat-labile enterotoxins (HLT) expressed by Vibrio cholerae (cholera toxin) and Escherichia coli (LT-I, LT-IIa, and LT-IIb) are potent systemic and mucosal adjuvants. Co-administration of the enterotoxins with a foreign antigen (Ag) produces an augmented immune response to that antigen. Although each enterotoxin has potent adjuvant properties, the means by which the enterotoxins induce various immune responses are distinctive for each adjuvant. Various mutants have been engineered to dissect the functions of the enterotoxins required for their adjuvanticity. The capacity to strongly bind to one or more specific ganglioside receptors appears to drive the distinctive immunomodulatory properties associated with each enterotoxin. Mutant enterotoxins with ablated or altered ganglioside binding affinities have been employed to investigate the role of gangliosides in enterotoxin-dependent immunomodulation. PMID:17931161

  8. Identification of a Gene within a Pathogenicity Island of Enterotoxigenic Escherichia coli H10407 Required for Maximal Secretion of the Heat-Labile Enterotoxin

    PubMed Central

    Fleckenstein, James M.; Lindler, Luther E.; Elsinghorst, Eric A.; Dale, James B.

    2000-01-01

    Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile (LT) and heat-stable enterotoxins and the colonization factor antigens. ETEC causes illnesses that range from mild diarrhea to severe cholera-like disease. These differences in disease severity are not readily accounted for by our current understanding of ETEC pathogenesis. Here we demonstrate that Tia, a putative adhesin of ETEC H10407, is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described E. coli pathogenicity islands. Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia. The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses. An in-frame deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation in a rabbit ileal loop model of infection. Although previous studies have suggested that E. coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island. PMID:10768971

  9. Escherichia coli heat-labile enterotoxin B subunit is a more potent mucosal adjuvant than its vlosely related homologue, the B subunit of cholera toxin.

    PubMed

    Millar, D G; Hirst, T R; Snider, D P

    2001-05-01

    Although cholera toxin (Ctx) and Escherichia coli heat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits. Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB. EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes. These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants. PMID:11292779

  10. Type II heat-labile enterotoxins from 50 diverse Escherichia coli isolates belong almost exclusively to the LT-IIc family and may be prophage encoded.

    PubMed

    Jobling, Michael G; Holmes, Randall K

    2012-01-01

    Some enterotoxigenic Escherichia coli (ETEC) produce a type II heat-labile enterotoxin (LT-II) that activates adenylate cyclase in susceptible cells but is not neutralized by antisera against cholera toxin or type I heat-labile enterotoxin (LT-I). LT-I variants encoded by plasmids in ETEC from humans and pigs have amino acid sequences that are ≥ 95% identical. In contrast, LT-II toxins are chromosomally encoded and are much more diverse. Early studies characterized LT-IIa and LT-IIb variants, but a novel LT-IIc was reported recently. Here we characterized the LT-II encoding loci from 48 additional ETEC isolates. Two encoded LT-IIa, none encoded LT-IIb, and 46 encoded highly related variants of LT-IIc. Phylogenetic analysis indicated that the predicted LT-IIc toxins encoded by these loci could be assigned to 6 subgroups. The loci corresponding to individual toxins within each subgroup had DNA sequences that were more than 99% identical. The LT-IIc subgroups appear to have arisen by multiple recombinational events between progenitor loci encoding LT-IIc1- and LT-IIc3-like variants. All loci from representative isolates encoding the LT-IIa, LT-IIb, and each subgroup of LT-IIc enterotoxins are preceded by highly-related genes that are between 80 and 93% identical to predicted phage lysozyme genes. DNA sequences immediately following the B genes differ considerably between toxin subgroups, but all are most closely related to genomic sequences found in predicted prophages. Together these data suggest that the LT-II loci are inserted into lambdoid type prophages that may or may not be infectious. These findings raise the possibility that production of LT-II enterotoxins by ETEC may be determined by phage conversion and may be activated by induction of prophage, in a manner similar to control of production of Shiga-like toxins by converting phages in isolates of enterohemmorhagic E. coli. PMID:22242186

  11. Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng.

    PubMed

    Kang, Tae-Jin; Lee, Won-Seok; Choi, Eun-Gyung; Kim, Jae-Whune; Kim, Bang-Geul; Yang, Moon-Sik

    2006-01-24

    The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period. PMID:16174540

  12. Evaluation of heat-labile enterotoxins type IIa and type IIb in the pathogenicity of enterotoxigenic Escherichia coli for neonatal pigs.

    PubMed

    Casey, Thomas A; Connell, Terry D; Holmes, Randall K; Whipp, Shannon C

    2012-09-14

    Type II heat-labile enterotoxins (LT-II) have been reported in Escherichia coli isolates from humans, animals, food and water samples. The goal here was to determine the specific roles of the antigenically distinguishable LT-IIa and LT-IIb subtypes in pathogenesis and virulence of enterotoxigenic E. coli (ETEC) which has not been previously reported. The prevalence of genes encoding for LT-II was determined by colony blot hybridization in a collection of 1648 E. coli isolates from calves and pigs with diarrhea or other diseases and from healthy animals. Only five isolates hybridized with the LT-II probe and none of these isolates contained genes for other enterotoxins or adhesins associated with porcine or bovine ETEC. Ligated intestinal loops in calves, pigs, and rabbits were used to determine the potential of purified LT-IIa and LT-IIb to cause intestinal secretion. LT-IIa and LT-IIb caused significant secretion in the intestinal loops in calves but not in the intestinal loops of rabbits or pigs. In contrast, neonatal pigs inoculated with isogenic adherent E. coli containing the cloned genes for LT-I, LT-IIa or LT-IIb developed severe watery diarrhea with weight loss that was significantly greater than pigs inoculated with the adherent, non-toxigenic parental or vector only control strains. The results demonstrate that the incidence of LT-II appeared to be very low in porcine and bovine E. coli. However, a potential role for these enterotoxins in E. coli-mediated diarrhea in animals was confirmed because purified LT-IIa and LT-IIb caused fluid secretion in bovine intestinal loops and adherent isogenic strains containing cloned genes encoding for LT-IIa or LT-IIb caused severe diarrhea in neonatal pigs. PMID:22480773

  13. Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the immunogenicity of rabies virus-like particles in mice and dogs.

    PubMed

    Qi, Yinglin; Kang, Hongtao; Zheng, Xuexing; Wang, Hualei; Gao, Yuwei; Yang, Songtao; Xia, Xianzhu

    2015-01-01

    Rabies remains an important worldwide public health threat, so safe, effective, and affordable vaccines are still being sought. Virus-like particle-based vaccines targeting various viral pathogens have been successfully produced, licensed, and commercialized. Here, we designed and constructed two chimeric rabies virus-like particles (cRVLPs) containing rabies virus (RABV) glycoprotein (G), matrix (M) protein, and membrane-anchored flagellin (EVLP-F) or Escherichia coli heat-labile enterotoxin B subunit (EVLP-L) as molecular adjuvants to enhance the immune response against rabies. The immunogenicity and potential of cRVLPs as novel rabies vaccine were evaluated by intramuscular vaccination in mouse and dog models. Mouse studies demonstrated that both EVLP-F and EVLP-L induced faster and larger virus-neutralizing antibodies (VNAs) responses and elicited greater numbers of CD4(+) and CD8(+) T cells secreting IFN-γ or IL-4 compared with a standard rabies VLP (sRVLP) containing only G and M. Moreover, cRVLPs recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. EVLP-F induced a strong, specific IgG2a response but not an IgG1 response, suggesting the activation of Th1 class immunity; in contrast, Th2 class immunity was observed with EVLP-L. The significantly enhanced humoral and cellular immune responses induced by cRVLPs provided complete protection against lethal challenge with RABV. Most importantly, dogs vaccinated with EVLP-F or EVLP-L exhibited increased VNA titers in sera and enhanced IFN-γ and IL-4 secretion from peripheral blood mononuclear cells. Taken together, these results illustrate that when incorporated into sRVLP, membrane-anchored flagellin, and heat-labile enterotoxin B subunit possess strong adjuvant activity. EVLP-F and EVLP-L induce significantly enhanced RABV-specific humoral and cellular immune responses in both mouse and dog. Therefore, these cRVLPs may be developed as safe and more efficacious rabies vaccine

  14. Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the immunogenicity of rabies virus-like particles in mice and dogs

    PubMed Central

    Qi, Yinglin; Kang, Hongtao; Zheng, Xuexing; Wang, Hualei; Gao, Yuwei; Yang, Songtao; Xia, Xianzhu

    2015-01-01

    Rabies remains an important worldwide public health threat, so safe, effective, and affordable vaccines are still being sought. Virus-like particle-based vaccines targeting various viral pathogens have been successfully produced, licensed, and commercialized. Here, we designed and constructed two chimeric rabies virus-like particles (cRVLPs) containing rabies virus (RABV) glycoprotein (G), matrix (M) protein, and membrane-anchored flagellin (EVLP-F) or Escherichia coli heat-labile enterotoxin B subunit (EVLP-L) as molecular adjuvants to enhance the immune response against rabies. The immunogenicity and potential of cRVLPs as novel rabies vaccine were evaluated by intramuscular vaccination in mouse and dog models. Mouse studies demonstrated that both EVLP-F and EVLP-L induced faster and larger virus-neutralizing antibodies (VNAs) responses and elicited greater numbers of CD4+ and CD8+ T cells secreting IFN-γ or IL-4 compared with a standard rabies VLP (sRVLP) containing only G and M. Moreover, cRVLPs recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. EVLP-F induced a strong, specific IgG2a response but not an IgG1 response, suggesting the activation of Th1 class immunity; in contrast, Th2 class immunity was observed with EVLP-L. The significantly enhanced humoral and cellular immune responses induced by cRVLPs provided complete protection against lethal challenge with RABV. Most importantly, dogs vaccinated with EVLP-F or EVLP-L exhibited increased VNA titers in sera and enhanced IFN-γ and IL-4 secretion from peripheral blood mononuclear cells. Taken together, these results illustrate that when incorporated into sRVLP, membrane-anchored flagellin, and heat-labile enterotoxin B subunit possess strong adjuvant activity. EVLP-F and EVLP-L induce significantly enhanced RABV-specific humoral and cellular immune responses in both mouse and dog. Therefore, these cRVLPs may be developed as safe and more efficacious rabies vaccine

  15. Mutations in the A subunit affect yield, stability, and protease sensitivity of nontoxic derivatives of heat-labile enterotoxin.

    PubMed

    Magagnoli, C; Manetti, R; Fontana, M R; Giannelli, V; Giuliani, M M; Rappuoli, R; Pizza, M

    1996-12-01

    Heat-labile toxin (LT) is a protein related to cholera toxin, produced by enterotoxigenic Escherichia coli strains, that is organized as an AB5 complex. A number of nontoxic derivatives of LT, useful for new or improved vaccines against diarrheal diseases or as mucosal adjuvants, have been constructed by site-directed mutagenesis. Here we have studied the biochemical properties of the nontoxic mutants LT-K7 (Arg-7-->Lys), LT-D53 (Val-53-->Asp), LT-K63 (Ser-63-->Lys), LT-K97 (Val-97-->Lys), LT-K104 (Tyr-104-->Lys), LT-K114 (Ser-114-->Lys), and LT-K7/K97 (Arg-7-->Lys and Val-97-->Lys). We have found that mutations in the A subunit may have profound effects on the ability to form the AB5 structure and on the stability and trypsin sensitivity of the purified proteins. Unstable mutants, during long-term storage at 4 degrees C, showed a decrease in the amount of the assembled protein in solution and a parallel appearance of soluble monomeric B subunit. This finding suggests that the stability of the B pentamer is influenced by the A subunit which is associated with it. Among the seven nontoxic mutants tested, LT-K63 was found to be efficient in AB5 production, extremely stable during storage, resistant to proteolytic attack, and very immunogenic. In conclusion, LT-K63 is a good candidate for the development of antidiarrheal vaccines and mucosal adjuvants. PMID:8945604

  16. Simultaneous Exposure to Escherichia coli Heat-Labile and Heat-Stable Enterotoxins Increases Fluid Secretion and Alters Cyclic Nucleotide and Cytokine Production by Intestinal Epithelial Cells

    PubMed Central

    Read, Lisa T.; Hahn, Rachel W.; Thompson, Carli C.; Bauer, David L.; Norton, Elizabeth B.

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrheal disease and death, especially in children in developing countries. ETEC causes disease by colonizing the small intestine and producing heat-labile toxin (LT), heat-stable toxin (ST), or both LT and ST (LT+ST). The majority of ETEC strains produce both ST and LT. Despite the prevalence of LT+ST-producing organisms, few studies have examined the physiologic or immunologic consequences of simultaneous exposure to these two potent enterotoxins. In the current report, we demonstrate that when LT and ST are both present, they increase water movement into the intestinal lumen over and above the levels observed with either toxin alone. As expected, cultured intestinal epithelial cells increased their expression of intracellular cyclic GMP (cGMP) when treated with ST and their expression of intracellular cyclic AMP (cAMP) when treated with LT. When both toxins were present, cGMP levels but not cAMP levels were synergistically elevated compared with the levels of expression caused by the corresponding single-toxin treatment. Our data also demonstrate that the levels of inflammatory cytokines produced by intestinal epithelial cells in response to LT are significantly reduced in animals exposed to both enterotoxins. These findings suggest that there may be complex differences between the epithelial cell intoxication and, potentially, secretory outcomes induced by ETEC strains expressing LT+ST compared with strains that express LT or ST only. Our results also reveal a novel mechanism wherein ST production may reduce the hosts' ability to mount an effective innate or adaptive immune response to infecting organisms. PMID:25287923

  17. Oral immunisation of mice with a recombinant rabies virus vaccine incorporating the heat-labile enterotoxin B subunit of Escherichia coli in an attenuated Salmonella strain.

    PubMed

    Wang, Xuelin; Liu, Juan; Wu, Xiuping; Yu, Lu; Chen, Haiying; Guo, Heng; Zhang, Maolin; Li, Huiping; Liu, Xue; Sun, Shumin; Zhao, Lijing; Zhang, Xinyue; Gao, Lifang; Liu, Mingyuan

    2012-10-01

    To investigate effective new rabies vaccines, a fusion protein consisting of the rabies virus (RV) glycoprotein and the heat-labile enterotoxin B subunit of Escherichia coli (LTB) was successfully constructed and delivered in a live attenuated Salmonella strain LH430. Mice were immunised with LH430 carrying pVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB. The antibody titres of mice immunised with oral LH430 carrying pVAX1-G-LTB or pVAX1-ori-G-LTB were significantly higher than those of pVAX1-G-immunised mice. The results of the challenge with the rabies virus standard strain (CVS-11) showed that the LH430 strain carrying the G-LTB gene induced immunity and elevated IL-2 levels in immunised mice ((∗∗)P<0.01), whereas LH430 carrying pVAX1-G did not contribute to protection. These results show that LH430 carrying recombinant G-LTB could provide overall immunity against challenge with CVS-11 and should be considered to be a potential rabies vaccine. PMID:22019192

  18. Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB.

    PubMed

    Marchioro, Silvana Beutinger; Fisch, Andressa; Gomes, Charles K; Jorge, Sérgio; Galli, Vanessa; Haesebrouck, Freddy; Maes, Dominiek; Dellagostin, Odir; Conceição, Fabricio R

    2014-09-17

    A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (P<0.05). The adjuvant rLTB enhanced the immune response in both fused and co-administered antigens; however, better results were obtained with the chimeric protein. This multi-antigen is a promising vaccine candidate that may help control M. hyopneumoniae infection. PMID:25091529

  19. Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin.

    PubMed

    Liu, Di; Guo, Hua; Zheng, Wenyun; Zhang, Na; Wang, Tianwen; Wang, Ping; Ma, Xingyuan

    2016-06-01

    Heat-labile enterotoxin (LT) is a protein toxin produced by enterotoxigenic Escherichia coli (ETEC). As a bacterial toxin, LT holotoxin can enter intestinal epithelial cells and cause diarrhea. In addition, LT is also a powerful mucosal adjuvant capable of enhancing the strong immune responses to co-administered antigens. However, the LT immunological mechanism is still not clear in some aspects, especially with the respect to how the LTA subunit functions alone. Here, we discovered that the A2 domain of LTA could carry a fluorescent protein into cells, whose function is similar to a cell-penetrating peptide. The transmembrane-transporting ability of the A2 domain is non-specific in its cell-penetrating function, which was shown through testing with different cell types. Moreover, the LTA2 fusion protein penetrated a fluorescently labeled cell membrane that identified LTA2 internalization through membrane transport pathways, and showed it finally localized in the endoplasmic reticulum. Furthermore, low-temperature stress and pharmacological agent treatments showed that the LTA2 internalization route is a temperature-dependent process involving the clathrin-mediated endocytosis and the macropinocytosis pathways. These results could explain the internalization of the LTA subunit alone without the LTB pentamer, contributing to a better understanding of LTA working as a mucosal adjuvant; they also suggest that the A2 domain could be used as a novel transport vehicle for research and treatment of disease. PMID:26960316

  20. A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

    PubMed

    Pizza, M; Fontana, M R; Giuliani, M M; Domenighini, M; Magagnoli, C; Giannelli, V; Nucci, D; Hol, W; Manetti, R; Rappuoli, R

    1994-12-01

    Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli. PMID:7964489

  1. Heat-labile enterotoxin of Escherichia coli and its site-directed mutant LTK63 enhance the proliferative and cytotoxic T-cell responses to intranasally co-immunized synthetic peptides.

    PubMed

    Partidos, C D; Salani, B F; Pizza, M; Rappuoli, R

    1999-04-15

    The adjuvanticity of heat-labile enterotoxin (LT) of Escherichia coli and its non-toxic mutant LTK63 was assessed and compared for intranasal immunization of synthetic peptides. Mice immunized intranasally with LT, or its mutant LTK63, generated strong systemic proliferative and cytotoxic T-cell responses to co-administered synthetic peptides. The wild LT toxin promoted higher peptide-specific proliferative and cytotoxic T-cell responses than the LTK63 mutant. Moreover, the wild-type LT toxin was shown to promote peptide-specific memory CTL responses which were detectable 1 year after intranasal priming. Both LT and LTK63 molecules were shown to be immunogenic, with serum antibody subclasses being predominantly IgG1 and to a lesser extent IgG2a. These findings demonstrate that cellular immune responses to small synthetic peptide antigens administered by the intranasal route can be potentiated with the use of mucosal adjuvants. Moreover, the ability of LT and LTK63 to promote both CD4+ and CD8+ T-cell responses will have relevance to the design and production of future mucosal vaccines. PMID:10369128

  2. Protective Mucosal Immunity to Ocular Herpes Simplex Virus Type 1 Infection in Mice by Using Escherichia coli Heat-Labile Enterotoxin B Subunit as an Adjuvant

    PubMed Central

    Richards, C. M.; Aman, A. T.; Hirst, T. R.; Hill, T. J.; Williams, N. A.

    2001-01-01

    The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 μg of rEtxB or above with 10 μg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 μg) with a trace (0.5 μg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 μg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed. PMID:11160664

  3. The catalytic A1 domains of cholera toxin and heat-labile enterotoxin are potent DNA adjuvants that evoke mixed Th1/Th17 cellular immune responses

    PubMed Central

    Bagley, Kenneth; Xu, Rong; Ota-Setlik, Ayuko; Egan, Michael; Schwartz, Jennifer; Fouts, Timothy

    2015-01-01

    DNA encoded adjuvants are well known for increasing the magnitude of cellular and/or humoral immune responses directed against vaccine antigens. DNA adjuvants can also tune immune responses directed against vaccine antigens to better protect against infection of the target organism. Two potent DNA adjuvants that have unique abilities to tune immune responses are the catalytic A1 domains of Cholera Toxin (CTA1) and Heat-Labile Enterotoxin (LTA1). Here, we have characterized the adjuvant activities of CTA1 and LTA1 using HIV and SIV genes as model antigens. Both of these adjuvants enhanced the magnitude of antigen-specific cellular immune responses on par with those induced by the well-characterized cytokine adjuvants IL-12 and GM-CSF. CTA1 and LTA1 preferentially enhanced cellular responses to the intracellular antigen SIVmac239-gag over those for the secreted HIVBaL-gp120 antigen. IL-12, GM-CSF and electroporation did the opposite suggesting differences in the mechanisms of actions of these diverse adjuvants. Combinations of CTA1 or LTA1 with IL-12 or GM-CSF generated additive and better balanced cellular responses to both of these antigens. Consistent with observations made with the holotoxin and the CTA1-DD adjuvant, CTA1 and LTA1 evoked mixed Th1/Th17 cellular immune responses. Together, these results show that CTA1 and LTA1 are potent DNA vaccine adjuvants that favor the intracellular antigen gag over the secreted antigen gp120 and evoke mixed Th1/Th17 responses against both of these antigens. The results also indicate that achieving a balanced immune response to multiple intracellular and extracellular antigens delivered via DNA vaccination may require combining adjuvants that have different and complementary mechanisms of action. PMID:26042527

  4. Comparative study on characterization of recombinant B subunit of E. coli heat-labile enterotoxin (rLTB) prepared from E. coli and P. patoris.

    PubMed

    Ma, Xingyuan; Yao, Bi; Zheng, Wenyun; Li, Linfeng

    2010-03-01

    Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-l fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6x His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at 30 degrees C, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at 37 degrees C. The expression level increased dramatically to 250- 300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6x His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GM1 gangliosides. The MW of LTB-6x His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system. PMID:20372026

  5. Protection of piglets against enteric colibacillosis by intranasal immunization with K88ac (F4ac) fimbriae and heat labile enterotoxin of Escherichia coli.

    PubMed

    Lin, Jun; Mateo, Kristina S; Zhao, Mojun; Erickson, Alan K; Garcia, Nuria; He, Dong; Moxley, Rodney A; Francis, David H

    2013-03-23

    Enterotoxigenic Escherichia coli (ETEC) is an important diarrheal agent of young domestic animals. Currently, there are no commercially available non-living vaccines to protect weaned pigs from the disease and no major veterinary biologics company markets a postweaning ETEC vaccine of any kind. While efforts have been made to develop a non-living postweaning ETEC vaccine for pigs, studies have been limited to the assessment of immune responses to experimental immunogens. In the present study, we describe a reproducible gnotobiotic piglet model of post-weaning ETEC diarrhea and efficacy tests in that model of subunit vaccines consisting of K88 (F4) fimbriae and/or heat labile enterotoxin (LT) delivered by the intranasal route. We also report antibody responses to the vaccine antigens. Piglets vaccinated with both antigens mounted a substantial immune response with serum and cecal antibody titers to K88 antigen significantly greater than those of controls. Serum anti-LT antibody titers were also significantly greater than those of controls. Piglets vaccinated with both antigens remained healthy following challenge with ETEC. At least some pigs vaccinated with either antigen alone, and most of the control piglets developed dehydrating diarrhea and suffered significant weight loss. The results of this study suggest that an intranasal vaccine consisting of both antigens is highly protective against a vigorous experimental challenge of pigs with K88+ ETEC, while that against either antigen alone is not. The current study provides a system whereby various ETEC antigens and/or combinations of antigens can be tested in exploring strategies for the development of vaccines for ETEC. PMID:23089483

  6. The catalytic A1 domains of cholera toxin and heat-labile enterotoxin are potent DNA adjuvants that evoke mixed Th1/Th17 cellular immune responses.

    PubMed

    Bagley, Kenneth; Xu, Rong; Ota-Setlik, Ayuko; Egan, Michael; Schwartz, Jennifer; Fouts, Timothy

    2015-01-01

    DNA encoded adjuvants are well known for increasing the magnitude of cellular and/or humoral immune responses directed against vaccine antigens. DNA adjuvants can also tune immune responses directed against vaccine antigens to better protect against infection of the target organism. Two potent DNA adjuvants that have unique abilities to tune immune responses are the catalytic A1 domains of Cholera Toxin (CTA1) and Heat-Labile Enterotoxin (LTA1). Here, we have characterized the adjuvant activities of CTA1 and LTA1 using HIV and SIV genes as model antigens. Both of these adjuvants enhanced the magnitude of antigen-specific cellular immune responses on par with those induced by the well-characterized cytokine adjuvants IL-12 and GM-CSF. CTA1 and LTA1 preferentially enhanced cellular responses to the intracellular antigen SIVmac239-gag over those for the secreted HIVBaL-gp120 antigen. IL-12, GM-CSF and electroporation did the opposite suggesting differences in the mechanisms of actions of these diverse adjuvants. Combinations of CTA1 or LTA1 with IL-12 or GM-CSF generated additive and better balanced cellular responses to both of these antigens. Consistent with observations made with the holotoxin and the CTA1-DD adjuvant, CTA1 and LTA1 evoked mixed Th1/Th17 cellular immune responses. Together, these results show that CTA1 and LTA1 are potent DNA vaccine adjuvants that favor the intracellular antigen gag over the secreted antigen gp120 and evoke mixed Th1/Th17 responses against both of these antigens. The results also indicate that achieving a balanced immune response to multiple intracellular and extracellular antigens delivered via DNA vaccination may require combining adjuvants that have different and complementary mechanisms of action. PMID:26042527

  7. Protease susceptibility and toxicity of heat-labile enterotoxins with a mutation in the active site or in the protease-sensitive loop.

    PubMed Central

    Giannelli, V; Fontana, M R; Giuliani, M M; Guangcai, D; Rappuoli, R; Pizza, M

    1997-01-01

    To generate nontoxic derivatives of Escherichia coli heat-labile enterotoxin (LT), site-directed mutagenesis has been used to change either the amino acid residues located in the catalytic site (M. Pizza, M. Domenighini, W. Hol, V. Giannelli, M. R. Fontana, M. M. Giuliani, C. Magagnoli, S. Peppoloni, R. Manetti, and R. Rappuoli, Mol. Microbiol. 14:51-60, 1994) or those located in the proteolytically sensitive loop that joins the A1 and A2 moieties of the A subunit (C. C. R. Grant, R. J. Messer, and W. J. Cieplack, Infect. Immun. 62:4270-4278, 1994; B. L. Dickinson and J. D. Clements, Infect. Immun. 63:1617-1623, 1995). In this work, we compared the in vitro and in vivo toxic properties and the resistance to protease digestion of the prototype molecules obtained by both approaches (LT-K63 and LT-R192G, respectively). As expected, LT-K63 was normally processed by proteases, while LT-R192G showed increased resistance to trypsin in vitro and was digested by trypsin only under denaturing conditions (3.5 M urea) or by intestinal proteases. No toxicity was detected with the LT-K63 mutant, even when 40 micrograms and 1 mg were used in the in vitro and in vivo assays, respectively. In marked contrast, LT-R192G showed only a modest (10-fold) reduction in toxicity in Y1 cells with a delay in the appearance of the toxic activity and had toxicity comparable to that of wild-type LT in the rabbit ileal loop assay. We conclude that mutagenesis of the active site generates molecules that are fully devoid of toxicity, while mutagenesis of the A1-A2 loop generates molecules that are resistant to trypsin in vitro but still susceptible to proteolytic activation by proteases other than trypsin, and therefore they may still be toxic in tissue culture and in vivo. PMID:8975934

  8. Salmonella enterica serovar enteritidis ghosts carrying the Escherichia coli heat-labile enterotoxin B subunit are capable of inducing enhanced protective immune responses.

    PubMed

    Jawale, Chetan V; Lee, John Hwa

    2014-06-01

    The Escherichia coli heat-labile enterotoxin B subunit (LTB) is a potent vaccine adjuvant. Salmonella enterica serovar Enteritidis ghosts carrying LTB (S. Enteritidis-LTB ghosts) were genetically constructed using a novel plasmid, pJHL187-LTB, designed for the coexpression of the LTB and E lysis proteins. S. Enteritidis-LTB ghosts were characterized using scanning electron microscopy to visualize their transmembrane tunnel structures. The expression of LTB in S. Enteritidis-LTB ghost preparations was confirmed by immunoblot and enzyme-linked immunosorbent assays. The parenteral adjuvant activity of LTB was demonstrated by immunizing chickens with either S. Enteritidis-LTB ghosts or S. Enteritidis ghosts. Chickens were intramuscularly primed at 5 weeks of age and subsequently boosted at 8 weeks of age. In total, 60 chickens were equally divided into three groups (n = 20 for each): group A, nonvaccinated control; group B, immunized with S. Enteritidis-LTB ghosts; and group C, immunized with S. Enteritidis ghosts. Compared with the nonimmunized chickens (group A), the immunized chickens (groups B and C) exhibited increased titers of plasma IgG and intestinal secretory IgA antibodies. The CD3(+) CD4(+) subpopulation of T cells was also significantly increased in both immunized groups. Among the immunized chickens, those in group B exhibited significantly increased titers of specific plasma IgG and intestinal secretory IgA (sIgA) antibodies compared with those in group C, indicating the immunomodulatory effects of the LTB adjuvant. Furthermore, both immunized groups exhibited decreased bacterial loads in their feces and internal organs. These results indicate that parenteral immunization with S. Enteritidis-LTB ghosts can stimulate superior induction of systemic and mucosal immune responses compared to immunization with S. Enteritidis ghosts alone, thus conferring efficient protection against salmonellosis. PMID:24671556

  9. Mucosal immunization of mice using CpG DNA and/or mutants of the heat-labile enterotoxin of Escherichia coli as adjuvants.

    PubMed

    McCluskie, M J; Weeratna, R D; Clements, J D; Davis, H L

    2001-06-14

    Cholera toxin (CT) and the Escherichia coli heat-labile enterotoxin (LT) are potent mucosal adjuvants in animals associated, at least in part, with their ability to induce cAMP. While toxicity generally precludes their use in humans, a number of different subunit or genetically detoxified mutants of CT and LT have been developed. Another type of adjuvant that has been shown to be effective at mucosal surfaces comprises synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN). We have previously demonstrated a synergy between CpG ODN and native toxins after intranasal (IN) administration to mice, and herein have examined whether this synergy is linked to the cAMP activity. The adjuvanticity of CpG ODN was evaluated with IN and oral delivery of tetanus toxoid or the hepatitis B surface antigen, relative to and in combination with native LT holotoxin (LTh), three active site mutants (LTS61F, LTA69G, LTE112K), a protease site mutant (LTR192G), and the B subunit of LT (LTB). At an equivalent dose, the adjuvants could generally be divided into two groups: one that included CpG ODN, LTh, LTR192G, and LTA69G which acted as strong adjuvants; and the second which comprised LTB, LTS61F, and LTE112K, which produced significantly weaker immune responses. When CpG ODN was co-administered with bacterial toxin-derivatives, in most cases, no synergy between CpG and the LT derivatives was found for strength of the humoral response. Nevertheless, for both routes and antigens, CpG ODN combined with any LT derivative induced a more Type 1-like response than LT derivative alone. These results suggest that while the synergy seen previously with native toxins may have been due in part to inherent cAMP activity, it may have also depended on the particular antigen used and the route of immunization. PMID:11395211

  10. Intranasal immunization with pneumococcal polysaccharide conjugate vaccines with nontoxic mutants of Escherichia coli heat-labile enterotoxins as adjuvants protects mice against invasive pneumococcal infections.

    PubMed

    Jakobsen, H; Schulz, D; Pizza, M; Rappuoli, R; Jónsdóttir, I

    1999-11-01

    Host defenses against Streptococcus pneumoniae depend largely on phagocytosis following opsonization by polysaccharide-specific immunoglobulin G (IgG) antibodies and complement. Since colonization of the respiratory mucosa is the first step in pneumococcal pathogenesis, mucosal immune responses may play a significant role. In addition to inducing systemic immune responses, mucosal vaccination with an effective adjuvant has the advantage of inducing mucosal IgA antibodies. The heat-labile enterotoxin (LT) of Escherichia coli is a well-studied mucosal adjuvant, and adjuvant activity of nontoxic LT mutants has been demonstrated for several protein antigens. We investigated the immunogenicity of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 in mice after intranasal (i.n.) immunization by using as an adjuvant the nontoxic LT mutant LT-K63 or LT-R72, which has minimal residual toxicity. Pneumococcal serotype-specific antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA), and vaccine-induced protection was evaluated by i.n. challenge with virulent pneumococci of the homologous serotype. When administered with LT mutants, i.n. immunization with both conjugates induced systemic and mucosal immune responses, and serum IgG antibody levels were significantly higher than after subcutaneous immunization. All mice immunized i.n. with PNC-1 and LT mutants were protected against bacteremia and cleared the pneumococci from the lung 24 h after i.n. challenge; pneumococcal density correlated significantly with serum IgG antibody levels. Similarly, the survival of mice immunized i.n. with PNC-3 and LT mutants was significantly prolonged. These results demonstrate that i.n. vaccination with PNC and potent adjuvants can protect mice against invasive and lethal pneumococcal infections, indicating that mucosal vaccination with PNC may be an alternative vaccination strategy for humans. PMID:10531245

  11. The Arg7Lys mutant of heat-labile enterotoxin exhibits great flexibility of active site loop 47-56 of the A subunit.

    PubMed

    van den Akker, F; Merritt, E A; Pizza, M; Domenighini, M; Rappuoli, R; Hol, W G

    1995-09-01

    The heat-labile enterotoxin from Escherichia coli (LT) is a member of the cholera toxin family. These and other members of the larger class of AB5 bacterial toxins act through catalyzing the ADP-ribosylation of various intracellular targets including Gs alpha. The A subunit is responsible for this covalent modification, while the B pentamer is involved in receptor recognition. We report here the crystal structure of an inactive single-site mutant of LT in which arginine 7 of the A subunit has been replaced by a lysine residue. The final model contains 103 residues for each of the five B subunits, 175 residues for the A1 subunit, and 41 residues for the A2 subunit. In this Arg7Lys structure the active site cleft within the A subunit is wider by approximately 1 A than is seen in the wild-type LT. Furthermore, a loop near the active site consisting of residues 47-56 is disordered in the Arg7Lys structure, even though the new lysine residue at position 7 assumes a position which virtually coincides with that of Arg7 in the wild-type structure. The displacement of residues 47-56 as seen in the mutant structure is proposed to be necessary for allowing NAD access to the active site of the wild-type LT. On the basis of the differences observed between the wild-type and Arg7Lys structures, we propose a model for a coordinated sequence of conformational changes required for full activation of LT upon reduction of disulfide bridge 187-199 and cleavage of the peptide loop between the two cysteines in the A subunit.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7669757

  12. Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity.

    PubMed

    Giuliani, M M; Del Giudice, G; Giannelli, V; Dougan, G; Douce, G; Rappuoli, R; Pizza, M

    1998-04-01

    Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala --> Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 microg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose-response experiments in mice, 20 microg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials. PMID:9529328

  13. Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant.

    PubMed

    Di Tommaso, A; Saletti, G; Pizza, M; Rappuoli, R; Dougan, G; Abrignani, S; Douce, G; De Magistris, M T

    1996-03-01

    Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose. PMID:8641809

  14. Effects of Site-Directed Mutagenesis of Escherichia coli Heat-Labile Enterotoxin on ADP-Ribosyltransferase Activity and Interaction with ADP-Ribosylation Factors

    PubMed Central

    A. Stevens, Linda; Moss, Joel; Vaughan, Martha; Pizza, Mariagrazia; Rappuoli, Rino

    1999-01-01

    Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsα, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. PMID:9864224

  15. Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

    PubMed Central

    Lee, C M; Chang, P P; Tsai, S C; Adamik, R; Price, S R; Kunz, B C; Moss, J; Twiddy, E M; Holmes, R K

    1991-01-01

    Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT. Images PMID:1902492

  16. Effects of site-directed mutagenesis of Escherichia coli heat-labile enterotoxin on ADP-ribosyltransferase activity and interaction with ADP-ribosylation factors.

    PubMed

    Stevens, L A; Moss, J; Vaughan, M; Pizza, M; Rappuoli, R

    1999-01-01

    Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. PMID:9864224

  17. Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells.

    PubMed

    Fekete, Peter Z; Mateo, Kristina S; Zhang, Weiping; Moxley, Rodney A; Kaushik, Radhey S; Francis, David H

    2013-06-28

    Previous studies in piglets indicate that heat labile enterotoxin (LT) expression enhances intestinal colonization by K88 adhesin-producing enterotoxigenic Escherichia coli (ETEC) as wild-type ETEC adhered to intestinal epithelium in substantially greater numbers than did non-toxigenic constructs. Enzymatic activity of the toxin was also shown to contribute to the adhesion of ETEC and non-ETEC bacteria to epithelial cells in culture. To further characterize the contribution of LT to host cell adhesion, a nontoxigenic, K88-producing E. coli was transformed with either the gene encoding for LT holotoxin, a catalytically-attenuated form of the toxin [LT(R192G)], or LTB subunits, and resultant changes in bacterial adherence to IPEC-J2 porcine intestinal epithelial cells were measured. Strains expressing LT holotoxin or mutants were able to adhere in significantly higher numbers to IPEC-J2 cells than was an isogenic, toxin-negative construct. LT+ strains were also able to significantly block binding of a wild-type LT+ ETEC strain to IPEC-J2 cells. Adherence of isogenic strains to IPEC-J2 cells was unaltered by cycloheximide treatment, suggesting that LT enhances ETEC adherence to IPEC-J2 cells independent of host cell protein synthesis. However, pretreating IPEC-J2 cells with LT promoted adherence of negatively charged latex beads (a surrogate for bacteria which carry a negative change), which adherence was inhibited by cycloheximide, suggesting LT may induce a change in epithelial cell membrane potential. Overall, these data suggest that LT may enhance ETEC adherence by promoting an association between LTB and epithelial cells, and by altering the surface charge of the host plasma membrane to promote non-specific adherence. PMID:23517763

  18. Influence of Host Interleukin-10 Polymorphisms on Development of Traveler's Diarrhea Due to Heat-Labile Enterotoxin-Producing Escherichia coli in Travelers from the United States Who Are Visiting Mexico▿

    PubMed Central

    Flores, Jose; DuPont, Herbert L.; Lee, Stephanie A.; Belkind-Gerson, Jaime; Paredes, Mercedes; Mohamed, Jamal A.; Armitige, Lisa Y.; Guo, Dong-Chuan; Okhuysen, Pablo C.

    2008-01-01

    Up to 60% of U.S. visitors to Mexico develop traveler's diarrhea (TD), mostly due to enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Distinct single-nucleotide polymorphisms (SNPs) within the interleukin-10 (IL-10) promoter have been associated with high, intermediate, or low production of IL-10. We conducted a prospective study to investigate the association of SNPs in the IL-10 promoter and the occurrence of TD in ETEC LT-exposed travelers. Sera from U.S. travelers to Mexico collected on arrival and departure were studied for ETEC LT seroconversion by using cholera toxin as the antigen. Pyrosequencing was performed to genotype IL-10 SNPs. Stools from subjects who developed diarrhea were also studied for other enteropathogens. One hundred twenty-one of 569 (21.3%) travelers seroconverted to ETEC LT, and among them 75 (62%) developed diarrhea. Symptomatic seroconversion was more commonly seen in subjects who carried a genotype producing high levels of IL-10; it was seen in 83% of subjects with the GG genotype versus 54% of subjects with the AA genotype at IL-10 gene position −1082 (P, 0.02), in 71% of those with the CC genotype versus 33% of those with the TT genotype at position −819 (P, 0.005), and in 71% of those with the CC genotype versus 38% of those with the AA genotype at position −592 (P, 0.02). Travelers with the GCC haplotype were more likely to have symptomatic seroconversion than those with the ATA haplotype (71% versus 38%; P, 0.002). Travelers genetically predisposed to produce high levels of IL-10 were more likely to experience symptomatic ETEC TD. PMID:18579697

  19. Attenuated Shigella flexneri 2a vaccine strain CVD 1204 expressing colonization factor antigen I and mutant heat-labile enterotoxin of enterotoxigenic Escherichia coli.

    PubMed

    Koprowski, H; Levine, M M; Anderson, R J; Losonsky, G; Pizza, M; Barry, E M

    2000-09-01

    A multivalent live oral vaccine against both Shigella spp. and enterotoxigenic Escherichia coli (ETEC) is being developed based on the hypothesis that protection can be achieved if attenuated shigellae express ETEC fimbrial colonization factors and genetically detoxified heat-labile toxin from a human ETEC isolate (LTh). Two detoxified derivatives of LTh, LThK63 and LThR72, were engineered by substitution-serine to lysine at residue 63, or lysine to arginine at residue 72. The genes encoding these two derivatives were cloned separately on expression plasmids downstream from the CFA/I operon. Following electroporation into S. flexneri 2a vaccine strain CVD 1204, coexpression of CFA/I and LThK63 or LThR72 was demonstrated by Western blot analysis, GM(1) binding assays, and agglutination with anti-CFA/I antiserum. Hemagglutination and electron microscopy confirmed surface expression of CFA/I. Guinea pigs immunized intranasally on days 0 and 15 with CVD 1204 expressing CFA/I and LThK63 or LThR72 exhibited high titers of both serum immunoglobulin G (IgG) and mucosal secretory IgA anti-CFA/I; 40% of the animals produced antibodies directed against LTh. All immunized guinea pigs also produced mucosal IgA (in tears) and serum IgG anti-S. flexneri 2a O antibodies. Furthermore, all immunized animals were protected from challenge with wild-type S. flexneri 2a. This prototype Shigella-ETEC hybrid vaccine demonstrates the feasibility of expressing multiple ETEC antigens on a single plasmid in an attenuated Shigella vaccine strain and engendering immune responses against both the heterologous antigens and vector strain. PMID:10948101

  20. Comparison of a live attenuated Salmonella Enteritidis vaccine candidate secreting Escherichia coli heat-labile enterotoxin B subunit with a commercial vaccine for efficacy of protection against internal egg contamination by Salmonella in hens

    PubMed Central

    Nandre, Rahul M.; Eo, Seong Kug; Park, Sang Youel; Lee, John Hwa

    2015-01-01

    This study compared a new live attenuated Salmonella Enteritidis vaccine candidate secreting Escherichia coli heat-labile enterotoxin B subunit (SE-LTB) with a commercial Salmonella Enteritidis (SE) vaccine for efficacy of protection against SE infection in laying hens. Chickens were divided into 3 groups of 20 each. Group A chickens were inoculated orally with phosphate-buffered saline and served as controls, group B chickens were inoculated orally with the vaccine candidate, and group C chickens were inoculated intramuscularly with a commercial vaccine, the primary inoculation in groups B and C being at 10 wk of age and the booster at 16 wk. Groups B and C showed significantly higher titers of plasma immunoglobulin G, intestinal secretory immunoglobulin A, and egg yolk immunoglobulin Y antibodies compared with the control group, and both vaccinated groups showed a significantly elevated cellular immune response. After virulent challenge, group B had significantly lower production of thin-shelled and/or malformed eggs and a significantly lower rate of SE contamination of eggs compared with the control group. Furthermore, the challenge strain was detected significantly less in all of the examined organs of group B compared with the control group. Group C had lower gross lesion scores only in the spleen and had lower bacterial counts only in the spleen, ceca, and ovary. These findings indicate that vaccination with the SE-LTB vaccine candidate can efficiently reduce internal egg and internal organ contamination by Salmonella and has advantages over the commercial vaccine. PMID:26130857

  1. Construction of Bifidobacterium infantis as a live oral vaccine that expresses antigens of the major fimbrial subunit (CfaB) and the B subunit of heat-labile enterotoxin (LTB) from enterotoxigenic Escherichia coli.

    PubMed

    Ma, Yongping; Luo, Yaolin; Huang, Xueping; Song, Fangzhou; Liu, Geli

    2012-02-01

    We sought to develop Bifidobacterium infantis (BI) as a vehicle for the expression of heterologous antigens. Two proteins of enterotoxigenic Escherichia coli (ETEC) were expressed in BI: CfaB, a major fimbrial subunit protein, and LTB, the B subunit of heat-labile enterotoxin. The expression of CfaB and LTB in BI was verified by electrophoretic analysis. Sprague-Dawley rats were then subjected to intragastric immunization with BI-CfaB and BI-LTB systems both separately and together. ELISA was used to characterize the serum and mucosal immune responses against ETEC antigens. The immunized rats were intraperitoneally challenged with wild-type ETEC H10407 to study the immune response in vivo. The serum titres of IgG and faecal IgA antibodies in the BI-CfaB plus BI-LTB mixed vaccination group were significantly greater than those in the other two groups, which were immunized with a single vaccine (P<0.05). However, no significant difference was seen between the two groups that received a single immunization. These results suggest that expressing CfaB and LTB in BI provides a probiotic system with immunogenic properties. Furthermore, the expression of LTB in BI preserved its mucosal adjuvant effect. So this study confirms that BI can be used as a novel oral vaccine expression system for a heterologous antigen and BI-LTB can provide mucosal adjuvant properties. PMID:22053005

  2. Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant.

    PubMed

    Marchetti, M; Rossi, M; Giannelli, V; Giuliani, M M; Pizza, M; Censini, S; Covacci, A; Massari, P; Pagliaccia, C; Manetti, R; Telford, J L; Douce, G; Dougan, G; Rappuoli, R; Ghiara, P

    1998-01-01

    We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: (1) protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans. PMID:9607006

  3. The adjuvant effect of a non-toxic mutant of heat-labile enterotoxin of Escherichia coli for the induction of measles virus-specific CTL responses after intranasal co-immunization with a synthetic peptide.

    PubMed

    Partidos, C D; Pizza, M; Rappuoli, R; Steward, M W

    1996-12-01

    The intranasal route has been shown to be effective for immunization. However, immunization via this route may require the use of potent and safe adjuvant. The construction of non-toxic mutants of heat labile enterotoxin of Escherichia coli (LT), which is a potent mucosal adjuvant, is a major breakthrough for the development of mucosal vaccines. In this study we have assessed the ability of an LT mutant (LTK63) to act as an adjuvant following intranasal co-immunization with a peptide corresponding to a measles virus cytotoxic T lymphocyte (CTL) epitope. LTK63 was more effective at potentiating the in vivo induction of peptide-specific and measles virus-specific CTL responses than was administration of the peptide in saline. A concentration of 10 micrograms/dose of LTK63 was found to be the most effective in potentiating the in vivo priming of peptide-specific and measles virus-specific CTL responses. These findings highlight the potential of the non-toxic mutant of LT as a safe mucosal adjuvant for use in humans. PMID:9014810

  4. Structure–activity correlations of variant forms of the B pentamer of Escherichia coli type II heat-labile enterotoxin LT-IIb with Toll-like receptor 2 binding

    SciTech Connect

    Cody, Vivian; Pace, Jim; Nawar, Hesham F.; King-Lyons, Natalie; Liang, Shuang; Connell, Terry D.; Hajishengallis, George

    2012-12-01

    Structural data for the S74D variant of the pentameric B subunit of type II heat-labile enterotoxin of Escherichia coli reveal a smaller pore opening that may explain its reduced Toll-like receptor binding affinity compared to that of the wild type enterotoxin. The explanation for the enhanced Toll-like receptor binding affinity of the S74A variant is more complex than simply being attributed to the pore opening. The pentameric B subunit of the type II heat-labile enterotoxin of Escherichia coli (LT-IIb-B{sub 5}) is a potent signaling molecule capable of modulating innate immune responses. It has previously been shown that LT-IIb-B{sub 5}, but not the LT-IIb-B{sub 5} Ser74Asp variant [LT-IIb-B{sub 5}(S74D)], activates Toll-like receptor (TLR2) signaling in macrophages. Consistent with this, the LT-IIb-B{sub 5}(S74D) variant failed to bind TLR2, in contrast to LT-IIb-B{sub 5} and the LT-IIb-B{sub 5} Thr13Ile [LT-IIb-B{sub 5}(T13I)] and LT-IIb-B{sub 5} Ser74Ala [LT-IIb-B{sub 5}(S74A)] variants, which displayed the highest binding activity to TLR2. Crystal structures of the Ser74Asp, Ser74Ala and Thr13Ile variants of LT-IIb-B{sub 5} have been determined to 1.90, 1.40 and 1.90 Å resolution, respectively. The structural data for the Ser74Asp variant reveal that the carboxylate side chain points into the pore, thereby reducing the pore size compared with that of the wild-type or the Ser74Ala variant B pentamer. On the basis of these crystallographic data, the reduced TLR2-binding affinity of the LT-IIb-B{sub 5}(S74D) variant may be the result of the pore of the pentamer being closed. On the other hand, the explanation for the enhanced TLR2-binding activity of the LT-IIb-B{sub 5}(S74A) variant is more complex as its activity is greater than that of the wild-type B pentamer, which also has an open pore as the Ser74 side chain points away from the pore opening. Data for the LT-IIb-B{sub 5}(T13I) variant show that four of the five variant side chains point to the outside

  5. LT-IIb(T13I), a non-toxic type II heat-labile enterotoxin, augments the capacity of a ricin toxin subunit vaccine to evoke neutralizing antibodies and protective immunity.

    PubMed

    Greene, Christopher J; Chadwick, Chrystal M; Mandell, Lorrie M; Hu, John C; O'Hara, Joanne M; Brey, Robert N; Mantis, Nicholas J; Connell, Terry D

    2013-01-01

    Currently, there is a shortage of adjuvants that can be employed with protein subunit vaccines to enhance protection against biological threats. LT-IIb(T13I) is an engineered nontoxic derivative of LT-IIb, a member of the type II subfamily of heat labile enterotoxins expressed by Escherichia coli, that possesses potent mucosal adjuvant properties. In this study we evaluated the capacity of LT-IIb(T13I) to augment the potency of RiVax, a recombinant ricin toxin A subunit vaccine, when co-administered to mice via the intradermal (i.d.) and intranasal (i.n.) routes. We report that co-administration of RiVax with LT-IIb(T13I) by the i.d. route enhanced the levels of RiVax-specific serum IgG antibodies (Ab) and elevated the ratio of ricin-neutralizing to non-neutralizing Ab, as compared to RiVax alone. Protection against a lethal ricin challenge was also augmented by LT-IIb(T13I). While local inflammatory responses elicited by LT-IIb(T13I) were comparable to those elicited by aluminum salts (Imject®), LT-IIb(T13I) was more effective than aluminum salts at augmenting production of RiVax-specific serum IgG. Finally, i.n. administration of RiVax with LT-IIb(T13I) also increased levels of RiVax-specific serum and mucosal Ab and enhanced protection against ricin challenge. Collectively, these data highlight the potential of LT-IIb(T13I) as an effective next-generation i.d., or possibly i.n. adjuvant for enhancing the immunogenicity of subunit vaccines for biodefense. PMID:23936344

  6. Oral immunization with an attenuated Salmonella Gallinarum mutant as a fowl typhoid vaccine with a live adjuvant strain secreting the B subunit of Escherichia coli heat-labile enterotoxin

    PubMed Central

    2013-01-01

    Background The Salmonella Gallinarum (SG) lon/cpxR deletion mutant JOL916 was developed as a live vaccine candidate for fowl typhoid (FT), and a SG mutant secreting an Escherichia coli heat-labile enterotoxin B subunit (LTB), designated JOL1229, was recently constructed as an adjuvant strain for oral vaccination against FT. In this study, we evaluated the immunogenicity and protective properties of the SG mutant JOL916 and the LTB adjuvant strain JOL1229 in order to establish a prime and boost immunization strategy for each strain. In addition, we compared the increase in body weight, the immunogenicity, the egg production rates, and the bacteriological egg contamination of these strains with those of SG 9R, a widely used commercial vaccine. Results Plasma IgG, intestinal secretory IgA (sIgA), and cell-mediated responses were significantly induced after a boost inoculation with a mixture of JOL916 and JOL1229, and significant reductions in the mortality of chickens challenged with a wild-type SG strain were observed in the immunized groups. There were no significant differences in increases in body weight, cell-mediated immune responses, or systemic IgG responses between our vaccine mixture and the SG 9R vaccine groups. However, there was a significant elevation in intestinal sIgA in chickens immunized with our mixture at 3 weeks post-prime-immunization and at 3 weeks post-boost-immunization, while sIgA levels in SG 9R-immunized chickens were not significantly elevated compared to the control. In addition, the SG strain was not detected in the eggs of chickens immunized with our mixture. Conclusion Our results suggest that immunization with the LTB-adjuvant strain JOL1229 can significantly increase the immune response, and provide efficient protection against FT with no side effects on body weight, egg production, or egg contamination. PMID:23647814

  7. Immunization with a Double-Mutant (R192G/L211A) of the Heat-Labile Enterotoxin of Escherichia coli Offers Partial Protection against Campylobacter jejuni in an Adult Mouse Intestinal Colonization Model.

    PubMed

    Albert, M John; Haridas, Shilpa; Ebenezer, Mathew; Raghupathy, Raj; Khan, Islam

    2015-01-01

    We have previously shown that antibodies to cholera toxin (CT) reacted with the major outer membrane proteins (MOMPs) from Campylobacter jejuni strains on Western blot. Further, oral immunization with CT significantly protected against challenge with C. jejuni in an adult mouse colonization model of infection. CT and the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli are structurally and functionally related. LT and its mutants including the double-mutant LT (R192G/L211A) (dmLT), are powerful mucosal adjuvants. Unlike LT which is reactogenic, dmLT has been shown to be safe for human use. In the current study, we determined whether rabbit anti-dmLT antibodies reacted with MOMPs from C. jejuni strains and whether immunization with dmLT would afford protection against C. jejuni. On Western blot, the MOMPs from C. jejuni 48 (Penner serotype O:19), C. jejuni 75 (O:3) and C. jejuni 111 (O:1,44) were probed with rabbit antibodies to dmLT or LT-E112K (a non-toxic LT mutant), which showed a lack of reaction. Adult BALB/c mice were orally immunized with dmLT and orally challenged with C. jejuni 48 or 111. Protection from colonization with the challenge bacteria was studied by enumerating Campylobacter colonies in feces daily for 9 days. Vaccination produced robust serum and stool antibody responses to dmLT and no antibody responses to C. jejuni MOMP. Vaccinated mice showed reduced colonization and excretion of both challenge strains compared to control mice. However, the differences were not statistically significant. The protective efficacy of the dmLT vaccine varied from 9.1% to 54.5%. The lack of cross-reaction between the MOMP and dmLT suggests that protection is not mediated by cross-reacting antibodies, but may be due to activation of innate immunity. As dmLT is safe for humans, it could be incorporated into a C. jejuni vaccine to enhance its efficacy. PMID:26540197

  8. Immunization with a Double-Mutant (R192G/L211A) of the Heat-Labile Enterotoxin of Escherichia coli Offers Partial Protection against Campylobacter jejuni in an Adult Mouse Intestinal Colonization Model

    PubMed Central

    Albert, M. John; Haridas, Shilpa; Ebenezer, Mathew; Raghupathy, Raj; Khan, Islam

    2015-01-01

    We have previously shown that antibodies to cholera toxin (CT) reacted with the major outer membrane proteins (MOMPs) from Campylobacter jejuni strains on Western blot. Further, oral immunization with CT significantly protected against challenge with C. jejuni in an adult mouse colonization model of infection. CT and the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli are structurally and functionally related. LT and its mutants including the double-mutant LT (R192G/L211A) (dmLT), are powerful mucosal adjuvants. Unlike LT which is reactogenic, dmLT has been shown to be safe for human use. In the current study, we determined whether rabbit anti-dmLT antibodies reacted with MOMPs from C. jejuni strains and whether immunization with dmLT would afford protection against C. jejuni. On Western blot, the MOMPs from C. jejuni 48 (Penner serotype O:19), C. jejuni 75 (O:3) and C. jejuni 111 (O:1,44) were probed with rabbit antibodies to dmLT or LT-E112K (a non-toxic LT mutant), which showed a lack of reaction. Adult BALB/c mice were orally immunized with dmLT and orally challenged with C. jejuni 48 or 111. Protection from colonization with the challenge bacteria was studied by enumerating Campylobacter colonies in feces daily for 9 days. Vaccination produced robust serum and stool antibody responses to dmLT and no antibody responses to C. jejuni MOMP. Vaccinated mice showed reduced colonization and excretion of both challenge strains compared to control mice. However, the differences were not statistically significant. The protective efficacy of the dmLT vaccine varied from 9.1% to 54.5%. The lack of cross-reaction between the MOMP and dmLT suggests that protection is not mediated by cross-reacting antibodies, but may be due to activation of innate immunity. As dmLT is safe for humans, it could be incorporated into a C. jejuni vaccine to enhance its efficacy. PMID:26540197

  9. Attenuated Escherichia coli strains expressing the colonization factor antigen I (CFA/I) and a detoxified heat-labile enterotoxin (LThK63) enhance clearance of ETEC from the lungs of mice and protect mice from intestinal ETEC colonization and LT-induced fluid accumulation.

    PubMed

    Byrd, Wyatt; Boedeker, Edgar C

    2013-03-15

    Although enterotoxigenic Escherichia coli (ETEC) infections are important causes of infantile and traveler's diarrhea there is no licensed vaccine available for those at-risk. Our goal is to develop a safe, live attenuated ETEC vaccine. We used an attenuated E. coli strain (O157:H7, Δ-intimin, Stx1-neg, Stx2-neg) as a vector (ZCR533) to prepare two vaccine strains, one strain expressing colonization factor antigen I (ZCR533-CFA/I) and one strain expressing CFA/I and a detoxified heat-labile enterotoxin (ZCR533-CFA/I+LThK63) to deliver ETEC antigens to mucosal sites in BALB/c mice. Following intranasal and intragastric immunization with the vaccine strains, serum IgG and IgA antibodies were measured to the CFA/I antigen, however, only serum IgG antibodies were detected to the heat-labile enterotoxin. Intranasal administration of the vaccine strains induced respiratory and intestinal antibody responses to the CFA/I and LT antigens, while intragastric administration induced only intestinal antibody responses with no respiratory antibodies detected to the CFA/I and LT antigens. Mice immunized intranasally with the vaccine strains showed enhanced clearance of wild-type (wt) ETEC bacteria from the lungs. Mice immunized intranasally and intragastrically with the vaccine strains were protected from intestinal colonization following oral challenge with ETEC wt bacteria. Mice immunized intragastrically with the ZCR533-CFA/I+LThK63 vaccine strain had less fluid accumulate in their intestine following challenge with ETEC wt bacteria or with purified LT as compared to the sham mice indicating that the immunized mice were protected from LT-induced intestinal fluid accumulation. Thus, mice intragastrically immunized with the ZCR533-CFA/I+LThK63 vaccine strain were able to effectively neutralize the activity of the LT enterotoxin. However, no difference in intestinal fluid accumulation was detected in the mice immunized intranasally with the vaccine strain as compared to the sham

  10. Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants.

    PubMed

    Douce, G; Turcotte, C; Cropley, I; Roberts, M; Pizza, M; Domenghini, M; Rappuoli, R; Dougan, G

    1995-02-28

    A nontoxic mutant (LTK7) of the Escherichia coli heat-labile enterotoxin (LT) lacking ADP-ribosylating activity but retaining holotoxin formation was constructed. By using site-directed mutagenesis, the arginine at position 7 of the A subunit was replaced with lysine. This molecule, which was nontoxic in several assays, was able to bind to eukaryotic cells and acted as a mucosal adjuvant for co-administered proteins; BALB/c mice immunized intranasally with LTK7 and ovalbumin developed high levels of serum and local antibodies to ovalbumin and toxin. In addition, mice immunized intranasally with fragment C of tetanus toxin and LTK7 were protected against lethal challenge with tetanus toxin. Thus nontoxic mutants of heat-labile toxin can act as effective intranasal mucosal adjuvants. PMID:7878032

  11. Cistrons encoding Escherichia coli heat-labile toxin.

    PubMed Central

    Dallas, W S; Gill, D M; Falkow, S

    1979-01-01

    The structure and products of the two cistrons encoding the Escherichia coli heat-labile toxin (LT) were studied. The LT deoxyribonucleic acid (DNA) region had been isolated as part of a DNA fragment from the plasmid P307, and this fragment was joined to the cloning vector pBR313. Deletion mutations of various lengths were introduced into the LT DNA region and into the adjacent DNA sequences. Analysis of the deletions indicated that the maximum size of the LT DNA region was 1.2 x 10(6) daltons. Two proteins of 11,500 daltons and 25,500 daltons had been shown to be encoded by the LT DNA region. The functions of these LT gene products were investigated. The 11,500-dalton protein had an adsorption activity for Y-1 adrenal cells, and this protein was shown to form aggregates of four or five monomers. The 25,500-dalton protein was shown to have an adenylate cyclase-activating activity. The two cistrons encoding for each of the LT proteins have been located on a genetic map of the LT DNA region. Both cistrons are probably transcribed from the same promoter. Images PMID:383697

  12. Characterization of a mutant Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral adjuvant.

    PubMed

    Norton, Elizabeth B; Lawson, Louise B; Freytag, Lucy C; Clements, John D

    2011-04-01

    Despite the fact that the adjuvant properties of the heat-labile enterotoxins of Escherichia coli (LT) and Vibrio cholerae (CT) have been known for more than 20 years, there are no available oral vaccines containing these molecules as adjuvants, primarily because they are both very potent enterotoxins. A number of attempts with various degrees of success have been made to reduce or eliminate the enterotoxicity of LT and CT so they can safely be used as oral adjuvants or immunogens. In this report we characterize the structural, enzymatic, enterotoxic, and adjuvant properties of a novel mutant of LT, designated LT(R192G/L211A), or dmLT. dmLT was not sensitive to trypsin activation, had reduced enzymatic activity for induction of cyclic AMP in Caco-2 cells, and exhibited no enterotoxicity in the patent mouse assay. Importantly, dmLT retained the ability to function as an oral adjuvant for a coadministered antigen (tetanus toxoid) and to elicit anti-LT antibodies. In vitro and in vivo data suggest that the reduced enterotoxicity of this molecule compared to native LT or the single mutant, LT(R192G), is a consequence of increased sensitivity to proteolysis and rapid intracellular degradation in mammalian cells. In conclusion, dmLT is a safe and powerful detoxified enterotoxin with the potential to function as a mucosal adjuvant for coadministered antigens and to elicit anti-LT antibodies without undesirable side effects. PMID:21288994

  13. Characterization of a Mutant Escherichia coli Heat-Labile Toxin, LT(R192G/L211A), as a Safe and Effective Oral Adjuvant ▿

    PubMed Central

    Norton, Elizabeth B.; Lawson, Louise B.; Freytag, Lucy C.; Clements, John D.

    2011-01-01

    Despite the fact that the adjuvant properties of the heat-labile enterotoxins of Escherichia coli (LT) and Vibrio cholerae (CT) have been known for more than 20 years, there are no available oral vaccines containing these molecules as adjuvants, primarily because they are both very potent enterotoxins. A number of attempts with various degrees of success have been made to reduce or eliminate the enterotoxicity of LT and CT so they can safely be used as oral adjuvants or immunogens. In this report we characterize the structural, enzymatic, enterotoxic, and adjuvant properties of a novel mutant of LT, designated LT(R192G/L211A), or dmLT. dmLT was not sensitive to trypsin activation, had reduced enzymatic activity for induction of cyclic AMP in Caco-2 cells, and exhibited no enterotoxicity in the patent mouse assay. Importantly, dmLT retained the ability to function as an oral adjuvant for a coadministered antigen (tetanus toxoid) and to elicit anti-LT antibodies. In vitro and in vivo data suggest that the reduced enterotoxicity of this molecule compared to native LT or the single mutant, LT(R192G), is a consequence of increased sensitivity to proteolysis and rapid intracellular degradation in mammalian cells. In conclusion, dmLT is a safe and powerful detoxified enterotoxin with the potential to function as a mucosal adjuvant for coadministered antigens and to elicit anti-LT antibodies without undesirable side effects. PMID:21288994

  14. Heterogenic virulence in a diarrheagenic Escherichia coli: evidence for an EPEC expressing heat-labile toxin of ETEC.

    PubMed

    Dutta, Sanjucta; Pazhani, Gururaja P; Nataro, James P; Ramamurthy, Thandavarayan

    2015-01-01

    We have encountered an Escherichia coli strain isolated from a child with acute diarrhea. This strain harbored eae and elt genes encoding for E. coli attaching and effacing property and heat-labile enterotoxin of EPEC and ETEC, respectively. Due to the presence of these distinct virulence factors, we named this uncommon strain as EPEC/ETEC hybrid. The elt gene was identified in a conjugally transferable plasmid of the EPEC/ETEC hybrid. In addition, several virulence genes in the locus of enterocyte effacement have been identified, which confirms that the EPEC/ETEC has an EPEC genetic background. The hybrid nature of this strain was further confirmed by using tissue culture assays. In the multi locus sequence typing (MLST) analysis, the EPEC/ETEC belonged to the sequence type ST328 and was belonging to ST278 Cplx. Sequence analysis of the plasmid DNA revealed presence of six large contigs with several insertion sequences. A phage integrase gene and the prophages of gp48 and gp49 have been found in the upstream of eltAB. In the downstream of elt, an urovirulence loci adhesion encoding (pap) cluster containing papG, and papC were also identified. Similar to other reports, we have identified a heterogenic virulence in a diarrheagenic E. coli but with different combination of genes. PMID:25465159

  15. Participation of ABH glycoconjugates in the secretory response to Escherichia coli heat-labile toxin in rabbit intestine.

    PubMed

    Galván, E M; Roth, G A; Monferran, C G

    1999-08-01

    The ability of membrane ABH blood group-active glycoconjugates to act as receptors of the heat-labile enterotoxin of Escherichia coli (LTh) was studied in vitro and in vivo when GM1 was blocked by the cholera toxin B subunit. Rabbits were classified as AB or H based on intestinal ABH-antigenic activities. Brush border membranes from AB rabbits contained 4 times more LTh binding sites than the H ones. LTh interaction could be inhibited by lectins that recognize ABH determinants. LTh induced a similar dose-dependent secretory response in ligated ileal loops of both types of animals. Anti-AB antibodies and Ulex europaeus I lectin could significantly reduce the fluid accumulation in AB and H rabbits, respectively. LTh caused adenylate cyclase activation even when GM1 was blocked, and this effect was abolished by the addition of specific ABH ligands. These results suggest that ABH glycoconjugates are involved in the host secretory response to LTh in rabbit intestine. PMID:10395858

  16. Seroepidemiology of heat-labile enterotoxigenic Escherichia coli and Norwalk virus infections in Panamanians, Canal Zone residents, Apache Indians, and United States Peace Corps volunteers.

    PubMed Central

    Ryder, R W; Greenberg, H; Singh, N; Oro, G; de Guardia, A; Sack, R B; Kapikian, A Z

    1982-01-01

    Serum antibody titrations against the heat-labile enterotoxin (LT) of Escherichia coli were carried out on Panamanians, U.S. citizens resident in the Panama Canal Zone, Apache Indians living on the reservation in Whiteriver, Arizona, and Peace Corps volunteers before they traveled overseas. Antibody titers to Norwalk virus were also carried out on serum from Panamanian and Canal Zone residents. A high prevalence of low-titer LT antibodies was found in infants and adults from Panama, the Canal Zone, and Whiteriver. Panamanian children aged 1 to 5 years had the highest LT antibody titers. Peace Corps volunteers had a low prevalence and titer of LT antibodies. Prevalence and titer of antibodies to Norwalk virus were generally higher in Panamanians compared with Canal Zone residents of the same age. In the populations we studied, various modes of transmission and mechanisms of immunity likely explain the differences which we observed in antibody prevalence and titer to these two enteric pathogens. PMID:6290396

  17. Radiation-induced heat-labile sites that convert into DNA double-strand breaks

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

  18. Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

    PubMed Central

    Andrade, Fernanda B.; Nepomuceno, Roberto; Silva, Anderson; Munhoz, Danielle D.; Yamamoto, Bruno B.; Luz, Daniela; Abreu, Patrícia A. E.; Horton, Denise S. P. Q.; Elias, Waldir P.; Ramos, Oscar H. P.; Piazza, Roxane M. F.

    2015-01-01

    Background Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains. Methods and Findings Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains. Conclusion The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis. PMID:26154103

  19. Pet, an Autotransporter Enterotoxin from Enteroaggregative Escherichia coli

    PubMed Central

    Eslava, Carlos; Navarro-García, Fernando; Czeczulin, John R.; Henderson, Ian R.; Cravioto, Alejandro; Nataro, James P.

    1998-01-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a β-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenic E. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects. PMID:9632580

  20. Design and characterization of a chimeric multiepitope construct containing CfaB, heat-stable toxoid, CssA, CssB, and heat-labile toxin subunit B of enterotoxigenic Escherichia coli: a bioinformatic approach.

    PubMed

    Zeinalzadeh, Narges; Salmanian, Ali Hatef; Ahangari, Ghasem; Sadeghi, Mahdi; Amani, Jafar; Bathaie, S Zahra; Jafari, Mahyat

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are the most common cause of bacterial diarrhea in children in developing countries and travelers to these areas. Enterotoxins and colonization factors (CFs) are two key virulence factors in ETEC pathogenesis, and the heterogeneity of the CFs is the bottleneck in reaching an effective vaccine. In this study, a candidate subunit vaccine, which is composed of CfaB, CssA and CssB, structural subunits of colonization factor antigen I and CS6 CFs, labile toxin subunit B, and the binding subunit of heat-labile and heat-stable toxoid, was designed to provide broad-spectrum protection against ETEC. The different features of chimeric gene, its mRNA stability, and chimeric protein properties were analyzed by using bioinformatic tools. The optimized chimeric gene was chemically synthesized and expressed successfully in a prokaryotic host. The purified protein was used for assessment of bioinformatic data by experimental methods. PMID:24372617

  1. Evaluation of commercial antisera for serotyping heat-labile antigens of Campylobacter jejuni and Campylobacter coli.

    PubMed Central

    Nicholson, M A; Patton, C M

    1993-01-01

    Commercial antisera for serotyping 22 heat-labile antigens of Campylobacter jejuni and Campylobacter coli were evaluated by using 66 isolates from human and nonhuman sources. Test results were compared with results of tests using antisera produced at the Centers for Disease Control (CDC), Atlanta, Ga. All strains (three isolates of each of the 22 serotypes) were typeable with the CDC antisera. Of 66 test strains, 39 (59%) were typed as the same serotype with both sets of antisera. Twenty-four strains (36%), including two heat-labile serotype reference strains, were nonreactive with the commercial antisera, and three strains (4.5%) were typed as serotypes different from those obtained with CDC antisera. Five of the 22 commercial antisera correctly serotyped all homologous strains. Our study indicated that two polyvalent antiserum pools, 7 unabsorbed antisera, and 16 absorbed monovalent antisera are weak and need modification to enhance their antibody titers. Further studies are necessary to explain the antigenic change to a different serotype in three strains. PMID:8463402

  2. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    SciTech Connect

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  3. Staphylococcal enterotoxin B-induced microRNA-155 targets SOCS1 to promote acute inflammatory lung injury.

    PubMed

    Rao, Roshni; Rieder, Sadiye Amcaoglu; Nagarkatti, Prakash; Nagarkatti, Mitzi

    2014-07-01

    Staphylococcal enterotoxin B (SEB) causes food poisoning in humans. It is considered a biological weapon, and inhalation can trigger lung injury and sometimes respiratory failure. Being a superantigen, SEB initiates an exaggerated inflammatory response. While the role of microRNAs (miRNAs) in immune cell activation is getting increasing recognition, their role in the regulation of inflammatory disease induced by SEB has not been studied. In this investigation, we demonstrate that exposure to SEB by inhalation results in acute inflammatory lung injury accompanied by an altered miRNA expression profile in lung-infiltrating cells. Among the miRNAs that were significantly elevated, miR-155 was the most overexpressed. Interestingly, miR-155(-/-) mice were protected from SEB-mediated inflammation and lung injury. Further studies revealed a functional link between SEB-induced miR-155 and proinflammatory cytokine gamma interferon (IFN-γ). Through the use of bioinformatics tools, suppressor of cytokine signaling 1 (SOCS1), a negative regulator of IFN-γ, was identified as a potential target of miR-155. While miR-155(-/-) mice displayed increased expression of Socs1, the overexpression of miR-155 led to its suppression, thereby enhancing IFN-γ levels. Additionally, the inhibition of miR-155 resulted in restored Socs1expression. Together, our data demonstrate an important role for miR-155 in promoting SEB-mediated inflammation in the lungs through Socs1 suppression and suggest that miR-155 may be an important target in preventing SEB-mediated inflammation and tissue injury. PMID:24778118

  4. New Surface-Associated Heat-Labile Colonization Factor Antigen (CFA/II) Produced by Enterotoxigenic Escherichia coli of Serogroups O6 and O8

    PubMed Central

    Evans, Dolores G.; Evans, Doyle J.

    1978-01-01

    Enterotoxigenic Escherichia coli (ETEC) belonging to serogroups O6 and O8 do not possess the H-10407-type colonization factor antigen (CFA/I). However, these frequently isolated ETEC were found to possess a second and distinct heat-labile surface-associated colonization factor antigen, termed CFA/II. Whereas CFA/I mediates mannose-resistant hemagglutination of human group A erythrocytes, CFA/II does not. CFA/II mediates mannose-resistant hemagglutination of bovine erythrocytes, and mannose-resistant hemagglutination is rapid only at reduced temperature (4°C). Because CFA/II, like CFA/I, is spontaneously lost by many ETEC isolates in the laboratory, it was possible to produce specific anti-CFA/II serum by preparing antiserum against living cells of a prototype strain (PB-176) and adsorbing this serum with living and heat-treated cells of its CFA/II-negative derivative strain PB-176-P. This serum, which neutralized the colonization factor activity of CFA/II-positive strains in infant rabbits, was employed to confirm the presence of CFA/II on ETEC which exhibited mannose-resistant hemagglutination of bovine but not human erythrocytes. CFA/II, like CFA/I, mediates adherence of the bacteria to the mucosal surface of the small intestine, as demonstrated by indirect immunofluorescence. CFA/II appears to be an important virulence factor for humans since CFA/II-positive ETEC are frequently isolated from diarrhea cases, particularly travelers' diarrhea, in Mexico; these ETEC were not uncommon in a collection of isolates from Bangladesh. The O6:H16 strain of ETEC responsible for an outbreak of diarrhea in the United States was also shown to be CFA/II positive. CFA/I and CFA/II were never found on the same serotypes of ETEC, but 98% of the heat-stable and heat-labile enterotoxin-producing ETEC belonging to the frequently isolated serogroups O6, O8, O15, O25, O63, and O78 were positive for either CFA/I or CFA/II. Images PMID:80383

  5. Multiplex PCR detection of enterotoxin genes in Aeromonas spp. from suspect food samples in northern Taiwan.

    PubMed

    Chang, Yu-Chang; Wang, Jan-Yi; Selvam, Ammaiyappan; Kao, Shu-Chen; Yang, Shang-Shyng; Shih, Daniel Yang-Chih

    2008-10-01

    Aeromonads possess an array of virulence factors and are causative agents of a number of human infections. Among them, genes of one cytotoxic (Act) and two cytotonic (Alt, Ast) enterotoxins are implicated in a human diarrheal disease. A rapid, specific, simultaneous detection of these enterotoxin genes in suspected food poisoning samples is not yet reported. Hence, a multiplex PCR assay was designed to amplify the cytotoxic (act), heat-labile cytotonic (alt), and heat-stable cytotonic (ast) enterotoxin genes of aeromonads. The PCR assay was tested with 133 Aeromonas spp. isolated from suspect food poisoning samples and retail samples of poultry and fish from wet markets in and around Taipei, Northern Taiwan. The Aeromonas spp. isolates were divided into six genotypes based on absence or presence of one or more enterotoxin genes. Of these 133 isolates, Aeromonas caviae (52.5%) and Aeromonas hydrophila (43.4%) were the most frequently isolated species from food poisoning samples and retail samples, respectively. Among the species, A. hydrophila had a significantly higher proportion for harboring three enterotoxin genes than had the others, whereas Aeromonas encheleia, considered a nonpathogen, was found harboring three enterotoxin genes. The multiplex PCR assays are rapid and specific, and provide a useful tool for the detection and genotyping of enterotoxin genes of aeromonads. PMID:18939759

  6. Adhesin degradation accelerates delivery of heat-labile toxin by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Kansal, Rita; Bartels, Scott R; Hamilton, David J; Shaaban, Salwa; Fleckenstein, James M

    2011-08-26

    Many enteric pathogens, including enterotoxigenic Escherichia coli (ETEC), produce one or more serine proteases that are secreted via the autotransporter (or type V) bacterial secretion pathway. These molecules have collectively been referred to as SPATE proteins (serine protease autotransporter of the Enterobacteriaceae). EatA, an autotransporter previously identified in ETEC, possesses a functional serine protease motif within its secreted amino-terminal passenger domain. Although this protein is expressed by many ETEC strains and is highly immunogenic, its precise function is unknown. Here, we demonstrate that EatA degrades a recently characterized adhesin, EtpA, resulting in modulation of bacterial adhesion and accelerated delivery of the heat-labile toxin, a principal ETEC virulence determinant. Antibodies raised against the passenger domain of EatA impair ETEC delivery of labile toxin to epithelial cells suggesting that EatA may be an effective target for vaccine development. PMID:21757737

  7. Heat-labile enterotoxigenic Escherichia coli and intestinal protozoa in asymptomatic travellers.

    PubMed

    Echeverria, P; Cross, J H

    1977-12-01

    Thirty-two asymptomatic travellers who had recently journeyed in the Near, Middle, and Far East and had experienced a high incidence of diarrhoeal disease were screened for heat-labile enterotoxigenic Escherichia coli (ent+ E. coli) and other bacterial and parasitic pathogens. Six percent were colonized with ent+ E. coli and while other bacterial pathogens were not found, the intestinal protozoa Giardia lamblia (13%), Entamoeba histolytica (6%), Entamoeba coli (6%), Endolimax nana (6%), and Entamoeba hartmanni (3%) were detected in the stools. Ent+ E. coli, G. lamblia and E. histolytica should be considered in the differential diagnosis of gastrointestinal disease in travellers returning from the Orient. Furthermore, these travellers may be a potential source for the introduction of ent+ E. coli into communities where such organisms are relatively rare. PMID:351820

  8. Hereditary heat-labile hexosaminidase B: its implication for recognizing Tay-Sachs genotypes.

    PubMed

    Navon, R; Nutman, J; Kopel, R; Gaber, L; Gadoth, N; Goldman, B; Nitzan, M

    1981-11-01

    Two pairs of alleles, at the two loci of hexosaminidase (HEX), were found to segregate in an Arab inbred family: the normal and the mutant Tay-Sachs (TSD) alleles of HEX A, and the normal and a mutant allele of HEX B. Since the mutant HEX B is heat labile, no reliable identification of TSD genotypes can be obtained in its presence, as long as the proportions of HEX A and B are estimated by the routinely used heat-inactivation method. The genotypes may be correctly identified in such cases by separation of the two isoenzymes on ion-exchange chromatography, estimating their individual activities, and calculating the ratio between them. Of the nine genotype combinations possible with these two pairs of alleles, five have been identified in the reported family by this procedure. PMID:6459736

  9. Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and analysis of its immunogenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Expression of the heat-labile toxin B subunit of enterotoxigenic Escherichia coli (LT) B was directed to the endoplasmic reticulum (ER) of soybean seed storage parenchyma cells for immunogen sequestration in de novo synthesized, ER-derived protein accretions in transgenic seed. Pentameric LTB accumu...

  10. Heat-labile- and heat-stable-toxoid fusions (LTR₁₉₂G-STaP₁₃F) of human enterotoxigenic Escherichia coli elicit neutralizing antitoxin antibodies.

    PubMed

    Liu, Mei; Ruan, Xiaosai; Zhang, Chengxian; Lawson, Steve R; Knudsen, David E; Nataro, James P; Robertson, Donald C; Zhang, Weiping

    2011-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Adhesins and enterotoxins, including heat-labile (LT) and heat-stable (STa) toxins, are the key virulence factors. Antigenic adhesin and LT antigens have been used in developing vaccines against ETEC diarrhea. However, STa has not been included because of its poor immunogenicity and potent toxicity. Our recent study showed that porcine-type STa toxoids became immunogenic and elicited neutralizing anti-STa antibodies after being genetically fused to a full-length porcine-type LT toxoid, LT(R₁₉₂G) (W. Zhang et al., Infect. Immun. 78:316-325, 2010). In this study, we mutated human-type LT and STa genes, which are highly homologous to porcine-type toxin genes, for a full-length LT toxoid (LT(R₁₉₂)) and a full-length STa toxoid (STa(P₁₃F)) and genetically fused them to produce LT₁₉₂-STa₁₃ toxoid fusions. Mice immunized with LT₁₉₂-STa₁₃ fusion antigens developed anti-LT and anti-STa IgG (in serum and feces) and IgA antibodies (in feces). Moreover, secretory IgA antibodies from immunized mice were shown to neutralize STa and cholera toxins in T-84 cells. In addition, we fused the STa₁₃ toxoid at the N terminus and C terminus, between the A1 and A2 peptides, and between the A and B subunits of LT₁₉₂ to obtain different fusions in order to explore strategies for enhancing STa immunogenicity. This study demonstrated that human-type LT₁₉₂-STa₁₃ fusions induce neutralizing antitoxin antibodies and provided important information for developing toxoid vaccines against human ETEC diarrhea. PMID:21788385

  11. Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC)

    PubMed Central

    Norton, Elizabeth B.; Branco, Luis M.; Clements, John D.

    2015-01-01

    Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit’s potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines

  12. Evaluating the A-Subunit of the Heat-Labile Toxin (LT) As an Immunogen and a Protective Antigen Against Enterotoxigenic Escherichia coli (ETEC).

    PubMed

    Norton, Elizabeth B; Branco, Luis M; Clements, John D

    2015-01-01

    Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit's potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines

  13. The discovery of cholera - like enterotoxins produced by Escherichia coli causing secretory diarrhoea in humans

    PubMed Central

    Sack, R. Bradley

    2011-01-01

    Non-vibrio cholera has been recognized as a clinical entity for as long as cholera was known to be caused by Vibrio cholerae. Until 1968, the aetiologic agent of this syndrome was not known. Following a series of studies in patients with non-vibrio cholera it was found that these patients had large concentrations of Escherichia coli in the small bowel and stools which produced cholera toxin-like enterotoxins, and had fluid and electrolyte transport abnormalities in the small bowel similar to patients with documented cholera. Furthermore, these patients developed antibodies to the cholera-like enterotoxin. Later studies showed that these strains, when fed to volunteers produced a cholera-like disease and that two enterotoxins were found to be produced by these organisms: a heat-labile enterotoxin (LT) which is nearly identical to cholera toxin, and a heat-stable enterotoxin (ST), a small molecular weight polypeptide. E. coli that produced one or both of these enterotoxins were designated enterotoxigenic E. coli (ETEC). ETEC are now known not only to cause a severe cholera-like illness, but to be the most common bacterial cause of acute diarrhoea in children in the developing world, and to be the most common cause of travellers’ diarrhoea in persons who visit the developing world. PMID:21415491

  14. Enterotoxin production in relation to taxonomic grouping and source of isolation of Aeromonas species.

    PubMed

    Turnbull, P C; Lee, J V; Miliotis, M D; Van de Walle, S; Koornhof, H J; Jeffery, L; Bryant, T N

    1984-02-01

    A total of 19 of 20 (95%) strains of Aeromonas hydrophila biovar hydrophila and 16 of 17 (94%) strains of Aeromonas sobria isolated from a variety of clinical and environmental sources were found to be enterotoxin positive. Only 2 of 18 (11%) A. hydrophila biovar anaerogenes and 2 of 13 (15%) unidentified Aeromonas strains from a similar variety of sources produced enterotoxin. No association was apparent between the source of isolation, in particular diarrheal stools, and enterotoxigenicity; 41% of the isolates from diarrheal stools were enterotoxin negative. A strong correlation was noted between ability to produce enterotoxin and positive results in six characters: lysine decarboxylase and Voges-Proskauer reactions, production of gas from glucose, gluconate oxidation, xanthine hydrolysis, and hemolysis of human erythrocytes. In the majority of cases (35 of 39 strains), enterotoxigenicity was detected using cell-free filtrates of brain heart infusion broth cultures grown at 36 degrees C for 15; however, the other four positive isolates were detected after growth in the same broth at 30 degrees C or in Casamino Acids-yeast extract broth at 30 or 37 degrees C. It is recommended that for enterotoxin tests, strains should be grown in both media at both temperatures. The infant mouse test was found to be a simple and reliable method for detection of the enterotoxin. The toxin proved to be heat labile and not neutralized by cholera antitoxin. PMID:6699147

  15. Staphylococcal Enterotoxins

    PubMed Central

    Pinchuk, Irina V.; Beswick, Ellen J.; Reyes, Victor E.

    2010-01-01

    Staphylococcus aureus (S. aureus) is a Gram positive bacterium that is carried by about one third of the general population and is responsible for common and serious diseases. These diseases include food poisoning and toxic shock syndrome, which are caused by exotoxins produced by S. aureus. Of the more than 20 Staphylococcal enterotoxins, SEA and SEB are the best characterized and are also regarded as superantigens because of their ability to bind to class II MHC molecules on antigen presenting cells and stimulate large populations of T cells that share variable regions on the β chain of the T cell receptor. The result of this massive T cell activation is a cytokine bolus leading to an acute toxic shock. These proteins are highly resistant to denaturation, which allows them to remain intact in contaminated food and trigger disease outbreaks. A recognized problem is the emergence of multi-drug resistant strains of S. aureus and these are a concern in the clinical setting as they are a common cause of antibiotic-associated diarrhea in hospitalized patients. In this review, we provide an overview of the current understanding of these proteins. PMID:22069679

  16. Toxicity and immunogenicity of Enterotoxigenic Escherichia coli heat-labile and heat-stable toxoid fusion 3xSTa(A14Q)-LT(S63K/R192G/L211A) in a murine model.

    PubMed

    Zhang, Chengxian; Knudsen, David E; Liu, Mei; Robertson, Donald C; Zhang, Weiping

    2013-01-01

    Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STa(P13F)) fused at the N- or C-terminus, or inside the A subunit of LT(R192G) elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STa(A14Q)) and a triple-mutant LT toxoid (LT(S63K/R192G/L211A), tmLT), constructed a toxoid fusion (3xSTa(A14Q)-tmLT) that carried 3 copies of STa(A14Q) for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTa(A14Q)-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea. PMID:24146989

  17. Moist-heat sterilization and the chemical stability of heat-labile parenteral solutions.

    PubMed

    Li, L C; Parasrampuria, J; Bommireddi, A; Pec, E; Dudleston, A; Mayoral, J

    1998-01-01

    The impact of moist-heat sterilization (autoclaving) on the chemical stability of parenteral solutions was examined using two heat-labile products, clindamycin phosphate and succinylcholine chloride injections, as examples. A nonisothermal kinetic model was used to predict the extent of product degradation during autoclaving. The predicted results were found to be in close agreement with the experimental data. For the same peak temperature, a greater loss of product was shown by using a cycle with a higher F0. On the other hand, a higher peak-temperature cycle resulted in less product degradation for the same F0 value. The benefit of a high-temperature cycle was further illustrated by the fact that less chemical degradation for both products was produced by a 122 degrees C cycle with an F0 of 11 as compared to that which occurred during a 116.5 degrees C cycle with an F0 of 8. Although clindamycin phosphate was found to be highly unstable during a conventional autoclaving process, predicted data indicate that a UHT (Ultra-High Temperature) process may be used to sterilize this product with acceptable degradation. PMID:15605602

  18. Cholera-like enterotoxins and Regulatory T cells.

    PubMed

    Basset, Christelle; Thiam, Fatou; Martino, Cyrille Di; Holton, John; Clements, John D; Kohli, Evelyne

    2010-07-01

    Cholera toxin (CT) and the heat-labile enterotoxin of E. coli (LT), as well as their non toxic mutants, are potent mucosal adjuvants of immunization eliciting mucosal and systemic responses against unrelated co-administered antigens in experimental models and in humans (non toxic mutants). These enterotoxins are composed of two subunits, the A subunit, responsible for an ADP-ribosyl transferase activity and the B subunit, responsible for cell binding. Paradoxically, whereas the whole toxins have adjuvant properties, the B subunits of CT (CTB) and of LT (LTB) have been shown to induce antigen specific tolerance when administered mucosally with antigens in experimental models as well as, recently, in humans, making them an attractive strategy to prevent or treat autoimmune or allergic disorders. Immunomodulation is a complex process involving many cell types notably antigen presenting cells and regulatory T cells (Tregs). In this review, we focus on Treg cells and cholera-like enterotoxins and their non toxic derivates, with regard to subtype, in vivo/in vitro effects and possible role in the modulation of immune responses to coadministered antigens. PMID:22069660

  19. Typing of heat-stable and heat-labile antigens of Campylobacter jejuni and Campylobacter coli by coagglutination.

    PubMed Central

    Wong, K H; Skelton, S K; Patton, C M; Feeley, J C; Morris, G

    1985-01-01

    A coagglutination system has been devised for typing heat-stable and heat-labile antigens of Campylobacter jejuni and C. coli. The use of protein A-positive Staphylococcus aureus cells carrying Campylobacter sp. serotype antibody and the treatment of Campylobacter sp. cells with DNase in the antigen suspension permitted rapid and specific coagglutination of rough (autoagglutinable) as well as smooth cultures. Cells of S. aureus were sensitized with Campylobacter sp. serotype antisera. Four to five types of sensitized S. aureus cells were pooled. A strain of Campylobacter sp. was first tested with the pools and then typed with the individual reagents of the reactive pool. After the described procedures, 68 serotype strains tested blindly as unknowns were correctly typed according to their heat-stable or heat-labile antigens. The two most commonly used typing schemes which are based separately on the heat-stable or the heat-labile antigens as assayed by passive hemagglutination and slide agglutination, respectively, can be utilized simultaneously in the coagglutination system for strain characterization. The coagglutination system is simple, yields results rapidly, conserves typing reagents, and offers the flexibility of formulating the pools of reagents according to the experimental design or the prevalence of serotypes in a geographic location. It should be a practical system for the typing of Campylobacter spp. in public health or clinical laboratories. PMID:3998098

  20. Parenteral Adjuvant Effects of an Enterotoxigenic Escherichia coli Natural Heat-Labile Toxin Variant

    PubMed Central

    Braga, Catarina J. M.; Rodrigues, Juliana F.; Medina-Armenteros, Yordanka; Farinha-Arcieri, Luís E.; Ventura, Armando M.; Boscardin, Silvia B.; Sbrogio-Almeida, Maria E.; Ferreira, Luís C. S.

    2014-01-01

    Native type I heat-labile toxins (LTs) produced by enterotoxigenic Escherichia coli (ETEC) strains exert strong adjuvant effects on both antibody and T cell responses to soluble and particulate antigens following co-administration via mucosal routes. However, inherent enterotoxicity and neurotoxicity (following intra-nasal delivery) had reduced the interest in the use of these toxins as mucosal adjuvants. LTs can also behave as powerful and safe adjuvants following delivery via parenteral routes, particularly for activation of cytotoxic lymphocytes. In the present study, we evaluated the adjuvant effects of a new natural LT polymorphic form (LT2), after delivery via intradermal (i.d.) and subcutaneous (s.c.) routes, with regard to both antibody and T cell responses. A recombinant HIV-1 p24 protein was employed as a model antigen for determination of antigen-specific immune responses while the reference LT (LT1), produced by the ETEC H10407 strain, and a non-toxigenic LT form (LTK63) were employed as previously characterized LT types. LT-treated mice submitted to a four dose-base immunization regimen elicited similar p24-specific serum IgG responses and CD4+ T cell activation. Nonetheless, mice immunized with LT1 or LT2 induced higher numbers of antigen-specific CD8+ T cells and in vivo cytotoxic responses compared to mice immunized with the non-toxic LT derivative. These effects were correlated with stronger activation of local dendritic cell populations. In addition, mice immunized with LT1 and LT2, but not with LTK63, via s.c. or i.d. routes developed local inflammatory reactions. Altogether, the present results confirmed that the two most prevalent natural polymorphic LT variants (LT1 or LT2) display similar and strong adjuvant effects for subunit vaccines administered via i.d. or s.c. routes. PMID:24432018

  1. Identification of a New Enterotoxin as Enterotoxin C

    PubMed Central

    Bergdoll, Merlin S.; Borja, Concordia R.; Avena, Remedios M.

    1965-01-01

    Bergdoll, Merlin S. (University of Chicago, Chicago, Ill.), Concordia R. Borja, and Remedios M. Avena. Identification of a new enterotoxin as enterotoxin C. J. Bacteriol. 90:1481–1485. 1965.—Identification of a new enterotoxin was accomplished by purification of the enterotoxins produced by staphylococcal strains 137 and 361 and by the preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel-diffusion plates. The enterotoxin has been designated enterotoxin C, and staphylococcal strain 137 (ATCC 19095) was selected as the prototype strain. Images PMID:4954560

  2. Nonimmunoglobulin fraction of human milk inhibits bacterial adhesion (hemagglutination) and enterotoxin binding of Escherichia coli and Vibrio cholerae.

    PubMed Central

    Holmgren, J; Svennerholm, A M; Ahrén, C

    1981-01-01

    Human milk and colostrum samples were divided into an immunoglobulin and a nonimmunoglobulin fraction by immunosorbent chromatography. The ability of these fractions to inhibit bacterial cell adhesion and enterotoxin receptor binding of Vibrio cholerae and various Escherichia coli isolates was then tested by in vitro assays. The strongest effect was generally seen with the nonimmunoglobulin fractions, which were shown to significantly inhibit E. coli cell adhesion (hemagglutination) mediated by CFA/I, CFA/II, or K88 fimbriae (but not type 1 pili) and V. cholerae hemagglutination, as well as the binding of cholera toxin and E. coli heat-labile enterotoxin to GM1 ganglioside. Also, the immunoglobulin fractions had significant inhibitory activity in some of these systems. The results are interpreted to suggest that human milk and colostrum may contain secreted structure analogs of the cell receptors for some bacterial adhesions and enterotoxins; this might contribute to the protective effect of milk against enteric infections. PMID:7021421

  3. Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065

    SciTech Connect

    Zsido, T.J.; Woynarowski, J.M.; Baker, R.M.; Gawron, L.S.; Beerman, T.A. )

    1991-04-16

    CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand break. The authors have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, They observe thermally induced breakage of ({sup 14}C)thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing ({sup 3}H)thymidine-labeled untreated cells with the ({sup 14}C)thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10{sup 6} base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The results of this study demonstrate that CC-1065 does generate heat-labile sites with the cellular DNA of intact cells and suggest that a mechanism of cytotoxic action of CC-1065 involves formation of covalent adducts to DNA.

  4. Escherichia coli K88ac fimbriae expressing heat-labile and heat-stable (STa) toxin epitopes elicit antibodies that neutralize cholera toxin and STa toxin and inhibit adherence of K88ac fimbrial E. coli.

    PubMed

    Zhang, Chengxian; Zhang, Weiping

    2010-12-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, (8)LCSEYRNTQIYTIN(21)) and an STa toxoid epitope ((5)CCELCCNPQCAGCY(18)) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC. PMID:20980482

  5. Characterization of heat-labile toxin-subunit B from Escherichia coli by liquid chromatography-electrospray ionization-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    PubMed

    Sospedra, I; De Simone, C; Soriano, J M; Mañes, J; Ferranti, P; Ritieni, A

    2012-11-01

    The possibilities of characterizing the heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) by liquid chromatography electrospray mass spectrometry (LC/ESI-MS) and matrix-assisted laser desorption with time-of-flight mass spectrometry (MALDI-TOF-MS) were investigated. The B subunit from recombinant E. coli (expression in Pichia pastoris) can be detected by LC/ESI-MS expressed in P. pastoris and the charge envelope signals can be observed; LC/ESI-MS and MALDI-TOF-MS analysis allowed the acquisition of labile toxin subunit B (LTB) molecular weight and preliminary structural characterization of LTB toxin. MALDI-TOF analysis after reduction and alkylation of the protein evidenced the presence of one disulfide bond in the structure of the protein. Confirmatory analysis was carried out by detection of most of the tryptic fragments of the B subunit by MALDI-TOF-MS, obtaining total coverage of the protein sequence. Possible biovariations in the toxin can mostly be determined by sequencing, where an increase of molecular mass in the N-terminal side of the protein was identified. This modification may be due to an O-GlcNAc-1-phosphorylation. PMID:22921353

  6. Escherichia coli K88ac Fimbriae Expressing Heat-Labile and Heat-Stable (STa) Toxin Epitopes Elicit Antibodies That Neutralize Cholera Toxin and STa Toxin and Inhibit Adherence of K88ac Fimbrial E. coli▿

    PubMed Central

    Zhang, Chengxian; Zhang, Weiping

    2010-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Bacterial adhesins and heat-labile (LT) and heat-stable (ST) enterotoxins are the virulence determinants in ETEC diarrhea. It is believed that vaccines inducing anti-adhesin immunity to inhibit bacterial adherence and anti-toxin immunity to eliminate toxin activity would provide broad-spectrum protection against ETEC. In this study, an ETEC fimbrial adhesin was used as a platform to express LT and STa for adhesin-toxin fusion antigens to induce anti-toxin and anti-adhesin immunity. An epitope from the B subunit of LT toxin (LTP1, 8LCSEYRNTQIYTIN21) and an STa toxoid epitope (5CCELCCNPQCAGCY18) were embedded in the FaeG major subunit of E. coli K88ac fimbriae. Constructed K88ac-toxin chimeric fimbriae were harvested and used for rabbit immunization. Immunized rabbits developed anti-K88ac, anti-LT, and anti-STa antibodies. Moreover, induced antibodies not only inhibited adherence of K88ac fimbrial E. coli to porcine small intestinal enterocytes but also neutralized cholera toxin and STa toxin. Data from this study demonstrated that K88ac fimbriae expressing LT and STa epitope antigens elicited neutralizing anti-toxin antibodies and anti-adhesin antibodies and suggested that E. coli fimbriae could serve as a platform for the development of broad-spectrum vaccines against ETEC. PMID:20980482

  7. Genetic Fusions of Heat-Labile (LT) and Heat-Stable (ST) Toxoids of Porcine Enterotoxigenic Escherichia coli Elicit Neutralizing Anti-LT and Anti-STa antibodies ▿

    PubMed Central

    Zhang, Weiping; Zhang, Chengxian; Francis, David H.; Fang, Ying; Knudsen, David; Nataro, James P.; Robertson, Donald C.

    2010-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT192(R→G) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa11(N→K), STa12(P→F), and STa13(A→Q)] and used the full-length pLT192 as an adjuvant to carry the pSTa toxoid for pLT192:pSTa-toxoid fusion antigens. Rabbits immunized with pLT192:pSTa12 or pLT192:pSTa13 fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT192:pSTa13 fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans. PMID:19858307

  8. Sequence Variability in Staphylococcal Enterotoxin Genes seb, sec, and sed

    PubMed Central

    Johler, Sophia; Sihto, Henna-Maria; Macori, Guerrino; Stephan, Roger

    2016-01-01

    Ingestion of staphylococcal enterotoxins preformed by Staphylococcus aureus in food leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. There are five major staphylococcal enterotoxins: SEA, SEB, SEC, SED, and SEE. While variants of these toxins have been described and were linked to specific hosts or levels or enterotoxin production, data on sequence variation is still limited. In this study, we aim to extend the knowledge on promoter and gene variants of the major enterotoxins SEB, SEC, and SED. To this end, we determined seb, sec, and sed promoter and gene sequences of a well-characterized set of enterotoxigenic Staphylococcus aureus strains originating from foodborne outbreaks, human infections, human nasal colonization, rabbits, and cattle. New nucleotide sequence variants were detected for all three enterotoxins and a novel amino acid sequence variant of SED was detected in a strain associated with human nasal colonization. While the seb promoter and gene sequences exhibited a high degree of variability, the sec and sed promoter and gene were more conserved. Interestingly, a truncated variant of sed was detected in all tested sed harboring rabbit strains. The generated data represents a further step towards improved understanding of strain-specific differences in enterotoxin expression and host-specific variation in enterotoxin sequences. PMID:27258311

  9. Multiplex PCR method for detection of three Aeromonas enterotoxin genes.

    PubMed

    Kingombe, Cesar I Bin; D'Aoust, Jean-Yves; Huys, Geert; Hofmann, Lisa; Rao, Mary; Kwan, Judy

    2010-01-01

    A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (act), heat-labile (alt), and heat-stable (ast) enterotoxin genes in Aeromonas spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including act, alt, act/alt, act/alt/ast, act/alt/148-bp amplicon, alt/ast, and alt/148-bp amplicon. The alt gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the act/alt, act/alt/ast, and alt/ast gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the Aeromonas salmonicida A449 genome sequence (GenBank accession number CP000644). This amplicon was named Aeromonas salmonicida subsp. salmonicida hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the act and alt genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The act and alt genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The act/alt gene pattern occurred in 315 isolates (59%), whereas the ast gene was always linked to strains exhibiting the act/alt/ast and alt/ast gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three Aeromonas enterotoxin genes in a single assay. PMID:19933350

  10. Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 or PARP

    SciTech Connect

    Stenerlöw, Bo; Karlsson, Karin H.; Radulescu, Irina; Rydberg, Bjorn; Stenerlow, Bo

    2008-04-29

    Ionizing radiation induces a variety of different DNA lesions: in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have previously shown that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites (HLS) within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of HLS on DSB induction and repair, four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for bi-phasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements the fraction of fast rejoining decreased to less than 50% of the total. However, neither the half-times of the fast (t{sub 1/2} = 7-8 min) or slow (t{sub 1/2} = 2.5 h) DSB rejoining were changed significantly. At t=0 the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSB/cell/Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all tested cells, including M059K cells treated with wortmannin or DNA-PKcs defect M059J cells. Furthermore, cells lacking XRCC1 or Poly(ADP-ribose) polymerase-1 (PARP-1) rejoined both total DSBs and heat-released DSBs similar to normal cells. In summary, the presence of heat-labile sites have a substantial impact on DSB induction yields and DSB rejoining rates measured by pulsed-field gel electrophoresis, and HLS repair is independent of DNA-PKcs, XRCC1 and PARP.

  11. Genetically Detoxified Mutants of Heat-Labile Toxin from Escherichia coli Are Able To Act as Oral Adjuvants

    PubMed Central

    Douce, Gill; Giannelli, Valentina; Pizza, Mariagrazia; Lewis, David; Everest, Paul; Rappuoli, Rino; Dougan, Gordon

    1999-01-01

    Detoxified mutants of the Escherichia coli heat-labile toxin (LT) act as mucosal adjuvants to intranasally presented coadministered antigens. Here, we compare the adjuvant activity of a panel of detoxified derivatives of LT, using both intranasal (i.n.) and oral (p.o.) routes of administration. The mutants used as adjuvants varied in sensitivity to proteases and toxicity. With keyhole limpet hemocyanin (KLH) as the bystander antigen, the immune responses to i.n. immunizations were consistently higher than the equivalent p.o.-delivered proteins. LT-G192, a mutant which demonstrates a 10-fold reduction in toxicity in vitro, demonstrated wild-type adjuvant activity both i.n. and p.o., inducing similar titers of KLH specific antibody in the sera and immunoglobulin A in local mucosal secretions as wild-type LT. In line with previous data, the nontoxic holotoxoid LT-K63 induced intermediate immune responses in both the serum and mucosal secretions which were lower than those achieved with wild-type LT but at least 10-fold higher than those measured when the antigen was administered with LT-B. Although significant levels of local and systemic anti-KLH antibodies were induced following p.o. immunization with LT-K63, cellular proliferative responses to KLH was poor or undetectable. In contrast, LT and LT-G192 induced significant T-cell responses to KLH following p.o. immunization. These proliferating cells secreted both gamma interferon and interleukin-5, suggesting that the type of immune response induced following p.o. coimmunization with LT and purified protein is a mixed Th1/Th2 response. PMID:10456880

  12. Allele variants of enterotoxigenic Escherichia coli heat-labile toxin are globally transmitted and associated with colonization factors.

    PubMed

    Joffré, Enrique; von Mentzer, Astrid; Abd El Ghany, Moataz; Oezguen, Numan; Savidge, Tor; Dougan, Gordon; Svennerholm, Ann-Mari; Sjöling, Åsa

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total-toxin production, secretion, and stability using GM1-enzyme-linked immunosorbent assays (GM1-ELISA) and in silico protein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P < 0.001), which may suggest greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally. PMID:25404692

  13. Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants.

    PubMed

    Douce, G; Giannelli, V; Pizza, M; Lewis, D; Everest, P; Rappuoli, R; Dougan, G

    1999-09-01

    Detoxified mutants of the Escherichia coli heat-labile toxin (LT) act as mucosal adjuvants to intranasally presented coadministered antigens. Here, we compare the adjuvant activity of a panel of detoxified derivatives of LT, using both intranasal (i.n.) and oral (p.o.) routes of administration. The mutants used as adjuvants varied in sensitivity to proteases and toxicity. With keyhole limpet hemocyanin (KLH) as the bystander antigen, the immune responses to i. n. immunizations were consistently higher than the equivalent p.o. -delivered proteins. LT-G192, a mutant which demonstrates a 10-fold reduction in toxicity in vitro, demonstrated wild-type adjuvant activity both i.n. and p.o., inducing similar titers of KLH specific antibody in the sera and immunoglobulin A in local mucosal secretions as wild-type LT. In line with previous data, the nontoxic holotoxoid LT-K63 induced intermediate immune responses in both the serum and mucosal secretions which were lower than those achieved with wild-type LT but at least 10-fold higher than those measured when the antigen was administered with LT-B. Although significant levels of local and systemic anti-KLH antibodies were induced following p.o. immunization with LT-K63, cellular proliferative responses to KLH was poor or undetectable. In contrast, LT and LT-G192 induced significant T-cell responses to KLH following p.o. immunization. These proliferating cells secreted both gamma interferon and interleukin-5, suggesting that the type of immune response induced following p.o. coimmunization with LT and purified protein is a mixed Th1/Th2 response. PMID:10456880

  14. Signaling of Escherichia coli enterotoxin on supramolecular redox bilayer vesicles

    SciTech Connect

    Cheng, Q.; Peng, T.; Stevens, R.C.

    1999-07-21

    Electron transport in supramolecular assemblies containing redox centers has been a subject of great interest. Depending on spatial arrangement of redox moieties in macromolecular structures, transport of electrons may occur via a diffusion mechanism or electron hopping between the neighboring redox sites. While research has largely dealt with 3-D redox polymers, some 2-D systems such as self-assembled and Langmuir-Blodgett monolayers have been exploited as well. The authors describe here a new interfacial architecture that combines the high redox concentration in 3-D polymers and controllable structure and functionality of the 2-D monolayer systems. The new interface utilizes structurally defined redox liposomes engineered with biomolecular recognition capability by incorporating cell surface receptor G{sub M1} into the bilayer membrane. The design allows for direct inspection of the dependency of electron transport on the state and extent of biomolecular recognition that has taken place on the vesicles and, thus, provides a method for direct measurement of E. coli heat-labile enterotoxin binding by electrochemistry.

  15. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011-2012, Russia.

    PubMed

    Kartsev, Nikolay N; Fursova, Nadezhda K; Pachkunov, Dmitry M; Bannov, Vasiliy A; Eruslanov, Boris V; Svetoch, Edward A; Dyatlov, Ivan A

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  16. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011–2012, Russia

    PubMed Central

    Kartsev, Nikolay N.; Fursova, Nadezhda K.; Pachkunov, Dmitry M.; Bannov, Vasiliy A.; Eruslanov, Boris V.; Svetoch, Edward A.; Dyatlov, Ivan A.

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011–2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates—to two drugs, one isolate—to three drugs, two isolates—to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  17. Vitamin B12 production by Citrobacter freundii or Klebsiella pneumoniae during tempeh fermentation and proof of enterotoxin absence by PCR.

    PubMed

    Keuth, S; Bisping, B

    1994-05-01

    The influence of some fermentation parameters on vitamin B12 formation by strains of Citrobacter freundii and Klebsiella pneumoniae isolated from Indonesian tempeh samples during tempeh fermentation was investigated. A decrease in fermentation temperature from 32 to 24 degrees C led to a decrease in vitamin B12 formation. Inoculation of soybeans with different numbers of cells of C. freundii at the beginning of solid-substrate fermentation showed that only the velocity of vitamin formation and not the final amount of vitamin formed depended on the number of cells. The addition of cobalt and 5,6-dimethylbenzimidazole increased the vitamin B12 content of tempeh. Nevertheless, levels of incorporation of the two precursors into the vitamin B12 molecule were very low. Neither C. freundii nor K. pneumoniae possessed the genes encoding the enterotoxins Shiga-like toxin SLT IIA, heat-labile enterotoxin LT Ih, and heat-stable enterotoxin ST Ih, as indicated by PCR. This result supports the suggested use of these two strains to form vitamin B12 during tempeh fermentation in Indonesia. PMID:8017933

  18. Role of Heat-Stable Enterotoxins in the Induction of Early Immune Responses in Piglets after Infection with Enterotoxigenic Escherichia coli

    PubMed Central

    Schauvliege, Stijn; Gasthuys, Frank; van der Meulen, Jan; Dubreuil, J. Daniel; Goddeeris, Bruno M.; Niewold, Theo; Cox, Eric

    2012-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat - labile (LT) enterotoxins are cause of post – weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac+, LT+ STa+ STb+ ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17. PMID:22815904

  19. Repair and enterotoxin synthesis by Staphylococcus aureus after thermal shock.

    PubMed Central

    Hernández, F J; Goyache, J; Orden, J A; Blanco, J L; Doménech, A; Suárez, G; Gómez-Lucía, E

    1993-01-01

    To study repair and enterotoxin synthesis, four staphylococcal strains (FRI-100, FRI-137, FRI-472, and S6) were subjected to sublethal heat treatment, transferred to four liquid repair media (1% powdered skim milk in distilled water, complex medium, M9 minimal salt medium, and saline solution), and then incubated at different temperatures. Powdered skim milk proved to be the most efficient medium for promoting the repair of injured cells, particularly at 37 degrees C. Minimal salt medium also gave good results. Salt tolerance also increased at 4 degrees C, although it did not reach normal values. After 6 h of incubation at 37 degrees C in powdered skim milk, strain FRI-100 synthesized detectable amounts of enterotoxin A. After 10 h of incubation in the same medium at the same temperature, enterotoxins were detected in all of the strains. PMID:8517746

  20. Production of Antiserum for Staphylococcal Enterotoxin

    PubMed Central

    Casman, E. P.; Bennett, R. W.

    1964-01-01

    Immunization of rabbits with approximately 95% pure enterotoxin B and approximately 20 and 50% pure enterotoxin A yielded specific antisera which required no and little absorption, respectively, when used in the slide double-diffusion test. Methods for purifying enterotoxin A and for the production and absorption of antisera are presented. Images FIG. 1 FIG. 2 PMID:14201091

  1. Mutants of Escherichia coli Heat-Labile Toxin Act as Effective Mucosal Adjuvants for Nasal Delivery of an Acellular Pertussis Vaccine: Differential Effects of the Nontoxic AB Complex and Enzyme Activity on Th1 and Th2 Cells

    PubMed Central

    Ryan, Elizabeth J.; McNeela, Edel; Murphy, Geraldine A.; Stewart, Helen; O'hagan, Derek; Pizza, Mariagrazia; Rappuoli, Rino; Mills, Kingston H. G.

    1999-01-01

    Mucosal delivery of vaccines is dependent on the identification of safe and effective adjuvants that can enhance the immunogenicity of protein antigens administered by nasal or oral routes. In this study we demonstrate that two mutants of Escherichia coli heat-labile toxin (LT), LTK63, which lacks ADP-ribosylating activity, and LTR72, which has partial enzyme activity, act as potent mucosal adjuvants for the nasal delivery of an acellular pertussis (Pa) vaccine. Both LTK63 and LTR72 enhanced antigen-specific serum immunoglobulin G (IgG), secretory IgA, and local and systemic T-cell responses. Furthermore, using the murine respiratory challenge model for infection with Bordetella pertussis, we demonstrated that a nasally delivered diphtheria, tetanus, and acellular pertussis (DTPa) combination vaccine formulated with LTK63 as an adjuvant conferred a high level of protection, equivalent to that generated with a parenterally delivered DTPa vaccine formulated with alum. This study also provides significant new information on the roles of the binding and enzyme components of LT in the modulation of Th1 and Th2 responses. LTK63, which lacks enzyme activity, promoted T-cell responses with a mixed Th1–Th2 profile, but LTR72, which retains partial enzyme activity, and the wild-type toxin, especially at low dose, induced a more polarized Th2-type response and very high IgA and IgG antibody titers. Our findings suggest that the nontoxic AB complex has broad adjuvant activity for T-cell responses and that the ADP-ribosyltransferase activity of the A subunit also appears to modulate cytokine production, but its effect on T-cell subtypes, as well as enhancing, may be selectively suppressive. PMID:10569737

  2. Mutants of Escherichia coli heat-labile toxin act as effective mucosal adjuvants for nasal delivery of an acellular pertussis vaccine: differential effects of the nontoxic AB complex and enzyme activity on Th1 and Th2 cells.

    PubMed

    Ryan, E J; McNeela, E; Murphy, G A; Stewart, H; O'hagan, D; Pizza, M; Rappuoli, R; Mills, K H

    1999-12-01

    Mucosal delivery of vaccines is dependent on the identification of safe and effective adjuvants that can enhance the immunogenicity of protein antigens administered by nasal or oral routes. In this study we demonstrate that two mutants of Escherichia coli heat-labile toxin (LT), LTK63, which lacks ADP-ribosylating activity, and LTR72, which has partial enzyme activity, act as potent mucosal adjuvants for the nasal delivery of an acellular pertussis (Pa) vaccine. Both LTK63 and LTR72 enhanced antigen-specific serum immunoglobulin G (IgG), secretory IgA, and local and systemic T-cell responses. Furthermore, using the murine respiratory challenge model for infection with Bordetella pertussis, we demonstrated that a nasally delivered diphtheria, tetanus, and acellular pertussis (DTPa) combination vaccine formulated with LTK63 as an adjuvant conferred a high level of protection, equivalent to that generated with a parenterally delivered DTPa vaccine formulated with alum. This study also provides significant new information on the roles of the binding and enzyme components of LT in the modulation of Th1 and Th2 responses. LTK63, which lacks enzyme activity, promoted T-cell responses with a mixed Th1-Th2 profile, but LTR72, which retains partial enzyme activity, and the wild-type toxin, especially at low dose, induced a more polarized Th2-type response and very high IgA and IgG antibody titers. Our findings suggest that the nontoxic AB complex has broad adjuvant activity for T-cell responses and that the ADP-ribosyltransferase activity of the A subunit also appears to modulate cytokine production, but its effect on T-cell subtypes, as well as enhancing, may be selectively suppressive. PMID:10569737

  3. Avirulent K88 (F4)+ Escherichia coli strains constructed to express modified enterotoxins protect young piglets from challenge with a virulent enterotoxigenic Escherichia coli strain that expresses the same adhesion and enterotoxins.

    PubMed

    Santiago-Mateo, Kristina; Zhao, Mojun; Lin, Jun; Zhang, Weiping; Francis, David H

    2012-10-12

    Virulence of enterotoxigenic Escherichia coli (ETEC) is associated with fimbrial adhesins and enterotoxins such as heat-labile (LT) and/or heat-stable (ST) enterotoxins. Previous studies using a cell culture model suggest that exclusion of ETEC from attachment to epithelial cells requires expression of both an adhesin such as K88 (F4) fimbriae, and LT. To test the ability of non-pathogenic E. coli constructs to exclude virulent ETEC sufficiently to prevent clinical disease, we utilized a piglet ETEC challenge model. Thirty-nine 5-day-old piglets were inoculated with a placebo (control), or with either of the three K88(+)E. coli strains isogenic with regard to modified LT expression: 8017 (pBR322 plasmid vector control), non-toxigenic mutant 8221 (LT(R192G)) in pBR322, or 8488, with the LT gene fused to the STb gene in pBR322 (LT(R192G)-STb). Piglets were challenged with virulent ETEC Strain 3030-2 (K88(+)/LT/STb) 24h post-inoculation. K88ac receptor-positive piglets in the control group developed diarrhea and became dehydrated 12-24h post-challenge. Piglets inoculated with 8221 or 8488 did not exhibit clinical signs of ETEC disease; most piglets inoculated with 8017 showed diarrhea. Control pigs exhibited significant weight loss, increased blood total protein, and higher numbers of colony-forming units of 3030-2 E. coli in washed ileum and jejunum than treated pigs. This study shows for the first time that pre-inoculation with an avirulent strain expressing adhesive fimbriae and a non-toxic form of LT provides significant short term protection from challenge with a virulent ETEC strain that expresses the same fimbrial adhesion and enterotoxin. PMID:22541162

  4. Cooperative role of antibodies against heat-labile toxin and the EtpA Adhesin in preventing toxin delivery and intestinal colonization by enterotoxigenic Escherichia coli.

    PubMed

    Roy, Koushik; Hamilton, David J; Fleckenstein, James M

    2012-10-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonization in vivo and toxin delivery to epithelial cells in vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development. PMID:22875600

  5. Influence of heat-labile serum components in the presence of OmpA on the outer membrane of Salmonella gallinarum.

    PubMed

    Vega-Manriquez, X; Huerta-Ascencio, L; Martínez-Gómez, D; López-Vidal, Y; Verdugo-Rodríguez, A

    2016-03-01

    Salmonella gallinarum is the causative agent of fowl typhoid. Being a Gram-negative bacteria, its outer membrane proteins (OMP) can be regulated by different microenvironments. S. gallinarum was cultured under the following conditions: nutrient broth (NB), NB supplemented with serum from specific pathogen-free birds (NBS) and NB with serum incubated at 56 °C prior to incubation with the bacteria (NBSD); OMP were subsequently extracted. Several changes were observed in the apparent expression of OMP, mainly a decrease in an OMP with a size of 30 kDa, approximately, under the NBS condition. In contrast, the same event was not observed in NB and NBSD when using one- and two-dimensional polyacrylamide gels (SDS-PAGE). Using the OMP with a size of 30 kDa, approximately, as antigen in indirect ELISA, we were able to differentiate serum from healthy and vaccinated birds, as well as birds infected with S. gallinarum and S. enteritidis. The amino-terminal of this protein was sequenced, showing 100 % identity with OmpA of S. typhimurium. Subsequently, we designed primers to amplify the gene by PCR. The partial sequence of the amplified gene showed 100 % identity with OmpA of S. gallinarum. (1) Heat-labile serum components influence the presence of OmpA in the OM of S. gallinarum; (2) by the way of ELISA, OmpA allows to specifically differentiate healthy from diseased birds. PMID:26597854

  6. Intranasal immunization with live recombinant Lactococcus lactis combined with heat-labile toxin B subunit protects chickens from highly pathogenic avian influenza H5N1 virus.

    PubMed

    Lei, Han; Peng, Xiaojue; Shu, Handing; Zhao, Daxian

    2015-01-01

    Development of safe and effective vaccines to prevent highly pathogenic avian influenza H5N1 virus infection is a challenging goal. Lactococcus lactis (L. lactis) is an ideal delivery vector for vaccine development, and it has been shown previously that oral immunization of encapsulated secretory L. lactis-hemagglutinin (HA) could provide complete protection against homologous H5N1 virus challenge in the mice model. While intranasal immunization is an appealing approach, it is now reported that secretory L. lactis-HA combined with mucosal adjuvant heat-labile toxin B subunit (LTB) could provide protective immunity in the chicken model. As compared to intranasal immunization with L. lactis-HA alone, L. lactis-HA combined with LTB (L. lactis-HA + LTB) could elicit robust neutralizing antibody responses and mucosal IgA responses, as well as strong cellular immune responses in the vaccinated chickens. Importantly, intranasal immunization with L. lactis-HA + LTB could provide 100% protection against H5N1 virus challenge. Taken together, these results suggest that intranasal immunization with L. lactis-HA + LTB can be considered as an effective approach for preventing and controlling infection of H5N1 virus in poultry during an avian influenza A/H5N1 pandemic. PMID:24861477

  7. The LT1 and LT2 variants of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) are associated with major ETEC lineages.

    PubMed

    Joffré, Enrique; Sjöling, Åsa

    2016-01-01

    The heat-labile toxin (LT) is one of the major virulence factors of enterotoxigenic Escherichia coli (ETEC). We recently described that 20 polymorphic LT variants are present in ETEC strains isolated globally. Two of the variants, LT1 and LT2, are particularly common and we found that they were associated with clonal ETEC lineages that express the colonization factors (CFs), CFA/I, CS1+CS3, CS2+CS3, and CS5+CS6. ETEC expressing these CFs are frequently found among ETEC strains isolated from cases with diarrhea. ETEC expressing the colonization factors CS1+CS3, and CS2+CS3 are found in 2 discrete clonal lineages and express the LT1 variant and heat stable toxin (STh). Although they clearly are virulent they neither produce, nor secrete, high amounts of LT toxin. On the other hand ETEC strains expressing LT, STh, CFA/I and LT, STh, CS5+CS6, carry the LT2 variant and produce and secrete significantly more LT toxin. Despite differences in toxin production, LT1 and LT2 are found in ETEC lineages that have managed to spread globally confirming that these variants are important for ETEC virulence. PMID:26939855

  8. The LT1 and LT2 variants of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) are associated with major ETEC lineages

    PubMed Central

    Joffré, Enrique; Sjöling, Åsa

    2016-01-01

    ABSTRACT The heat-labile toxin (LT) is one of the major virulence factors of enterotoxigenic Escherichia coli (ETEC). We recently described that 20 polymorphic LT variants are present in ETEC strains isolated globally. Two of the variants, LT1 and LT2, are particularly common and we found that they were associated with clonal ETEC lineages that express the colonization factors (CFs), CFA/I, CS1+CS3, CS2+CS3, and CS5+CS6. ETEC expressing these CFs are frequently found among ETEC strains isolated from cases with diarrhea. ETEC expressing the colonization factors CS1+CS3, and CS2+CS3 are found in 2 discrete clonal lineages and express the LT1 variant and heat stable toxin (STh). Although they clearly are virulent they neither produce, nor secrete, high amounts of LT toxin. On the other hand ETEC strains expressing LT, STh, CFA/I and LT, STh, CS5+CS6, carry the LT2 variant and produce and secrete significantly more LT toxin. Despite differences in toxin production, LT1 and LT2 are found in ETEC lineages that have managed to spread globally confirming that these variants are important for ETEC virulence. PMID:26939855

  9. E. coli heat-stable enterotoxin and guanylyl cyclase C: new functions and unsuspected actions.

    PubMed Central

    Giannella, Ralph A.; Mann, Elizabeth A.

    2003-01-01

    Some E. coli cause diarrhea by elaborating heat-labile and heat-stable (ST) enterotoxins which stimulate intestinal secretion. E. coli ST's are small peptides which bind to intestinal luminal epithelial cell receptors. The ST receptor, one of a family of receptor-cyclases called guanylyl cyclase C (GC-C), is a membrane spanning protein containing an extracellular binding domain and intracellular protein kinase and catalytic domains. The intestine synthesizes and secretes homologous peptides, guanylin and uroguanylin. The kidney also synthesizes uroguanylin. ST, guanylin or uroguanylin binding to GC-C results in increased cGMP, phosphorylation of the CFTR Cl- channel and secretion. Proguanylin and prouroguanylin circulate in blood and bind to receptors in intestine, kidney, liver, brain etc. In the kidney, they stimulate the excretion of Na+ and K+. Study of GC-C "knock-out" mice reveal that GC-C is important to intestinal salt and water secretion, duodenal bicarbonate secretion, recovery from CCl4-induced liver injury, and to intestinal polyp formation in Min mice lacking GC-C. PMID:12813912

  10. Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells

    PubMed Central

    Beltrán, Ana R.; Carraro-Lacroix, Luciene R.; Bezerra, Camila N. A.; Cornejo, Marcelo; Norambuena, Katrina; Toledo, Fernando; Araos, Joaquín; Pardo, Fabián; Leiva, Andrea; Sanhueza, Carlos; Malnic, Gerhard; Sobrevia, Luis; Ramírez, Marco A.

    2015-01-01

    The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa–decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting

  11. Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.

    PubMed

    Beltrán, Ana R; Carraro-Lacroix, Luciene R; Bezerra, Camila N A; Cornejo, Marcelo; Norambuena, Katrina; Toledo, Fernando; Araos, Joaquín; Pardo, Fabián; Leiva, Andrea; Sanhueza, Carlos; Malnic, Gerhard; Sobrevia, Luis; Ramírez, Marco A

    2015-01-01

    The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human

  12. A live attenuated Salmonella Enteritidis secreting detoxified heat labile toxin enhances mucosal immunity and confers protection against wild-type challenge in chickens.

    PubMed

    Lalsiamthara, Jonathan; Kamble, Nitin Machindra; Lee, John Hwa

    2016-01-01

    A live attenuated Salmonella Enteritidis (SE) capable of constitutively secreting detoxified double mutant Escherichia coli heat labile toxin (dmLT) was developed. The biologically adjuvanted strain was generated via transformation of a highly immunogenic SE JOL1087 with a plasmid encoding dmLT gene cassette; the resultant strain was designated JOL1641. A balanced-lethal host-vector system stably maintained the plasmid via auxotrophic host complementation with a plasmid encoded aspartate semialdehyde dehydrogenase (asd) gene. Characterization by western blot assay revealed the dmLT subunit proteins in culture supernatants of JOL1641. For the investigation of adjuvanticity and protective efficacy, chickens were immunized via oral or intramuscular routes with PBS, JOL1087 and JOL1641. Birds immunized with JOL1641 showed significant (P ≤ 0.05) increases in intestinal SIgA production at the 1(st) and 2(nd) weeks post-immunization via oral and intramuscular routes, respectively. Interestingly, while both strains showed significant splenic protection via intramuscular immunization, JOL1641 outperformed JOL1087 upon oral immunization. Oral immunization of birds with JOL1641 significantly reduced splenic bacterial counts. The reduction in bacterial counts may be correlated with an adjuvant effect of dmLT that increases SIgA secretion in the intestines of immunized birds. The inclusion of detoxified dmLT in the strain did not cause adverse reactions to birds, nor did it extend the period of bacterial fecal shedding. In conclusion, we report here that dmLT could be biologically incorporated in the secretion system of a live attenuated Salmonella-based vaccine, and that this construction is safe and could enhance mucosal immunity, and protect immunized birds against wild-type challenge. PMID:27262338

  13. Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1.

    PubMed

    Liu, Lin; Ma, Yongping; Zhou, Huicong; Wu, Mingjun

    2016-01-01

    The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system. PMID:27618897

  14. The staphylococcal enterotoxin (SE) family

    PubMed Central

    Krakauer, Teresa; Stiles, Bradley G

    2013-01-01

    Staphylococcus aureus plays an important role in numerous human cases of food poisoning, soft tissue, and bone infections, as well as potentially lethal toxic shock. This common bacterium synthesizes various virulence factors that include staphylococcal enterotoxins (SEs). These protein toxins bind directly to major histocompatibility complex class II on antigen-presenting cells and specific Vβ regions of T-cell receptors, resulting in potentially life-threatening stimulation of the immune system. Picomolar concentrations of SEs ultimately elicit proinflammatory cytokines that can induce fever, hypotension, multi-organ failure, and lethal shock. Various in vitro and in vivo models have provided important tools for studying the biological effects of, as well as potential vaccines/therapeutics against, the SEs. This review succinctly presents known physical and biological properties of the SEs, including various intervention strategies. In particular, SEB will often be portrayed as per biodefense concerns dating back to the 1960s. PMID:23959032

  15. Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (STa) and heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) retain broad anti-CFA and antitoxin antigenicity.

    PubMed

    Ruan, Xiaosai; Sack, David A; Zhang, Weiping

    2015-01-01

    Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent

  16. In vitro inhibition of ETEC K88 adhesion by pea hulls and of LT enterotoxin binding by faba bean hulls.

    PubMed

    Becker, P M; van der Meulen, J; Jansman, A J M; van Wikselaar, P G

    2012-12-01

    Enterotoxigenic Escherichia coli (ETEC) expressing K88 (F4) adhesins are associated with post-weaning diarrhoea in piglets. Different grain fractions from pea (Pisum sativum) and faba bean (Vicia faba) were tested in vitro for their capacity to counteract aetiological factors, which contribute to the development of diarrhoea. In detail, adhesion of E. coli O149:K91:K88ac (ETEC K88ac) to grain legume products, intended to impair the colonization of the host, was studied as well as interference with receptor binding of the pathogen's heat-labile enterotoxin LT, intended to reduce toxin-inflicted gut cell damage. When comparing different pea and faba bean products tested for their binding capacity of ETEC K88ac, especially pea hulls, but also whole pea meal, starch-enriched and protein-enriched pea meal, and digestion-resistant pea hull and meal fractions showed a higher binding of ETEC K88ac than faba bean products. In contrast to the ETEC K88ac adhesion results, bean hulls proved more effective than pea hulls in preventing GM1 receptor binding of LT. Previous small intestinal segment perfusion experiments we performed with ETEC K88ac-challenged piglets indicated that both pea and bean hulls have the potential for successful application in diarrhoea prophylaxis and treatment, which is in agreement with and refined by our detection of their different modes of functioning. PMID:21929729

  17. MHC class I-restricted cytotoxic lymphocyte responses induced by enterotoxin-based mucosal adjuvants.

    PubMed

    Simmons, C P; Mastroeni, P; Fowler, R; Ghaem-maghami, M; Lycke, N; Pizza, M; Rappuoli, R; Dougan, G

    1999-12-15

    The ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257-264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257-264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257-264. Both 51Cr release assays and IFN-gamma enzyme-linked immunospot assays indicated that IFN-gamma-/- and IL-12 p40-/- gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-gamma and probably by a mechanism unrelated to cross-priming. PMID:10586042

  18. Bacterial Toxins-Staphylococcal Enterotoxin B.

    PubMed

    Fries, Bettina C; Varshney, Avanish K

    2013-12-01

    Staphylococcal enterotoxin B is one of the most potent bacterial superantigens that exerts profound toxic effects upon the immune system, leading to stimulation of cytokine release and inflammation. It is associated with food poisoning, nonmenstrual toxic shock, atopic dermatitis, asthma, and nasal polyps in humans. Currently, there is no treatment or vaccine available. Passive immunotherapy using monoclonal antibodies made in several different species has shown significant inhibition in in vitro studies and reduction in staphylococcal enterotoxin B-induced lethal shock in in vivo studies. This should encourage future endeavors to develop these antibodies as therapeutic reagents. PMID:26184960

  19. Enterotoxin formation by different toxigenic types of Clostridium perfringens.

    PubMed Central

    Skjelkvålé, R; Duncan, C L

    1975-01-01

    Sixty-nine strains of Clostridium perfringens of different toxigenic types were investigated for enterotoxin production. Enterotoxin was definitively detected only in strains of types A and C. This is the first report where enterotoxin production has been demonstrated in a toxigenic type other than type A. The exterotoxin-positive type C strains were isolated from cases of enteritis necroticans ("pig bel+) in New Guinea. The major enterotoxin from type C showed a reaction of complete identity with enterotoxin from type A in immunodiffusion using anti-enterotoxin serum prepared against the latter; it induced erythema when injected intradermally into depilated guinea pigs and caused fluid accumulation in the rabbit ileal loop. The results indicate that the major enterotoxin from type C was serologically and biologically similar to enterotoxin from type A. In some C was serologically and biologically similar to enterotoxin from type A. In some type C strains, an enterotoxin was detected that showed a reaction of partial serological identity. Spore coat proteins were extracted from 14-strains by alkaline dithiothreitol, and the extracts were assayed for enterotoxin-like spore protein. Enterotoxin could be extracted from type A and type C spores, and all positive strains showed a reaction of complete identity in immunodiffusion with enterotoxin obtained from cell extracts of type A. Disc immunoelectrophoresis demonstrated that two distinct components that reacted serologically with anti-enterotoxin serum were present in both the cell extract and in extracted spore protein from one type C strain. These distinct components differed in molecular weight. Images PMID:163799

  20. Cholera toxin, and the related nontoxic adjuvants mmCT and dmLT, promote human Th17 responses via cyclic AMP-protein kinase A and inflammasome-dependent IL-1 signaling.

    PubMed

    Larena, Maximilian; Holmgren, Jan; Lebens, Michael; Terrinoni, Manuela; Lundgren, Anna

    2015-04-15

    We have examined the molecular pathways involved in the adjuvant action of cholera toxin (CT) and two novel nontoxic molecules, multiple-mutated CT (mmCT) and double-mutant heat-labile toxin (dmLT) on human T cell responses. Human PBMCs or isolated monocytes were stimulated in vitro with CT, mmCT, or dmLT plus a polyclonal stimulus (staphylococcal enterotoxin B) or specific bacterial Ags, and effects on expression of cytokines and signaling molecules were determined. CT, mmCT, and dmLT strongly enhanced IL-17A and to a lesser extent IL-13 responses, but had little effect on IFN-γ production or cell proliferation. Intracellular cytokine staining revealed that the enhanced IL-17A production was largely confined to CD4(+) T cells and coculture experiments showed that the IL-17A promotion was effectively induced by adjuvant-treated monocytes. Relative to CT, mmCT and dmLT induced at least 100-fold lower levels of cAMP, yet this cAMP was enough and essential for the promotion of Th17 responses. Thus, inhibition of cAMP-dependent protein kinase A was abolished, and stimulation with a cAMP analog mimicked the adjuvant effect. Furthermore, CT, mmCT, and dmLT induced IL-1β production and caspase-1 activation in monocytes, which was associated with increased expression of key proinflammatory and inflammasome-related genes, including NLRP1, NLRP3, and NLRC4. Inflammasome inhibition with a specific caspase-1 inhibitor, or blocking of IL-1 signaling by IL-1 receptor antagonist, abrogated the Th17-promoting effect. We conclude that CT, mmCT, and dmLT promote human Th17 responses via cAMP-dependent protein kinase A and caspase-1/inflammasome-dependent IL-1 signaling. PMID:25786687

  1. Ability of SPI2 mutant of S. typhi to effectively induce antibody responses to the mucosal antigen enterotoxigenic E. coli heat labile toxin B subunit after oral delivery to humans

    PubMed Central

    Khan, S.; Chatfield, S.; Stratford, R.; Bedwell, J.; Bentley, M.; Sulsh, S.; Giemza, R.; Smith, S.; Bongard, E.; Cosgrove, C.A.; Johnson, J.; Dougan, G.; Griffin, G.E.; Makin, J.; Lewis, D.J.M.

    2007-01-01

    We have evaluated an oral vaccine based on an Salmonella enteric serovar typhi (S. typhi) Ty2 derivative TSB7 harboring deletion mutations in ssaV (SPI-2) and aroC together with a chromosomally integrated copy of eltB encoding the B subunit of enterotoxigenic Escherichia coli heat labile toxin (LT-B) in volunteers. Two oral doses of 108 or 109 CFU were administered to two groups of volunteers and both doses were well tolerated, with no vaccinemia, and only transient stool shedding. Immune responses to LT-B and S. typhi lipopolysaccharide were demonstrated in 67 and 97% of subjects, respectively, without evidence of anti-carrier immunity preventing boosting of LT-B responses in many cases. Further development of this salmonella-based (spi-VEC) system for oral delivery of heterologous antigens appears warranted. PMID:17412462

  2. Natural Indoles, Indole-3-Carbinol (I3C) and 3,3’-Diindolylmethane (DIM), Attenuate Staphylococcal Enterotoxin B-Mediated Liver Injury by Downregulating miR-31 Expression and Promoting Caspase-2-Mediated Apoptosis

    PubMed Central

    Busbee, Philip B.; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells. PMID:25706292

  3. Natural indoles, indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM), attenuate staphylococcal enterotoxin B-mediated liver injury by downregulating miR-31 expression and promoting caspase-2-mediated apoptosis.

    PubMed

    Busbee, Philip B; Nagarkatti, Mitzi; Nagarkatti, Prakash S

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40 mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells. PMID:25706292

  4. [Biological models in studying of staphylococci enterotoxin toxicity].

    PubMed

    Shepeliakovskaia, A O; Semushina, S G; Murashev, A N; Adameĭko, I I; Brovko, F A

    2014-01-01

    Enterotoxins--superantigens--are the main toxic agents of staphylococci. Currently, an important role of these proteins is estabished in both toxicity itself--toxic shock and in the development of autoimmune diseases. Enterotoxin studies are carried out in several directions including the search for novel molecular targets, studies in cell tests and establishment of toxicity in animal models. Methods of studying toxicity in animal models: monkeys, mice and rabbits are examined in the review. Methods of animal priming to achieve lethality, features of using various lines of mice during analysis of individual enterotoxins are discussed. Methods of studying enterotoxin-neutralizing compounds in animal models are discussed. PMID:25536781

  5. Antigenicity and immunogenicity of fused B-subunit of heat labile toxin of Escherichia coli and colonization factor antigen I polyepitopes.

    PubMed

    Savar, Nastaran Sadat; Dashti, Amir; Darzi Eslam, Elham; Jahanian-Najafabadi, Ali; Jafari, Anis

    2014-11-01

    Linear B-cell epitopes ((93)AKEFEAAAL(101) and (66)PQLTDVLN(73)) of CfaB were genetically fused to ltb-(gly)5-cfaB(1-25). Sera of rabbits immunized with fusion proteins reacted strongly with solid-phase bound ETEC bacteria bearing CFA/I fimbriae. Sera failed to agglutinate or inhibit hemagglutination promoted by CFA/I-positive strain which may be due to solvent inaccessibility of epitope residues on intact fimbriae. PMID:25108290

  6. The enterotoxin of Bacteroides fragilis is a metalloprotease.

    PubMed Central

    Moncrief, J S; Obiso, R; Barroso, L A; Kling, J J; Wright, R L; Van Tassell, R L; Lyerly, D M; Wilkins, T D

    1995-01-01

    During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M(r), approximately 20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37 degrees C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional

  7. Genetic Fusions of Heat-Labile Toxoid (LT) and Heat-Stable Toxin b (STb) of Porcine Enterotoxigenic Escherichia coli Elicit Protective Anti-LT and Anti-STb Antibodies ▿

    PubMed Central

    Zhang, Weiping; Francis, David H.

    2010-01-01

    Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea causes a substantial economic loss to swine producers worldwide. The majority of ETEC strains causing porcine diarrhea, especially postweaning diarrhea (PWD), produce heat-labile toxin (LT) and heat-stable toxin b (STb). LT is commonly used in vaccine development, but STb has not been included because of its poor immunogenicity. As a virulence factor in porcine diarrhea, STb needs to be included as an antigen for development of broad-spectrum vaccines. In this study, we used an LT toxoid (LTR192G [hereafter, LT192]) derived from porcine ETEC to carry a mature STb peptide for LT192-STb fusions to enhance STb immunogenicity for potential vaccine application. Anti-LT and anti-STb antibodies were detected in immunized rabbits and pigs. In addition, when challenged with an STb-positive ETEC strain, all 10 suckling piglets borne by immunized gilts remained healthy, whereas 7 out 9 piglets borne by unimmunized gilts developed moderate diarrhea. This study indicates that the LT192-STb fusion enhanced anti-STb immunogenicity and suggests the LT192-STb fusion antigen can be used in future vaccine development against porcine ETEC diarrhea. PMID:20505006

  8. Modulation of innate and acquired immune responses by Escherichia coli heat-labile toxin: distinct pro- and anti-inflammatory effects of the nontoxic AB complex and the enzyme activity.

    PubMed

    Ryan, E J; McNeela, E; Pizza, M; Rappuoli, R; O'Neill, L; Mills, K H

    2000-11-15

    We have examined the roles of enzyme activity and the nontoxic AB complex of heat-labile toxin (LT) from Escherichia coli on its adjuvant and immunomodulatory properties. LTK63, an LT mutant that is completely devoid of enzyme activity, enhanced Th1 responses to coinjected Ags at low adjuvant dose. In contrast, LTR72, a partially detoxified mutant, enhanced Th2 responses and when administered intranasally to mice before infection with Bordetella pertussis suppressed Th1 responses and delayed bacterial clearance from the lungs. LTR72 or wild-type LT inhibited Ag-induced IFN-gamma production by Th1 cells, and LT enhanced IL-5 production by Th2 cells in vitro. Each of the toxins enhanced B7-1 expression on macrophages, but enhancement of B7-2 expression was dependent on enzyme activity. We also observed distinct effects of the nontoxic AB complex and enzyme activity on inflammatory cytokine production. LT and LTR72 suppressed LPS and IFN-gamma induced TNF-alpha and IL-12 production, but enhanced IL-10 secretion by macrophages in vitro and suppressed IL-12 production in vivo in a murine model of LPS-induced shock. In contrast, LTK63 augmented the production of IL-12 and TNF-alpha. Furthermore, LTK63 enhanced NF-kappaB translocation, whereas low doses of LTR72 or LT failed to activate NF-kappaB, but stimulated cAMP production. Thus, E. coli LT appears to be capable of suppressing Th1 responses and enhancing Th2 responses through the modulatory effects of enzyme activity on NF-kappaB activation and IL-12 production. In contrast, the nontoxic AB complex can stimulate acquired immune responses by activating components of the innate immune system. PMID:11067933

  9. Crystal structure of the superantigen staphylococcal enterotoxin type A.

    PubMed Central

    Schad, E M; Zaitseva, I; Zaitsev, V N; Dohlsten, M; Kalland, T; Schlievert, P M; Ohlendorf, D H; Svensson, L A

    1995-01-01

    Staphylococcal enterotoxins are prototype superantigens characterized by their ability to bind to major histocompatibility complex (MHC) class II molecules and subsequently activate a large fraction of T-lymphocytes. The crystal structure of staphylococcal enterotoxin type A (SEA), a 27 kDa monomeric protein, was determined to 1.9 A resolution with an R-factor of 19.9% by multiple isomorphous replacement. SEA is a two domain protein composed of a beta-barrel and a beta-grasp motif demonstrating the same general structure as staphylococcal enterotoxins SEB and TSST-1. Unique for SEA, however, is a Zn2+ coordination site involved in MHC class II binding. Four amino acids including Ser1, His187, His225 and Asp227 were found to be involved in direct coordination of the metal ion. SEA is the first Zn2+ binding enterotoxin that has been structurally determined. Images PMID:7628431

  10. Production of staphylococcal enterotoxin A in cream-filled cake.

    PubMed

    Anunciaçao, L L; Linardi, W R; do Carmo, L S; Bergdoll, M S

    1995-07-01

    Cakes were baked with normal ingredients and filled with cream, inoculated with different size enterotoxigenic-staphylococcal inocula. Samples of the cakes were incubated at room temperature and put in the refrigerator. Samples of cake and filling were taken at different times and analyzed for staphylococcal count and presence of enterotoxin. The smaller the inoculum, the longer the time required for sufficient growth (10(6)) to occur for production of detectable enterotoxin. Enterotoxin added to the cake dough before baking (210 degrees C, 45 min) did not survive the baking. The presence of enterotoxin in the contaminated cream filling indicated this as the cause of staphylococcal food poisoning from cream-filled cakes. Refrigeration of the cakes prevented the growth of the staphylococci. PMID:7577363

  11. Enterotoxin gene profiles among Staphylococcus aureus isolated from raw milk

    PubMed Central

    Nazari, R; Godarzi, H; Rahimi Baghi, F; Moeinrad, M

    2014-01-01

    Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the nine Staphylococcal enterotoxins (SEs) and enterotoxin gene profiles in S. aureus isolates derived from raw bovine milk. A total of 52 S. aureus isolates were obtained from 246 milk samples of 246 dairy cows from eight different farms in Qom, Iran. On the basis of cultural and biochemical properties as well as by amplification of the 23S rRNA specific to S. aureus, all isolates could be identified as S. aureus. Of the 52 isolates studied, 80.7% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene encoding for enterotoxin A (Sea) was the most frequent (16 isolates, 30.7%), followed by Seb (14 isolates, 26.9%) and Sed (8 isolates, 15.37%). Among the genes encoding the other enterotoxins, Seg and Seh were the most frequently observed (8 isolates each, 15.38%), followed by Sej (6 isolates, 11.5%) and Sei (1 isolates, 3.84%). With the recent identification of new SEs, the frequency of enterotoxigenic strains has increased, suggesting that the pathogenic potential of Staphylococci may be higher than previously thought. These results of enterotoxin genes positivity of milk-derived Staphylococci constitute a potential risk for consumers’ health. PMID:27175141

  12. Detection of Bacteroides fragilis Enterotoxin Gene by PCR

    PubMed Central

    Shetab, Razeq; Cohen, Stuart H.; Prindiville, Thomas; Tang, Yajarayma J.; Cantrell, Mary; Rahmani, Darush; Silva, Joseph

    1998-01-01

    Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases. PMID:9620408

  13. Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp).

    PubMed

    Tobias, Joshua; Von Mentzer, Astrid; Loayza Frykberg, Patricia; Aslett, Martin; Page, Andrew J; Sjöling, Åsa; Svennerholm, Ann-Mari

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located. PMID:27054573

  14. Stability of the Encoding Plasmids and Surface Expression of CS6 Differs in Enterotoxigenic Escherichia coli (ETEC) Encoding Different Heat-Stable (ST) Enterotoxins (STh and STp)

    PubMed Central

    Tobias, Joshua; Von Mentzer, Astrid; Loayza Frykberg, Patricia; Aslett, Martin; Page, Andrew J.; Sjöling, Åsa; Svennerholm, Ann-Mari

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located. PMID:27054573

  15. Staphylococcus aureus isolates from Irish domestic refrigerators possess novel enterotoxin and enterotoxin-like genes and are clonal in nature.

    PubMed

    Smyth, Davida S; Kennedy, Jean; Twohig, Jane; Miajlović, Helen; Bolton, Declan; Smyth, Cyril J

    2006-03-01

    A previous study carried out by the National Food Centre in Dublin on bacterial contamination of Irish domestic refrigeration systems revealed that 41% were contaminated with Staphylococcus aureus. One hundred fifty-seven S. aureus isolates were screened by multiplex PCR analysis for the presence of 15 staphylococcal enterotoxin and enterotoxin-like genes (sea-see, seg-sei, selj-selo, and selq) and the toxic shock toxin superantigen tst gene. Of the refrigerator isolates, 64.3% possessed more than one staphylococcal enterotoxin or staphylococcal enterotoxin-like gene. All bar one of the 101 staphylococcal enterotoxin or staphylococcal enterotoxin-like gene-positive strains possessed the egc locus bearing the seg, sei, selm, seln, and selo genes. Twelve random amplified polymorphic DNA (RAPD) types accounted for 119 (75.8%) of the strains, two of these types accounting for 25 (RAPD type 1, 15.9%) and 52 (RAPD type 5, 33.1%), respectively. All of the RAPD type 5 isolates possessed the egc gene cluster only. The RAPD type 5 amplicon profile was identical to that of S. aureus isolates associated with osteomyelitis in broiler chickens in Northern Ireland that also possessed the egc locus only. However, the RAPD type 5 domestic refrigerator and chicken isolates differed in penicillin G sensitivity, production of Protein A and staphylokinase, and crystal violet agar growth type. These findings highlight that the average Irish household refrigerator harbors potential enterotoxin-producing S. aureus that may or may not be of animal origin and, accordingly, is a potential reservoir for staphylococcal food poisoning. PMID:16541679

  16. Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese▿

    PubMed Central

    Duquenne, Manon; Fleurot, Isabelle; Aigle, Marina; Darrigo, Claire; Borezée-Durant, Elise; Derzelle, Sylviane; Bouix, Marielle; Deperrois-Lafarge, Véronique; Delacroix-Buchet, Agnès

    2010-01-01

    Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production. PMID:20061456

  17. Chromosomal locus for staphylococcal enterotoxin B.

    PubMed Central

    Shafer, W M; Iandolo, J J

    1978-01-01

    The genetic locus of staphylococcal enterotoxin B (SEB) was investigated in the Staphylococcus aureus food-poisoning isolates, strains S6 and 277. Direct neutral sucrose gradient centrifugation analysis of sodium dodecyl sulfate-sodium chloride-mediated cleared lysates demonstrated that strain S6 contained a single 37S plasmid. Transductional analysis revealed that the 37S plasmid in S6 encoded for cadmium resistance (Cad) but not SEB. Additionally, elimination of cadmium resistance in S6 provided a plasmid-negative derivative that produced SEB at the same level as the parent. Examination of strain 277 showed two plasmids, a 37S species encoding for penicillin resistance (Penr) and a 21S species containing the gene(s) responsible for tetracycline resistance (Tetr). Elimination of the 37S, penr plasmid in 277 had no effect on SEB production, whereas introduction of the 21S tetr plasmid via transformation into strain 8325 (SEB--) did not confer enterotoxigenesis upon the transformants. The data obtained in this investigation suggest that the SEB gene(s) in these food-poisoning isolates of S. aureus is chromosomal. Images PMID:669796

  18. Inverse relationship between heat stable enterotoxin-b induced fluid accumulation and adherence of F4ac-positive enterotoxigenic Escherichia coli in ligated jejunal loops of F4ab/ac fimbria receptor-positive swine.

    PubMed

    Erume, Joseph; Wijemanne, Prageeth; Berberov, Emil M; Kachman, Stephen D; Oestmann, Daniel J; Francis, David H; Moxley, Rodney A

    2013-01-25

    Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) increases bacterial adherence to porcine enterocytes in vitro and enhances small intestinal colonization in swine. Heat-stable enterotoxin-b (STb) is not known to affect colonization; however, through an induction of net fluid accumulation it might reduce bacterial adherence. The relationship between fluid accumulation and bacterial adherence in jejunal loops inoculated with ETEC strains that produce LT, STb, both, or neither toxin was studied. Ligated jejunal loops were constructed in weaned Yorkshire pigs in two independent experiments (Exp. 1, n=5, 8-week-old; Exp. 2, n=6, 6-8-week-old). Each pig was inoculated with six F4ac(+)E. coli strains: (1) LT(+), STb(+) parent (WAM2317); (2) STb(-) (ΔestB) mutant (MUN297); (3) MUN297 complemented with STb (MUN298); (4) LT(-) STb(-) (ΔeltAB ΔestB) mutant (MUN300); (5) MUN300 complemented with LT (MUN301); and (6) 1836-2 (non-enterotoxigenic, wild-type). Pigs were confirmed to be K88 (F4)ab/ac receptor-positive in Exp. 2 by testing for intestinal mucin-type glycoproteins and inferred to be receptor-positive in both Exp. 1 and 2 based on histopathologic evidence of bacterial adherence. Strains that produced STb induced marked fluid accumulation with the response (ml/cm) to WAM2317 and MUN298 significantly greater than that to the other strains (P<0.0001). Conversely, bacterial adherence scores based on immunohistochemistry and CFU/g of washed mucosa were both lowest in the strains that expressed STb and highest in those that did not. For the two experiments combined, the Pearson correlation coefficient (R) between fluid volume (ml/cm) and log CFU per gram was -0.57021 (P<0.0001); R(2)=0.3521 (n=197). These results support the hypothesis that enterotoxin-induced fluid accumulation flushes progeny organisms into the lumen of the bowel, thereby increasing the likelihood of fecal shedding and transmission of the pathogen to new hosts. PMID

  19. Contamination of beef products with staphylococcal classical enterotoxins in Egypt and Saudi Arabia

    PubMed Central

    Shawish, Reyad R.; Al-Humam, Naser A.

    2016-01-01

    Food-borne pathogens are of high concern for public health and food safety. Staphylococcus aureus food poisoning is one of the most economically devastating types of food poisoning globally. The purpose of this study was to detect staphylococcal classical enterotoxins (SEs) in processed beef from Kingdom of Saudi Arabia (KSA) and Egypt. In the present investigation a total of 250 random processed meat samples (50 each of minced meat, beef burger, beef sausage, beef kofta and beef luncheon) were collected from different super markets in the study area. Using conventional cultural methods, samples were cultured for isolation and identification of S. aureus. Multiplex PCR was used to detect SEs of the classical type SEA, SEB, SEC and SED from isolates. The percentage presence of S. aureus in minced meat, beef burger, beef sausage, beef kofta and beef luncheon was 38%, 22%, 30%, 32% and 12%, respectively. Multiplex PCR indicated that all examined samples contain different types of classical staphylococcal enterotoxins and only minced meat samples contained all four types of toxins. Multiplex PCR is efficient in detection of SEs from food and may be used in tracing of toxins to promote food hygiene. Implications of contamination of processed meat to food hygiene in the study area are highlighted. PMID:27088066

  20. Contamination of beef products with staphylococcal classical enterotoxins in Egypt and Saudi Arabia.

    PubMed

    Shawish, Reyad R; Al-Humam, Naser A

    2016-01-01

    Food-borne pathogens are of high concern for public health and food safety. Staphylococcus aureus food poisoning is one of the most economically devastating types of food poisoning globally. The purpose of this study was to detect staphylococcal classical enterotoxins (SEs) in processed beef from Kingdom of Saudi Arabia (KSA) and Egypt. In the present investigation a total of 250 random processed meat samples (50 each of minced meat, beef burger, beef sausage, beef kofta and beef luncheon) were collected from different super markets in the study area. Using conventional cultural methods, samples were cultured for isolation and identification of S. aureus. Multiplex PCR was used to detect SEs of the classical type SEA, SEB, SEC and SED from isolates. The percentage presence of S. aureus in minced meat, beef burger, beef sausage, beef kofta and beef luncheon was 38%, 22%, 30%, 32% and 12%, respectively. Multiplex PCR indicated that all examined samples contain different types of classical staphylococcal enterotoxins and only minced meat samples contained all four types of toxins. Multiplex PCR is efficient in detection of SEs from food and may be used in tracing of toxins to promote food hygiene. Implications of contamination of processed meat to food hygiene in the study area are highlighted. PMID:27088066

  1. Evaluation of an alternative extraction procedure for enterotoxin determination in dairy products.

    PubMed

    Meyrand, A; Atrache, V; Bavai, C; Montet, M P; Vernozy-Rozand, C

    1999-06-01

    A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products. PMID:10389254

  2. Raffinose increases sporulation and enterotoxin production by Clostridium perfringens type A.

    PubMed Central

    Labbe, R G; Rey, D K

    1979-01-01

    Replacement of starch with raffinose in Duncan and Strong sporulation medium improved percent sporulation in six of eight strains tested. Enterotoxin concentration in cell extracts was increased in the case of four of five known enterotoxin-positive strains. With strain NCTC 10240, levels of 0.3, 0.4, and 0.5% raffinose produced the highest enterotoxin concentration 300 to 320 micrograms of enterotoxin per mg of cell extract protein. At a level of 0.4% raffinose the highest enterotoxin concentration in cell extracts of NCTC 10240 occurred after 8 h of growth in Duncan and Strong medium. Enterotoxin produced in the presence of starch or raffinose by three separate strains all migrated at similar Rm by polyacrylamide gel electrophoresis. Images PMID:225991

  3. An Enterotoxin-Bearing Pathogenicity Island in Staphylococcus epidermidis.

    PubMed

    Madhusoodanan, Jyoti; Seo, Keun Seok; Remortel, Brian; Park, Joo Youn; Hwang, Sun Young; Fox, Lawrence K; Park, Yong Ho; Deobald, Claudia F; Wang, Dan; Liu, Song; Daugherty, Sean C; Gill, Ann Lindley; Bohach, Gregory A; Gill, Steven R

    2011-04-01

    Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage. PMID:21317317

  4. Staphylococcal enterotoxins bind H-2Db molecules on macrophages

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Iandolo, J. J.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

  5. Promotion

    PubMed Central

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed. PMID:24436683

  6. Sensitive, rapid, quantitative and in vitro method for the detection of biologically active staphylococcal enterotoxin type E

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is a major bacterial pathogen which causes clinical infections and food poisoning. This bacterium produces a group of enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis and function as superantigens that activa...

  7. Prevalence of enterotoxigenic Escherichia coli in some processed raw food from animal origin.

    PubMed

    Reis, M H; Vasconcelos, J C; Trabulsi, L R

    1980-01-01

    Eighteen of 1,200 colonies of Escherichia coli isolated from "keebe," hamburger, or sausage produced heat-labile enterotoxin. None of them produced heat-stable enterotoxin. The characteristics of 9 of the 18 strains are presented. PMID:6986850

  8. DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B (SEB) WITH SURFACE PLASMON RESONANCE BIOSENSOR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An automated and rapid method for detection of staphylococcal enterotoxins is needed by the food industry. We have developed a biosensor "Sandwich Assay" using a surface plasmon resonance (SPR) biosensor for the detection of Staphylococcus aureus enterotoxin B (SEB). The assay is based on the immobi...

  9. TNF as biomarker for rapid quantification of active Staphylococcus enterotoxin A in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is a major bacterial pathogen which causes clinical infection and food poisoning. This bacterium produces a group of twenty-one enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis and function as superantigens t...

  10. Surface plasmon resonance (SPR) detection of Staphylococcal Enterotoxin A in food samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An automated and rapid method for detection of staphylococcal enterotoxins (SE) is needed. A sandwich assay was developed using a surface plasmon resonance (SPR) biosensor for detection of staphylococcal enterotoxin A (SEA) at subpicomolar concentration. Assay conditions were optimized for capturing...

  11. Production, secretion and biological activity of Bacillus cereus enterotoxins.

    PubMed

    Senesi, Sonia; Ghelardi, Emilia

    2010-07-01

    Bacillus cereus behaves as an opportunistic pathogen frequently causing gastrointestinal diseases, and it is increasingly recognized to be responsible for severe local or systemic infections. Pathogenicity of B. cereus mainly relies on the secretion of a wide array of toxins and enzymes and also on the ability to undergo swarming differentiation in response to surface-sensing. In this report, the pathogenicity exerted by B. cereus toxins is described with particular attention to the regulatory mechanisms of production and secretion of HBL, Nhe and CytK enterotoxins. PMID:22069656

  12. Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications

    PubMed Central

    Freedman, John C.; Shrestha, Archana; McClane, Bruce A.

    2016-01-01

    Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination. PMID:26999202

  13. Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications.

    PubMed

    Freedman, John C; Shrestha, Archana; McClane, Bruce A

    2016-01-01

    Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination. PMID:26999202

  14. The Distribution of 18 Enterotoxin and Enterotoxin-Like Genes in Staphylococcus aureus Strains from Different Sources in East China.

    PubMed

    Cheng, Jinghua; Wang, Yan; Cao, Yongzhong; Yan, Wenguang; Niu, Xiaosai; Zhou, Liping; Chen, Jianhao; Sun, Ying; Li, Chenxi; Zhang, Xiaorong; Wu, Yantao

    2016-04-01

    The distribution of 18 staphylococcal enterotoxin (SE) or SE-like (SEl) genes in Staphylococcus aureus strains from different sources in east China was investigated. Among all 496 S. aureus strains, 291 strains carried one or more SE genes. The more frequently occurred genes were sea, seb, seg, selk, sell, selm, selo, and seq; the less frequent occurred genes were sec, selj, and ser. The classic SE genes and the enterotoxin gene cluster (egc) (seg, sei, selm, seln, selo, and/or selu) accounted for 25.67% and 61.68% of all detected genes, respectively. There were three gene clusters (egc, sea-sek-seq, and sed-sej-ser), of which the egc cluster was the important one that could generate novel complexes, and the sea-sek-seq cluster was a close relative to the hospital-acquired methicillin-resistant S. aureus. The SE gene distributions were different among strains of different sources and formed diverse toxin gene profiles. The human- and foodborne-origin strains harbored classic and novel SE and SEl genes, whereas animal-origin strains harbored egc and other novel SE and SEl genes mainly. The foodborne- and human-origin strains were the main dangerous factors of classic staphylococcal foodborne poisoning, whereas the strains (especially from animals) that carried egc and other novel genes mainly should be new potential dangerous factors for food safety. PMID:27074376

  15. Biochemistry of Vibrio cholerae virulence: purification of cholera enterotoxin by preparative disc electrophoresis.

    PubMed Central

    Lewis, A C; Richardson, S H; Sheridan, B

    1976-01-01

    Procedures for cholera enterotoxin purification previously developed in this labarotory were not applicable to large-scale purification, and these methods resulted in low yields of pure toxin. An efficient scheme has been developed whereby pure cholera enterotoxin can be obtained from 6 to 8 liters of culture supernatant fluid. This method consists of concentration by membrane ultrafiltration followed by gel filtration and cation-exchange chromatography. Pure cholera enterotoxin of high biological potency was obtained after a final step of preparative acrylamide gel electrophoresis. The degree of purity of the toxin-antigen as well as its biological activity were determined at various setps of purification. This alternate technique for purification is offered because of the widespread interest in cholera enterotoxin as a specific stimulator of adenyl cyclase. Images PMID:987751

  16. Neutralization of Staphylococcal Enterotoxin B by an Aptamer Antagonist

    PubMed Central

    Wang, Kaiyu; Gan, Longjie; Jiang, Li; Zhang, Xianhui; Yang, Xiangyue; Chen, Min

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a major virulence factor for staphylococcal toxic shock syndrome (TSS). SEB activates a large subset of the T lymphocytic population, releasing proinflammatory cytokines. Blocking SEB-initiated toxicity may be an effective strategy for treating TSS. Using a process known as systematic evolution of ligands by exponential enrichment (SELEX), we identified an aptamer that can antagonize SEB with nanomolar binding affinity (Kd = 64 nM). The aptamer antagonist effectively inhibits SEB-mediated proliferation and cytokine secretion in human peripheral blood mononuclear cells. Moreover, a PEGylated aptamer antagonist significantly reduced mortality in a “double-hit” mouse model of SEB-induced TSS, established via sensitization with d-galactosamine followed by SEB challenge. Therefore, our novel aptamer antagonist may offer potential therapeutic efficacy against SEB-mediated TSS. PMID:25624325

  17. Enterotoxin production by staphylococci isolated from healthy goats.

    PubMed Central

    Valle, J; Gomez-Lucia, E; Piriz, S; Goyache, J; Orden, J A; Vadillo, S

    1990-01-01

    The ability of 342 staphylococcal isolates from different anatomical sites in healthy goats to produce staphylococcal enterotoxins (SE) was investigated. SE were produced by 74.3% of the 70 coagulase-positive strains and by 22% of the coagulase-negative strains studied. Most enterotoxigenic strains were isolated from the skin of udders and teats and from milk. SEC was the SE type most frequently produced, either alone (67.9%) or in combination with others. Five coagulase-negative species not previously reported as SE producers were identified (Staphylococcus chromogenes, S. warneri, S. sciuri, S. saprophyticus, and S. lentus). SEA, SEB, and SEC were detected in the milk of 17 of the 133 healthy goats studied. These results suggest that the goat is an important reservoir of enterotoxigenic staphylococci, most of which produce SEC. PMID:2339886

  18. Effects of Staphylococcal Enterotoxins on Human Neutrophil Functions and Apoptosis

    PubMed Central

    Moulding, Dale A.; Walter, Catherine; Hart, C. Anthony; Edwards, Steven W.

    1999-01-01

    Staphylococcal enterotoxins have marked effects on the properties of T cells and monocytes and have recently been reported to affect neutrophil function. In this study, we investigated the abilities of staphylococcal enterotoxins A and B and toxic shock syndrome toxin 1 to affect respiratory burst activity and to delay apoptosis in human neutrophils. When cultures containing approximately 97% neutrophils were tested, the toxins all delayed neutrophil apoptosis in a dose-dependent manner and induced the expression of FcγRI on the neutrophil cell surface. These effects on apoptosis and expression of FcγRI were largely abrogated by the addition of a neutralizing anti-gamma interferon antibody. Similarly, the effects of these toxins on phorbol ester-induced chemiluminescence were decreased after neutralization of gamma interferon. These effects on neutrophil function were mimicked by the addition of conditioned medium from peripheral blood mononuclear cells incubated with the toxins, and again, neutralizing anti-gamma interferon antibodies largely negated the effects. However, when highly purified neutrophils prepared by immunodepletion of T cells and major histocompatibility complex class II-expressing cells were analyzed, the toxins were without effect on apoptosis and FcγRI expression, but granulocyte-macrophage colony-stimulating factor and gamma interferon could still delay apoptosis. These data indicate that these toxins have no direct effect on neutrophil apoptosis but can act indirectly via the production of T-cell-derived and monocyte-derived cytokines. It is noteworthy that such effects are detected in neutrophil suspensions containing only 3% contamination with T cells and other mononuclear cells. PMID:10225889

  19. The effects of irradiation and temperature on the immunological activity of staphylococcal enterotoxin A.

    PubMed

    Modi, N K; Rose, S A; Tranter, H S

    1990-08-01

    The effects of irradiation and temperature on purified staphylococcal enterotoxin A were investigated using sensitive ELISA systems. Thermal inactivation of staphylococcal enterotoxin A in phosphate-buffered saline was considerably faster at temperatures of 60, 70, 115 and 121 degrees C than at 90 and 100 degrees C. In gelatin phosphate buffer, staphylococcal enterotoxin A was completely inactivated by irradiation at 8.0 kGy; in a 15% mince slurry, however, 27-37% of staphylococcal enterotoxin A remained at this level of irradiation. Even at a dose of 23.7 kGy, 16-26% residual staphylococcal enterotoxin A could still be detected. Generally, increasing the mince concentration increased the protection against the effect of irradiation on staphylococcal enterotoxin A. However, the protective effect of mince at a concentration of 50% was less than at a mince concentration of 30%. Both irradiation and heat processing of food should only be used in conjunction with good manufacturing practices to prevent proliferation of microorganisms and toxin productions. PMID:2223523

  20. Bacteriological and Molecular Assessment of Staphylococcal Enterotoxin E in the Blood of Patients With Rheumatoid Arthritis

    PubMed Central

    Zahiri Yeganeh, Samaneh; Ataee, Ramezan Ali; Alishiri, Gholam Hossein; Movahedi, Monireh

    2015-01-01

    Background: Rheumatoid arthritis (RA) is the most common chronic inflammatory disease of unknown etiology. In this regard, the role of bacterial superantigens (as an effective agent) were considered. Objectives: This study aimed to assess staphylococcal enterotoxin E in the blood of patients with rheumatoid arthritis. Patients and Methods: A total of 83 blood samples of patients with RA were studied. All of patient’s blood samples have been cultured. Polymerase chain reaction (PCR) and ELISA methods have been used to assess the existence of staphylococcal enterotoxin E (entE). The data were analyzed through descriptive statistics. Results: During this study and after sequential sub cultures, only 5 bacterial strains were isolated. Based on the results of biochemical tests, just one case was detected as Staphylococcus aureus. The result of molecular diagnosis of enterotoxin E gene was 13.25%. The results of ELISA were 40.96% positive for staphylococcal enterotoxin E. Conclusions: In this study, staphylococcal enterotoxin E (superantigen E) was detected in the blood of patients with RA, but its origin is unknown, because no staphylococcus enterotoxin E producer was isolated. This finding could provide a good model for the diagnosis and treatment of RA. However, the results of this study have shown some evidence regarding endogenous origin of involved superantigens in patients with rheumatoid arthritis. PMID:25793096

  1. Sensitive quantitative and in vitro method as an alternative to animal bioassays for the detection of biologically active staphylococcal enterotoxin type E

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is a major bacterial pathogen which causes clinical infections and food poisoning. This bacterium produces a group of enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis and function as superantigens that activa...

  2. Detection of Staphylococcus Enterotoxin B at Picogram Levels Using Piezoelectric-Excited Millimeter-Sized Cantilever Sensors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report a highly sensitive and rapid method for thhe detection of Staphylocoiccus aureus enterotoxin B (SEB) at picogram levels using a piezolelectric-excited millimeter cantilever (PEMC) sensor. Affinity purified polyclonal antibody to staphylococcal enterotoxin B (anti-SEB) was immobilized on th...

  3. Modeling the effect of water activity, pH, and temperature on the probability of enterotoxin production by Staphylococcus aureus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is a foodborne pathogen widespread in the environment and found in various food products. This pathogen can produce enterotoxins that cause illnesses in humans. The objectives of this study were to develop a probability model of S. aureus enterotoxin production as affected by w...

  4. CD154 as a potential early molecular biomarker for rapid quantification analysis of active Staphylococcus enterotoxin A

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus is a major bacterial pathogen producing a group of twenty-one enterotoxins (SEs). These enterotoxins have two separate but related biological activities, They cause gastroenteritis, and they function as a superantigens that activate large numbers of T cells. In the current stud...

  5. SinR Controls Enterotoxin Expression in Bacillus thuringiensis Biofilms

    PubMed Central

    Økstad, Ole-Andreas; Verplaetse, Emilie; Gilois, Nathalie; Bennaceur, Imène; Perchat, Stéphane; Gominet, Myriam; Aymerich, Stéphane; Kolstø, Anne-Brit; Lereclus, Didier; Gohar, Michel

    2014-01-01

    The entomopathogen Bacillus thuringiensis produces dense biofilms under various conditions. Here, we report that the transition phase regulators Spo0A, AbrB and SinR control biofilm formation and swimming motility in B. thuringiensis, just as they control biofilm formation and swarming motility in the closely related saprophyte species B. subtilis. However, microarray analysis indicated that in B. thuringiensis, in contrast to B. subtilis, SinR does not control an eps operon involved in exopolysaccharides production, but regulates genes involved in the biosynthesis of the lipopeptide kurstakin. This lipopeptide is required for biofilm formation and was previously shown to be important for survival in the host cadaver (necrotrophism). Microarray analysis also revealed that the SinR regulon contains genes coding for the Hbl enterotoxin. Transcriptional fusion assays, Western blots and hemolysis assays confirmed that SinR controls Hbl expression, together with PlcR, the main virulence regulator in B. thuringiensis. We show that Hbl is expressed in a sustained way in a small subpopulation of the biofilm, whereas almost all the planktonic population transiently expresses Hbl. The gene coding for SinI, an antagonist of SinR, is expressed in the same biofilm subpopulation as hbl, suggesting that hbl transcription heterogeneity is SinI-dependent. B. thuringiensis and B. cereus are enteric bacteria which possibly form biofilms lining the host intestinal epithelium. Toxins produced in biofilms could therefore be delivered directly to the target tissue. PMID:24498128

  6. Two common structural motifs for TCR recognition by staphylococcal enterotoxins.

    PubMed

    Rödström, Karin E J; Regenthal, Paulina; Bahl, Christopher; Ford, Alex; Baker, David; Lindkvist-Petersson, Karin

    2016-01-01

    Superantigens are toxins produced by Staphylococcus aureus, called staphylococcal enterotoxins (abbreviated SEA to SEU). They can cross-link the T cell receptor (TCR) and major histocompatibility complex class II, triggering a massive T cell activation and hence disease. Due to high stability and toxicity, superantigens are potential agents of bioterrorism. Hence, antagonists may not only be useful in the treatment of disease but also serve as countermeasures to biological warfare. Of particular interest are inhibitors against SEA and SEB. SEA is the main cause of food poisoning, while SEB is a common toxin manufactured as a biological weapon. Here, we present the crystal structures of SEA in complex with TCR and SEE in complex with the same TCR, complemented with computational alanine-scanning mutagenesis of SEA, SEB, SEC3, SEE, and SEH. We have identified two common areas that contribute to the general TCR binding for these superantigens. This paves the way for design of single antagonists directed towards multiple toxins. PMID:27180909

  7. Two common structural motifs for TCR recognition by staphylococcal enterotoxins

    PubMed Central

    Rödström, Karin E. J.; Regenthal, Paulina; Bahl, Christopher; Ford, Alex; Baker, David; Lindkvist-Petersson, Karin

    2016-01-01

    Superantigens are toxins produced by Staphylococcus aureus, called staphylococcal enterotoxins (abbreviated SEA to SEU). They can cross-link the T cell receptor (TCR) and major histocompatibility complex class II, triggering a massive T cell activation and hence disease. Due to high stability and toxicity, superantigens are potential agents of bioterrorism. Hence, antagonists may not only be useful in the treatment of disease but also serve as countermeasures to biological warfare. Of particular interest are inhibitors against SEA and SEB. SEA is the main cause of food poisoning, while SEB is a common toxin manufactured as a biological weapon. Here, we present the crystal structures of SEA in complex with TCR and SEE in complex with the same TCR, complemented with computational alanine-scanning mutagenesis of SEA, SEB, SEC3, SEE, and SEH. We have identified two common areas that contribute to the general TCR binding for these superantigens. This paves the way for design of single antagonists directed towards multiple toxins. PMID:27180909

  8. ResDE-Dependent Regulation of Enterotoxin Gene Expression in Bacillus cereus: Evidence for Multiple Modes of Binding for ResD and Interaction with Fnr▿ †

    PubMed Central

    Esbelin, Julia; Armengaud, Jean; Zigha, Assia; Duport, Catherine

    2009-01-01

    In the food-borne pathogen Bacillus cereus F4430/73, the production of major virulence factors hemolysin BL (Hbl) and nonhemolytic enterotoxin (Nhe) is regulated through complex mechanisms. The two-component regulatory system ResDE is involved in the activation of hbl and nhe transcription. Here, the response regulator ResD and the sensor kinase ResE were overexpressed and purified, and autophosphorylation of ResE and transphosphorylation of ResD by ResE were demonstrated in vitro. ResD is mainly monomeric in solution, regardless of its phosphorylation state. ResD was shown to interact directly with promoter regions (p) of the enterotoxin regulator genes resDE, fnr, and plcR and the enterotoxin structural genes nhe and hbl, but with different affinities. Binding of ResD to pplcR, pnhe, and phbl was not dependent on the ResD phosphorylation status. In contrast, ResD phosphorylation significantly increased interactions between ResD and presDE and pfnr. Taken together, these results showed that phosphorylation of ResD results in a different target expression pattern. Furthermore, ResD and the redox activator Fnr were found to physically interact and simultaneously bind their target DNAs. We propose that unphosphorylated ResD acts as an antiactivator of Fnr, while phosphorylated ResD acts as a coactivator of Fnr. Finally, our findings represent the first molecular evidence of the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates B. cereus virulence. PMID:19395489

  9. Rapid whole protein quantitation of staphylococcal enterotoxins A and B by liquid chromatography/mass spectrometry.

    PubMed

    Sospedra, Isabel; Soler, Carla; Mañes, Jordi; Soriano, José Miguel

    2012-05-18

    Staphylococcus aureus is an important pathogen and has been indicated as the fifth causative agent of food-borne human illness throughout the world. Staphylococcal enterotoxins (SEs) are toxic compounds excreted mainly by strains of S. aureus. Among these toxins, enterotoxins A (SEA) and B (SEB) are both of the most prevalent compounds in staphylococcal food poisoning. In this work, reverse phase liquid chromatography coupled to ESI mass spectrometry (LC-ESI/MS) has been applied for its rapid identification and quantification. Limit of detection (LOD) values were 0.5 and 0.2 ng for SEA and SEB, respectively and limit of quantification (LOQ) value was 1 ng for both enterotoxins. SEA and SEB have been analyzed as intact proteins in milk and fruit juices. Analytical methods are essential for routine monitoring purposes and safeguard public health and the proposed technique can detect and quantify successfully SEA and SEB in food samples. PMID:22498351

  10. [Influence of various pectins on production of staphylococcal enterotoxins types A and B].

    PubMed

    Fluer, F S; Men'shikov, D D; Lazareva, E B; Prokhorov, V Ia; Vesnin, A V

    2007-01-01

    Experimental in vitro study of influence of 2% solution of pectins (red beet, apple, citrus, manufactured by "Vitaline" company, citrus high- and low-etherified pectins, manufactured by "Hercules" company, Unipectine OB 700, and biologically active supplement "Pecto") on growth of staphylococci and production by them of type A and B enterotoxins was performed. It was shown that red beet, citrus high- and low-etherified pectins, as well as biologically active supplement "Pecto" render bactericidal effect on staphylococci and inhibit synthesis of types A and B staphylococcal enterotoxins. Citrus pectin "Vitaline" and Unipectine OB 700 don't have such influence. The most effective pectins, which were able to inhibit synthesis of types A and B staphylococcal enterotoxins, were red beet, apple, and citrus low-etherified pectins as well as biologically active supplement "Pecto". PMID:18277535

  11. Enterotoxin-encoding genes in Staphylococcus spp. from bulk goat milk.

    PubMed

    Lyra, Daniele G; Sousa, Francisca G C; Borges, Maria F; Givisiez, Patrícia E N; Queiroga, Rita C R E; Souza, Evandro L; Gebreyes, Wondwossen A; Oliveira, Celso J B

    2013-02-01

    Although Staphylococcus aureus has been implicated as the main Staphylococcus species causing human food poisoning, recent studies have shown that coagulase-negative Staphylococcus could also harbor enterotoxin-encoding genes. Such organisms are often present in goat milk and are the most important mastitis-causing agents. Therefore, this study aimed to investigate the occurrence of enterotoxin-encoding genes among coagulase-positive (CoPS) and coagulase-negative (CoNS) staphylococci isolated from raw goat milk produced in the semi-arid region of Paraiba, the most important region for goat milk production in Brazil. Enterotoxin-encoding genes were screened in 74 staphylococci isolates (30 CoPS and 44 CoNS) by polymerase chain reaction targeting the genes sea, seb, sec, sed, see, seg, seh, and sei. Enterotoxin-encoding genes were found in nine (12.2%) isolates, and four different genes (sea, sec, seg, and sei) were identified amongst the isolates. The most frequent genes were seg and sei, which were often found simultaneously in 44.5% of the isolates. The gene sec was the most frequent among the classical genes, and sea was found only in one isolate. All CoPS isolates (n=7) harboring enterotoxigenic genes were identified as S. aureus. The two coagulase-negative isolates were S. haemolyticus and S. hominis subsp. hominis and they harbored sei and sec genes, respectively. A higher frequency of enterotoxin-encoding genes was observed amongst CoPS (23.3%) than CoNS (4.5%) isolates (p<0.05), reinforcing the importance of S. aureus as a potential foodborne agent. However, the potential risk posed by CoNS in goat milk should not be ignored because it has a higher occurrence in goat milk and enterotoxin-encoding genes were detected in some isolates. PMID:23441914

  12. Receptors and cGMP signalling mechanism for E. coli enterotoxin in opossum kidney

    SciTech Connect

    Forte, L.R.; Krause, W.J.; Freeman, R.H. Harry S. Truman Memorial Veterans Medical Center, Columbia, MO )

    1988-11-01

    Receptors for the heat-stable enterotoxin produced by Escherichia coli were found in the kidney and intestine of the North American opossum and in cultured renal cell lines. The enterotoxin markedly increased guanosine 3{prime},5{prime}-cyclic monophosphate (cGMP) production in slices of kidney cortex and medulla, in suspensions of intestinal mucosa, and in the opossum kidney (OK) and rat kangaroo kidney (PtK-2) cell lines. In contrast, atrial natriuretic factor elicited much smaller increases in cGMP levels of kidney, intestine, or cultured kidney cell lines. The enterotoxin receptors in OK cells had a molecular mass of approximately 120 kDa when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptors crosslinked with {sup 125}I-enterotoxin. The occurrence of receptors for the E. coli peptide in OK implies that these receptors may be involved in the regulation of renal tubular function in the opossum. E. coli enterotoxin caused a much larger increase in urine cGMP excretion than did atrial natriuretic factor when these peptides were injected intravenously into opossums. However, atrial natriuretic factor elicited a marked diuresis, natriuresis, and increased urinary excretion of calcium, phosphate, potassium, and magnesium. In contrast, the enterotoxin did not acutely influence OK fluid and electrolyte excretion. Thus the substantial increase in cGMP synthesis produced by the bacterial peptide in OK cortex and medulla in vitro and the increased renal excretion of cGMP in vivo were not associated with changes in electrolyte or water excretion. Whether cGMP represents a second messenger molecule in the kidney is an interesting question that was raised but not answered in this series of experiments.

  13. Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies*

    PubMed Central

    Dutta, Kaushik; Varshney, Avanish K.; Franklin, Matthew C.; Goger, Michael; Wang, Xiaobo; Fries, Bettina C.

    2015-01-01

    Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations. PMID:25572397

  14. Late Multiple Organ Surge in Interferon-Regulated Target Genes Characterizes Staphylococcal Enterotoxin B Lethality

    PubMed Central

    Ferreyra, Gabriela A.; Elinoff, Jason M.; Demirkale, Cumhur Y.; Starost, Matthew F.; Buckley, Marilyn; Munson, Peter J.; Krakauer, Teresa; Danner, Robert L.

    2014-01-01

    Background Bacterial superantigens are virulence factors that cause toxic shock syndrome. Here, the genome-wide, temporal response of mice to lethal intranasal staphylococcal enterotoxin B (SEB) challenge was investigated in six tissues. Results The earliest responses and largest number of affected genes occurred in peripheral blood mononuclear cells (PBMC), spleen, and lung tissues with the highest content of both T-cells and monocyte/macrophages, the direct cellular targets of SEB. In contrast, the response of liver, kidney, and heart was delayed and involved fewer genes, but revealed a dominant genetic program that was seen in all 6 tissues. Many of the 85 uniquely annotated transcripts participating in this shared genomic response have not been previously linked to SEB. Nine of the 85 genes were subsequently confirmed by RT-PCR in every tissue/organ at 24 h. These 85 transcripts, up-regulated in all tissues, annotated to the interferon (IFN)/antiviral-response and included genes belonging to the DNA/RNA sensing system, DNA damage repair, the immunoproteasome, and the ER/metabolic stress-response and apoptosis pathways. Overall, this shared program was identified as a type I and II interferon (IFN)-response and the promoters of these genes were highly enriched for IFN regulatory matrices. Several genes whose secreted products induce the IFN pathway were up-regulated at early time points in PBMCs, spleen, and/or lung. Furthermore, IFN regulatory factors including Irf1, Irf7 and Irf8, and Zbp1, a DNA sensor/transcription factor that can directly elicit an IFN innate immune response, participated in this host-wide SEB signature. Conclusion Global gene-expression changes across multiple organs implicated a host-wide IFN-response in SEB-induced death. Therapies aimed at IFN-associated innate immunity may improve outcome in toxic shock syndromes. PMID:24551153

  15. Novel Tissue Level Effects of the Staphylococcus aureus Enterotoxin Gene Cluster Are Essential for Infective Endocarditis

    PubMed Central

    Stach, Christopher S.; Vu, Bao G.; Merriman, Joseph A.; Herrera, Alfa; Cahill, Michael P.; Schlievert, Patrick M.; Salgado-Pabón, Wilmara

    2016-01-01

    Background Superantigens are indispensable virulence factors for Staphylococcus aureus in disease causation. Superantigens stimulate massive immune cell activation, leading to toxic shock syndrome (TSS) and contributing to other illnesses. However, superantigens differ in their capacities to induce body-wide effects. For many, their production, at least as tested in vitro, is not high enough to reach the circulation, or the proteins are not efficient in crossing epithelial and endothelial barriers, thus remaining within tissues or localized on mucosal surfaces where they exert only local effects. In this study, we address the role of TSS toxin-1 (TSST-1) and most importantly the enterotoxin gene cluster (egc) in infective endocarditis and sepsis, gaining insights into the body-wide versus local effects of superantigens. Methods We examined S. aureus TSST-1 gene (tstH) and egc deletion strains in the rabbit model of infective endocarditis and sepsis. Importantly, we also assessed the ability of commercial human intravenous immunoglobulin (IVIG) plus vancomycin to alter the course of infective endocarditis and sepsis. Results TSST-1 contributed to infective endocarditis vegetations and lethal sepsis, while superantigens of the egc, a cluster with uncharacterized functions in S. aureus infections, promoted vegetation formation in infective endocarditis. IVIG plus vancomycin prevented lethality and stroke development in infective endocarditis and sepsis. Conclusions Our studies support the local tissue effects of egc superantigens for establishment and progression of infective endocarditis providing evidence for their role in life-threatening illnesses. In contrast, TSST-1 contributes to both infective endocarditis and lethal sepsis. IVIG may be a useful adjunct therapy for infective endocarditis and sepsis. PMID:27124393

  16. Mechanisms mediating enhanced neutralization efficacy of staphylococcal enterotoxin B by combinations of monoclonal antibodies.

    PubMed

    Dutta, Kaushik; Varshney, Avanish K; Franklin, Matthew C; Goger, Michael; Wang, Xiaobo; Fries, Bettina C

    2015-03-13

    Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-β chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations. PMID:25572397

  17. Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice.

    PubMed

    Takeshita, W M; Gushiken, V O; Ferreira-Duarte, A P; Pinheiro-Torres, A S; Roncalho-Buck, I A; Squebola-Cola, D M; Mello, G C; Anhê, G F; Antunes, E; DeSouza, I A

    2015-09-15

    Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1μg), and at 4, 12 and 24h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. PMID:26091799

  18. A tripartite fusion, FaeG-FedF-LT(192)A2:B, of enterotoxigenic Escherichia coli (ETEC) elicits antibodies that neutralize cholera toxin, inhibit adherence of K88 (F4) and F18 fimbriae, and protect pigs against K88ac/heat-labile toxin infection.

    PubMed

    Ruan, Xiaosai; Liu, Mei; Casey, Thomas A; Zhang, Weiping

    2011-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT(192)) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT(192)A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT(192)A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT(192)A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrial E. coli strains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea. PMID:21813665

  19. Diagnostic importance of Clostridium perfringens enterotoxin analysis in recurring enteritis among elderly, chronic care psychiatric patients.

    PubMed Central

    Jackson, S G; Yip-Chuck, D A; Clark, J B; Brodsky, M H

    1986-01-01

    A series of Clostridium perfringens-related gastrointestinal outbreaks occurred over a period of several months among elderly, chronic care patients in a psychiatric hospital. Several serotypes of C. perfringens and many nontypeable isolates were found. The distribution of certain serotypes and the incidence of detection of enterotoxin in fecal extracts were related to wards on which patients were resident (six wards were involved). Several patients were reported to have chronic or recurring fecal incontinence or diarrhea or both. With a background of elevated spore counts of several serotypes and chronic diarrhea, only detection of enterotoxin could provide definitive evidence of C. perfringens etiology in gastoenteritis cases. PMID:2871043

  20. Clostridium perfringens Type A Enterotoxin Damages the Rabbit Colon

    PubMed Central

    Garcia, Jorge P.; Li, Jihong; Shrestha, Archana; Freedman, John C.; Beingesser, Juliann; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens enterotoxin causes the gastrointestinal (GI) symptoms of C. perfringens type A food poisoning and CPE-associated non-food-borne human GI diseases. It is well established that CPE induces fluid accumulation and severe tissue damage in ligated small intestinal loops of rabbits and other animals. However, a previous study had also reported that CPE binds to rabbit colonic cells yet does not significantly affect rabbit colonic loops. To the contrary, the current study determined that treatment with 50 or 100 μg/ml of CPE causes significant histologic lesions and luminal fluid accumulation in rabbit colonic loops. Interestingly, a CPE-neutralizing monoclonal antibody blocked the development of CPE-induced histologic damage but not luminal fluid accumulation in these loops. Similar luminal fluid accumulation, without significant histologic damage, also occurred after treatment of colonic loops with heat-inactivated CPE, antibody alone, or bovine serum albumin (BSA), indicating that increased osmolarity was causing or contributing to fluid accumulation in CPE-treated colonic loops. Comparative studies revealed the similar development of histologic damage and luminal fluid accumulation in both small intestinal loops and colonic loops after as little as a 1-h treatment with 50 μg/ml of CPE. Consistent with the CPE sensitivity of the small intestine and colon, Western blotting detected CPE binding and large-complex formation in both organs. In addition, Western blotting demonstrated the presence of the high-affinity CPE receptors claudin-3 and -4 in both organs of rabbits, consistent with the observed toxin binding. Collectively, these results offer support for the possible involvement of the colon in CPE-mediated GI disease. PMID:24643537

  1. Detection and expression of enterotoxin genes in plant-associated strains of Bacillus cereus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacillus cereus is an environmental microbe that commonly inhabits plants and soil. Twenty five plant-associated B. cereus isolates were obtained from apple, cacao, tomato, and potato. The isolates were screened for the presence and expression of enterotoxin B (BcET) components of the nonhemolytic e...

  2. Influence of starch source on sporulation and enterotoxin production by Clostridium perfringens type A.

    PubMed

    Labbe, R; Somers, E; Duncan, C

    1976-03-01

    Of 16 different starch preparations tested, Clostridium perfringes NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters. PMID:180885

  3. The olive compound 4-hydroxytyrosol inactivates Staphyloccoccus aureus bacteria and Staphylococcal enterotoxin A (SEA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The foodborne pathogen Staphylococcus aureus produces the virulent staphylococcal enterotoxin A (SEA), a single chain protein which consists of 233 amino acid residues with a molecular weight of 27,078 Da. SEA is a superantigen that is reported to contribute to animal (mastitis) and human (emesis, ...

  4. Genotypes and enterotoxin gene profiles of Staphylococcus aureus clinical isolates from China

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A total of 108 S. aureus isolates from 16 hospitals located in 14 different provinces in China were characterized for the profiles of 19 staphylococcal enterotoxin (SE) genes by PCR and genotyped by PFGE and MLST. Of these strains, 88.9% (96/108) harbored SE genes, in which tsst was the most prevale...

  5. In vitro cell based assay for activity analysis of staphylococcal enterotoxin A in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning. They function both as toxins that cause gastroenteritis after ingestion and as superantigens that non-specifically activate large numbers of T cells. Monkey or kitten bioassays were historically developed for analysis of SE act...

  6. Use of inactivated E.Coli enterotoxins to enhance respiratory mucosal adjuvanticity during vaccination in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to augment responses to respiratory vaccines in swine, various adjuvants were intranasally co-administered with an antigen to pigs. Detoxified E. coli enterotoxins LTK63 and LTR72 enhanced mucosal and systemic immunity to the model peptide, exhibiting their efficacy as mucosal adjuvants for...

  7. Importance of Flagella and Enterotoxins for Aeromonas Virulence in a Mouse Model

    EPA Science Inventory

    A genetic characterization of eight virulence factor genes, elastase, lipase, polar flagella (flaA/flaB, flaG), lateral flagella (lafA), and the enterotoxins alt, act, and ast, was performed using polymerase chain reaction with 55 drinking water and nine clinical isolates. When 1...

  8. Incidence and characterization of diarrheal enterotoxins of fecal Bacillus cereus isolates associated with diarrhea.

    PubMed

    Al-Khatib, Mariam Saleh; Khyami-Horani, Hala; Badran, Eman; Shehabi, Asem A

    2007-12-01

    A total of 490 stool specimens were collected from patients with diarrhea and healthy controls without diarrhea to investigate the incidence of Bacillus cereus and its enterotoxins. B. cereus was found more significant in stools of persons with diarrhea than without diarrhea (9.5% versus 1.8%, P < 0.05), and was also detected more frequent but not significant in individuals aged > or =1 year and in adults than in children aged <1 year (11% and 8% versus 7.8%, P > 0.05). The hemolytic enterotoxin HBL genes of B. cereus isolates (hblA, hblC, hblD) were detected in 58%, 58%, and 68%, respectively, whereas the nonhemolytic enterotoxin NHE genes (nheA, nheB, nheC) were detected more frequent in 71.%, 84%, and 90% of the isolates, respectively. This study suggests that B. cereus isolates harboring 1 or more enterotoxin gene(s) can be a potential cause of diarrhea in Jordanian population. PMID:17878069

  9. Techniques for rapid detection and quantification of active foodborne Staphylococcus Enterotoxin(Abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Staphylococcus aureus is an important bacterial pathogen and causative agent of foodborne illnesses.Staphylococcal enterotoxins(SEs)produced by this organism act upon the gastrointestinal tract and generate a superantigen immune response in low concentrations. Recent S. aureus foodborne ...

  10. From genome to toxicity: a combinatory approach highlights the complexity of enterotoxin production in Bacillus cereus

    PubMed Central

    Jeßberger, Nadja; Krey, Viktoria M.; Rademacher, Corinna; Böhm, Maria-Elisabeth; Mohr, Ann-Katrin; Ehling-Schulz, Monika; Scherer, Siegfried; Märtlbauer, Erwin

    2015-01-01

    In recent years Bacillus cereus has gained increasing importance as a food poisoning pathogen. It is the eponymous member of the B. cereus sensu lato group that consists of eight closely related species showing impressive diversity of their pathogenicity. The high variability of cytotoxicity and the complex regulatory network of enterotoxin expression have complicated efforts to predict the toxic potential of new B. cereus isolates. In this study, comprehensive analyses of enterotoxin gene sequences, transcription, toxin secretion and cytotoxicity were performed. For the first time, these parameters were compared in a whole set of B. cereus strains representing isolates of different origin (food or food poisoning outbreaks) and of different toxic potential (enteropathogenic and apathogenic) to elucidate potential starting points of strain-specific differential toxicity. While toxin gene sequences were highly conserved and did not allow for differentiation between high and low toxicity strains, comparison of nheB and hblD enterotoxin gene transcription and Nhe and Hbl protein titers revealed not only strain-specific differences but also incongruence between toxin gene transcripts and toxin protein levels. With one exception all strains showed comparable capability of protein secretion and so far, no secretion patterns specific for high and low toxicity strains were identified. These results indicate that enterotoxin expression is more complex than expected, possibly involving the orchestrated interplay of different transcriptional regulator proteins, as well as posttranscriptional and posttranslational regulatory mechanisms plus additional influences of environmental conditions. PMID:26113843

  11. The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment

    PubMed Central

    Wallin-Carlquist, Nina; Thorup Cohn, Marianne; Lindqvist, Roland; Barker, Gary C; Rådström, Peter

    2011-01-01

    The recent finding that the formation of staphylococcal enterotoxins in food is very different from that in cultures of pure Staphylococcus aureus sheds new light on, and brings into question, traditional microbial risk assessment methods based on planktonic liquid cultures. In fact, most bacteria in food appear to be associated with surfaces or tissues in various ways, and interaction with other bacteria through molecular signaling is prevalent. Nowadays it is well established that there are significant differences in the behavior of bacteria in the planktonic state and immobilized bacteria found in multicellular communities. Thus, in order to improve the production of high-quality, microbiologically safe food for human consumption, in situ data on enterotoxin formation in food environments are required to complement existing knowledge on the growth and survivability of S. aureus. This review focuses on enterotoxigenic S. aureus and describes recent findings related to enterotoxin formation in food environments, and ways in which risk assessment can take into account virulence behavior. An improved understanding of how environmental factors affect the expression of enterotoxins in foods will enable us to formulate new strategies for improved food safety. PMID:22030860

  12. Staphylococcus epidermidis and Staphylococcus haemolyticus: Molecular Detection of Cytotoxin and Enterotoxin Genes

    PubMed Central

    Pinheiro, Luiza; Ivo Brito, Carla; de Oliveira, Adilson; Yoshida Faccioli Martins, Patrícia; Cataneli Pereira, Valéria; Ribeiro de Souza da Cunha, Maria de Lourdes

    2015-01-01

    Although opportunistic pathogens, coagulase-negative staphylococci (CoNS), including Staphylococcus epidermidis and Staphylococcus haemolyticus, have long been regarded as avirulent organisms. The role of toxins in the development of infections caused by CoNS is still controversial. The objective of this study was to characterize the presence of enterotoxin and cytotoxin genes in S. epidermidis and S. haemolyticus isolates obtained from blood cultures. Cytotoxin genes were detected by PCR using novel species-specific primers. Among the 85 S. epidermidis and 84 S. haemolyticus isolates, 95.3% and 79.8%, respectively, carried at least one enterotoxin gene. The most frequent enterotoxin genes were sea (53.3%), seg (64.5%) and sei (67.5%). The seg gene was positively associated with S. epidermidis (p = 0.02), and this species was more toxigenic than S. haemolyticus. The hla/yidD gene was detected in 92.9% of S. epidermidis and the hla gene in 91.7% of S. haemolyticus isolates; hlb was detected in 92.9% of the S. epidermidis isolates and hld in 95.3%. Nosocomial Staphylococcus epidermidis and S. haemolyticus isolates exhibited a high toxigenic potential, mainly containing the non-classical enterotoxin genes seg and sei. The previously unreported detection of hla/yidD and hlb in S. epidermidis and S. haemolyticus using species-specific primers showed that these hemolysin genes differ between CoNS species and that they are highly frequent in blood culture isolates. PMID:26389954

  13. A novel electrochemical immunosensor based on magnetosomes for detection of staphylococcal enterotoxin B in milk.

    PubMed

    Wu, Longyun; Gao, Bo; Zhang, Fang; Sun, Xiulan; Zhang, Yinzhi; Li, Zaijun

    2013-03-15

    In this paper, a novel electrochemical immunosensor to detect staphylococcal enterotoxin B based on bio-magnetosomes, polyaniline nano-gold composite and 1,2-dimethyl-3-butylimidazolium hexafluorophosphate ionic liquid, was developed, and found to exhibit high sensitivity and stability. The specific antibody to staphylococcal enterotoxin B conjugated with the magnetosomes showed rapid immunoreactions and good dispersion, which contributed to the formation of a nanostructurally smooth and dense film on the surface of a gold electrode. Polyaniline nano-gold composite and 1,2-dimethyl-3-butylimidazolium hexafluorophosphate ionic liquid were used to modify the electrode as mediators to improve the electron transfer and offer an excellent biocompatible microenvironment for the antibody to retain its activity to enhance the response of the electrochemical sensor. Under optimal conditions, the developed immunosensor showed a good linear response in the range from 0.05 to 5 ng/mL (R(2)=0.9957) with a detection limit as low as 0.017 ng/mL, compared with the one without magnetosomes (0.05-5 ng/mL, 0.033 ng/mL), this developed immunosensor showed a wider response range and a reduced detection limit. And a good specificity with little adsorption to staphylococcal enterotoxin A, C and Na(+), K(+), Ca(2+) was obtained. Moreover, the immunosensor exhibited a good long-time stability at 4 °C reaching up to 60 days, which showed a relatively long working life. Meanwhile the immunosensor could be regenerated four times using NaOH elution. The sensor also displayed a good repeatability with a relative standard deviation of 5.02% for staphylococcal enterotoxin B detection (1 ng/mL, n=9). Furthermore, high recoveries in milk samples from 81% to 118% were achieved and successfully applied to milk sample detection. The obtained results demonstrate that the developed electrochemical immunosensor is a promising tool for the detection of staphylococcal enterotoxin B in food. PMID:23598138

  14. Effect of shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo.

    PubMed Central

    Fasano, A; Noriega, F R; Liao, F M; Wang, W; Levine, M M

    1997-01-01

    BACKGROUND: Shigella enterotoxin 1 is a novel enterotoxin elaborated by Shigella flexneri 2a that causes fluid accumulation in rabbit ileal loops and a rise in short circuit current in Ussing chambers. AIMS: To gain insights into the mechanism of action of shigella enterotoxin 1. METHODS: Supernatants from genetically engineered clones either overexpressing shigella enterotoxin 1 or producing deletion mutants of the toxin were tested in rabbit ileum both in vitro and in vivo. RESULTS: In rabbit ileum shigella enterotoxin 1 induced an irreversible rise in short circuit current that was not mediated by any of the recognised intracellular mediators of secretion. Deletion of 90% of the A subunit of the holotoxin ablated its enterotoxicity. In the in vivo perfusion model, the toxin induced a time dependent decrease in water absorption, whereas no changes were detected in the segment perfused with supernatants obtained from the deletion mutant. Finally, partially purified toxin induced a dose dependent increment in short circuit current that reached its plateau at a toxin concentration of 4 x 10(-6) M. CONCLUSIONS: Shigella enterotoxin 1 induces a time and dose dependent intestinal secretion in the rabbit animal model, suggesting that it may be responsible for the watery phase of Shigella flexneri 2a infection. Images PMID:9176079

  15. Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin.

    PubMed

    Bennett, V; Cuatrecasas, P

    1975-06-01

    The kinetics and properties of the activation of adenylate cyclase by cholera enterotoxin have been examined primarily in toad erythrocytes, but also in avian erythrocytes, rat fat cells and cultured melanoma cells. When cholera toxin is incubated with intact cells it stimulates adenylate cyclase activity, as measured in the subsequently isolated plasma membranes, according to a triphasic time course. This consists of a true lag period of about 30 min, followed by a stage of exponentially increasing adenylate cyclase activity which continues for 110 to 130 min, and finally a period of slow activation which may extend as long as 30 hr in cultured melanoma cells. The progressive activation of adenylate cyclase activity by cholera toxin is interrupted by cell lysis; continued incubation of the isolated membranes under nearly identical conditions does not lead to further activation of the enzyme. The delay in the action of the toxin is not grossly dependent of the number of toxin-receptor (GM1 ganglioside) complexes, and is still seen upon adding a second dose of toxin to partially stimulated cells. Direct measurements indicate negligible intracellular levels of biologically active radioiodinated toxin in either a soluble or a nuclear-bound form. The effects are not prevented by Actinomycin D (20 mug/ml), uromycin (30 mug/ml), cycloheximide (30 mug/ml), sodium fluoride (10 mM) or sodium azide (1 mM); KCN, however, almost completely prevents the action of cholera toxin. The action of the toxin is temperature dependent, occurring at very slow or negligible rates below certain critical temperatures, the values of which depend on the specific animal species. Thetransition for toad erythrocytes occurs at 15 to 17 degrees C, while rat adipocytes and turkey erythrocytes demonstrate a discontinuity at 26 to 30 degrees C. The temperature effects are evident during the lag period as well as during the exponential phase of activation. The rate of decay of the stimulated adenylate

  16. The effect of undissociated lactic acid on Staphylococcus aureus growth and enterotoxin A production.

    PubMed

    Rosengren, Asa; Lindblad, Mats; Lindqvist, Roland

    2013-03-15

    The potential of Staphylococcus aureus cheese isolates to grow and produce staphylococcal enterotoxin A under conditions typical for cheese making was investigated in three broth experiments. The effect of the concentration of undissociated lactic acid (HLac) in conjunction with specific pH values was studied by adjusting pH at a single concentration of lactic acid. First, the time-to-growth of S. aureus was modelled by using survival analysis and absorbance data obtained from an automated turbidity reader. The fitted model describes the time to growth and indicates the growth ⁄ no growth boundary of S. aureus as a function of HLac concentration, temperature and water activity. Second, growth rates and lag times of S. aureus were estimated after two different pre-treatments in skim milk at three HLac concentrations and two temperatures based on optical detection times of serial dilutions of bacterial solutions. Growth rates differed between strains, and increased with increasing temperature and decreasing HLac concentration. Preliminary results indicate that lag times were dependent on pre-treatment suggesting that the growth potential of S. aureus in cheese curd may be greater if milk is used immediately after milking compared to holding at 4°C after milking. Third, growth, inactivation, and enterotoxin A production of S. aureus strains were investigated at twelve combinations of HLac concentration and temperature. Concentrations of enterotoxin A increased linearly during the first four days, with a production rate increasing with increasing temperature and decreasing HLac concentration. Significant amounts of enterotoxin A were produced during extended incubation, up to 14days, but then initial pH had changed. This highlights a potential limitation of modelling based on the initial environmental conditions in batch experiments. In summary, ranges of time-to-growth, growth rates, lag times and enterotoxin A production rates of S. aureus in the presence of HLac

  17. Enterotoxin genes in coagulase-negative and coagulase-positive staphylococci isolated from bovine milk.

    PubMed

    de Freitas Guimarães, Felipe; Nóbrega, Diego Borin; Richini-Pereira, Virginia Bodelão; Marson, Pâmela Merlo; de Figueiredo Pantoja, José Carlos; Langoni, Helio

    2013-05-01

    The objective of this study was to isolate and identify the main staphylococcal species causing bovine mastitis in 10 Brazilian dairy herds and study their capability to produce enterotoxins. Herds were selected based on size and use of milking technology, and farms were visited once during the study. All mammary glands of all lactating cows were screened using the California Mastitis Test (CMT) and a strip cup. A single aseptic milk sample (20 mL) was collected from all CMT-positive quarters. Identification of Staphylococcus spp. was performed using conventional microbiology, and PCR was used to determine the presence of enterotoxin-encoding genes (sea, seb, sec, and sed). Of the 1,318 CMT-positive milk samples, Staphylococcus spp. were isolated from 263 (19.9%). Of these isolates, 135 (51%) were coagulase-positive staphylococci (CPS) and 128 (49%) were coagulase-negative staphylococci (CNS). Eighteen different species of CNS were isolated, among which S. warneri, S. epidermidis and S. hyicus were the most frequent. The distribution of Staphylococcus species was different among herds: S. epidermidis was found in 8 herds, S. warneri was found in 7 herds, and S. hyicus in 6 herds. Some of the CNS species (S. saprophyticus ssp. saprophyticus, S. auricularis, S. capitis, and S. chromogenes) were isolated in only one of the farms. Genes related to production of enterotoxins were found in 66% (n=85) of all CNS and in 35% of the CPS isolates. For both CNS and CPS isolates, the most frequently identified enterotoxin genes were sea, seb, and sec; the prevalence of sea differed between CPS (9.5%) and CNS (35.1%) isolates. Staphylococcus warneri isolates showed a greater percentage of sea than seb, sec, or sed, whereas S. hyicus isolates showed a greater percentage of sea than sec. Over 60% of CNS belonged to 3 major species, which carried 62.2 to 81.3% of the enterotoxin genes. The high prevalence highlights the potential for food poisoning caused by these species. For

  18. Predictions of T-cell receptor- and major histocompatibility complex-binding sites on staphylococcal enterotoxin C1.

    PubMed Central

    Hoffmann, M L; Jablonski, L M; Crum, K K; Hackett, S P; Chi, Y I; Stauffacher, C V; Stevens, D L; Bohach, G A

    1994-01-01

    We have focused on regions of staphylococcal enterotoxin C1 (SEC1) causing immunomodulation. N-terminal deletion mutants lacking residues 6 through 13 induced T-cell proliferation similar to that induced by native toxin. However, mutants with residues deleted between positions 19 and 33, although nonmitogenic themselves, were able to inhibit both SEC1-induced T-cell proliferation and binding of the native toxin to major histocompatibility complex (MHC) class II. Presumably, these deletions define a part of SEC1 that interacts with the T-cell receptor. Three synthetic peptides containing residues located in a region analogous to the alpha 5 groove of SEC3 had residual mitogenic activity or blocked T-cell proliferation induced by SEC1 and appear to recognize the same site as SEC1 on a receptor for the toxin, presumably MHC class II. We conclude that isolated portions of the SEC1 molecule can retain residual mitogenic activity but that the entire protein is needed to achieve maximal superantigenic stimulation. Our results, together with the results of other investigators, support a model in which SEC1 binds to an alpha helix of MHC class II through a central groove in the toxin and thereby promotes or stabilizes the interaction between antigen-presenting cells and T cells. Images PMID:8039910

  19. Staphylococcal enterotoxin A regulates bone marrow granulocyte trafficking during pulmonary inflammatory disease in mice

    SciTech Connect

    Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.; Pinheiro-Torres, A.S.; Roncalho-Buck, I.A.; Squebola-Cola, D.M.; Mello, G.C.; Anhê, G.F.; Antunes, E.; DeSouza, I.A.

    2015-09-15

    Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. - Highlights: • Airway exposure to SEA causes acute lung inflammation. • SEA induces accumulation of bone marrow (BM) in immature and mature neutrophils. • SEA increases BM granulocyte or BM PMN adhesion to ICAM-1 and VCAM-1, and MAC-1 expression. • SEA induces BM elevations of CXCL-1, INF-γ, TNF-α, GM-CSF, G-CSF and

  20. Biological and immunological properties of the carboxyl terminus of staphylococcal enterotoxin C1.

    PubMed

    Bohach, G A; Handley, J P; Schlievert, P M

    1989-01-01

    Comparisons of recently published primary sequences of staphylococcal and streptococcal pyrogenic toxins prompted an evaluation of biological and immunological properties of the C terminus of staphylococcal enterotoxin C1. The 59 N-terminal amino acids were deleted from the toxin by digestion with trypsin. The resulting fragment (Mr, 20,659) contained the remaining 180 C-terminal residues. This fragment (Trp F1) consisted of two polypeptide chains (Trp F1a and Trp F1b) linked by cysteine residues. Trp F1 was mitogenic, pyrogenic, and enhanced susceptibility of rabbits to lethal endotoxin shock. In addition, this fragment contained at least one antigenic epitope that cross-reacted with enterotoxin B. PMID:2909489

  1. Clostridium and Bacillus Binary Enterotoxins: Bad for the Bowels, and Eukaryotic Being

    PubMed Central

    Stiles, Bradley G.; Pradhan, Kisha; Fleming, Jodie M.; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R.

    2014-01-01

    Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin. PMID:25198129

  2. Clostridium and bacillus binary enterotoxins: bad for the bowels, and eukaryotic being.

    PubMed

    Stiles, Bradley G; Pradhan, Kisha; Fleming, Jodie M; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R

    2014-09-01

    Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin. PMID:25198129

  3. Unique regulatory mechanism of sporulation and enterotoxin production in Clostridium perfringens.

    PubMed

    Ohtani, Kaori; Hirakawa, Hideki; Paredes-Sabja, Daniel; Tashiro, Kosuke; Kuhara, Satoru; Sarker, Mahfuzur R; Shimizu, Tohru

    2013-06-01

    Clostridium perfringens causes gas gangrene and gastrointestinal (GI) diseases in humans. The most common cause of C. perfringens-associated food poisoning is the consumption of C. perfringens vegetative cells followed by sporulation and production of enterotoxin in the gut. Despite the importance of spore formation in C. perfringens pathogenesis, the details of the regulation of sporulation have not yet been defined fully. In this study, microarray and bioinformatic analyses identified a candidate gene (the RNA regulator virX) for the repression of genes encoding positive regulators (Spo0A and sigma factors) of C. perfringens sporulation. A virX mutant constructed in the food poisoning strain SM101 had a much higher sporulation efficiency than that of the wild type. The transcription of sigE, sigF, and sigK was strongly induced at 2.5 h of culture of the virX mutant. Moreover, the transcription of the enterotoxin gene was also strongly induced in the virX mutant. Western blotting confirmed that the levels of enterotoxin production were higher in the virX mutant than in the wild type. These observations indicated that the higher levels of sporulation and enterotoxin production in the virX mutant were specifically due to inactivation of the virX gene. Since virX homologues were not found in any Bacillus species but were present in other clostridial species, our findings identify further differences in the regulation of sporulation between Bacillus and certain Clostridium species. The virX RNA regulator plays a key role in the drastic shift in lifestyle of the anaerobic flesh eater C. perfringens between the vegetative state (for gas gangrene) and the sporulating state (for food poisoning). PMID:23585540

  4. An Enterotoxin-Bearing Pathogenicity Island in Staphylococcus epidermidis▿†

    PubMed Central

    Madhusoodanan, Jyoti; Seo, Keun Seok; Remortel, Brian; Park, Joo Youn; Hwang, Sun Young; Fox, Lawrence K.; Park, Yong Ho; Deobald, Claudia F.; Wang, Dan; Liu, Song; Daugherty, Sean C.; Gill, Ann Lindley; Bohach, Gregory A.; Gill, Steven R.

    2011-01-01

    Cocolonization of human mucosal surfaces causes frequent encounters between various staphylococcal species, creating opportunities for the horizontal acquisition of mobile genetic elements. The majority of Staphylococcus aureus toxins and virulence factors are encoded on S. aureus pathogenicity islands (SaPIs). Horizontal movement of SaPIs between S. aureus strains plays a role in the evolution of virulent clinical isolates. Although there have been reports of the production of toxic shock syndrome toxin 1 (TSST-1), enterotoxin, and other superantigens by coagulase-negative staphylococci, no associated pathogenicity islands have been found in the genome of Staphylococcus epidermidis, a generally less virulent relative of S. aureus. We show here the first evidence of a composite S. epidermidis pathogenicity island (SePI), the product of multiple insertions in the genome of a clinical isolate. The taxonomic placement of S. epidermidis strain FRI909 was confirmed by a number of biochemical tests and multilocus sequence typing. The genome sequence of this strain was analyzed for other unique gene clusters and their locations. This pathogenicity island encodes and expresses staphylococcal enterotoxin C3 (SEC3) and staphylococcal enterotoxin-like toxin L (SElL), as confirmed by quantitative reverse transcription-PCR (qRT-PCR) and immunoblotting. We present here an initial characterization of this novel pathogenicity island, and we establish that it is stable, expresses enterotoxins, and is not obviously transmissible by phage transduction. We also describe the genome sequence, excision, replication, and packaging of a novel bacteriophage in S. epidermidis FRI909, as well as attempts to mobilize the SePI element by this phage. PMID:21317317

  5. Staphylococcal Enterotoxin A Detection from Rheumatoid Arthritis Patients’ Blood and Synovial Fluid

    PubMed Central

    Ataee, Ramezan Ali; Kahani, Mahboobeh Sadat; Alishiri, Gholam Hossein; Ahamadi, Zyenab

    2016-01-01

    Introduction Direct detection of microbial super antigens in synovial fluid of patients with rheumatoid arthritis may be able to guide to the design of cost-effective therapies. The purpose of this study was to assess the existence of Staphylococcal enterotoxin A (superantigen A) in the synovial fluid of patients with RA by the PCR and ELISA methods. Methods This experimental study was conducted on the synovial fluid of 103 RA patients from Baqiyatallah University of Medical Sciences’ Rheumatology Clinic in Tehran, Iran in 2011–2014. Bacterial cultures, polymerase chain reaction with specific primer pairs and enzyme-linked immunosorbent assay (ELISA) methods were used. The PCR products were subjected to sequence as a confirmatory molecular method results. The data were descriptively analyzed by SPSS Version 19. Results The bacteriological study result indicated that, in four cases (3.8%) of the patients, bacterial strains were isolated. The result of PCR molecular method for staphylococcal enterotoxin A gene showed that, 42 of the patients (40.7%) tested positive for the ent A gene. The results of ELISA were positive for staphylococcal enterotoxin A (superantigen A) in 51 cases (49.51%) of the patients’ synovial fluids. The results indicated that the possibility of detecting superantigen A in the SF of RA patients, but the origin of the enterotoxin A gene remained unknown. Conclusions The findings of this study may be able to alter the actual theory on the pathogenesis, diagnosis, and treatment of RA patients. In addition, the results have shown the probability of an endogenous origin for the involved superantigen A in RA patients’ synovial fluids. PMID:27053990

  6. Bacteroides fragilis enterotoxin induces cytoskeletal changes and surface blebbing in HT-29 cells.

    PubMed Central

    Donelli, G; Fabbri, A; Fiorentini, C

    1996-01-01

    Certain strains of the anaerobic bacterium Bacteroides fragilis are known to produce an enterotoxin of about 20 kDa which is able to induce a fluid response in ligated intestinal loops and a cytotoxic response in HT-29 cells. It presents protease activity, belonging to a family of metalloproteases termed metzincins. In order to investigate the mode of action of the enterotoxin in cultured cells, we performed a study with HT-29 cells, using both fluoresence and electron microscopy. Treated cells underwent morphological changes, mainly consisting of the retraction of the cell body and the formation of numerous blebs on the cell surface. The microfilament system was reorganized, the F-actin being condensed as a ring at the cell periphery, whereas other cell organelles appeared to be unaffected. All these changes, clearly visible after 3 h of exposure to the toxin, were reversed within 24 h of treatment. By inhibiting the protease activity of the toxin with specific metal chelators, the cytoskeletal effects were also prevented. Thus, B. fragilis enterotoxin appears to act on cells by reversibly modifying the actin cytoskeleton, an effect probably dependent on its proteolytic activity. PMID:8557328

  7. Differential RNA regulation by staphylococcal enterotoxins A and B in murine macrophages

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Beharka, A. A.; Hart, M. E.; Smeltzer, M. S.; Iandolo, J. J.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Staphylococcal enterotoxin A (SEA) is significantly better than enterotoxin B (SEB) in activating tumor necrosis factor (TNF) secretion by B6MP102 cells. Both toxins bound to B6MP102 cells; however, SEB competed less effectively with SEA than SEA competed with SEB. This suggested that receptors unique to SEA were present on B6MP102 cells. Signal transduction occurred in response to both toxins. Within 30 s after addition, SEA and SEB significantly increased the F-actin concentration in B6MP102 cells. However, only SEA induced increased TNF mRNA levels. B6MP102 cells incubated with interferon-gamma and SEB secreted TNF. However, enhanced mRNA expression was delayed and the concentration of TNF secreted was less than that of B6MP102 cells stimulated with SEA. Although these data suggest that receptors unique to SEA are present on B6MP102 cells, they also indicate that staphylococcal enterotoxins differentially regulate TNF at the RNA level, perhaps because of differences in binding to the plasma membrane.

  8. Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

    PubMed Central

    Craven, S E; Blankenship, L C; McDonel, J L

    1981-01-01

    Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed. PMID:6266336

  9. An enterotoxin-negative strain of Yersinia enterocolitica serotype O:3 is capable of producing diarrhea in mice.

    PubMed Central

    Schiemann, D A

    1981-01-01

    A strain of Yersinia enterocolitica serotype O:3 that consistently produced heat-stable enterotoxin at 22 but not at 37 degrees C and another strain of the same serotype which did not produce enterotoxin at 22 degrees C were both positive for autoagglutination at 35 degrees C, a test that has been related to virulence in yersiniae. Both strains were infective for HeLa cells and produced guinea pig conjunctivitis. Mice infected with either strain through their drinking water developed diarrhea and excreted the organism in high numbers in the feces. A control strain of serotype O:3 positive for enterotoxin and HeLa cell infectivity but negative for autoagglutination was avirulent. Extracts of feces and intestines from mice with diarrhea were negative for enterotoxin. The results indicate that the heat-stable enterotoxin produced in vitro by some strains of Y. enterocolitica and measured by the infant mouse assay plays no role in pathogenesis as described by the mouse diarrhea model. PMID:7251137

  10. Comparative Bioinformatics and Experimental Analysis of the Intergenic Regulatory Regions of Bacillus cereus hbl and nhe Enterotoxin Operons and the Impact of CodY on Virulence Heterogeneity.

    PubMed

    Böhm, Maria-Elisabeth; Krey, Viktoria M; Jeßberger, Nadja; Frenzel, Elrike; Scherer, Siegfried

    2016-01-01

    Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5' intergenic regions (5' IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5' IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5' untranslated regions (5' UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5' UTR in B. cereus INRA C3 showed that the entire 331 bp 5' UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5' UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5' IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5' UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. PlcR binding sites are

  11. Comparative Bioinformatics and Experimental Analysis of the Intergenic Regulatory Regions of Bacillus cereus hbl and nhe Enterotoxin Operons and the Impact of CodY on Virulence Heterogeneity

    PubMed Central

    Böhm, Maria-Elisabeth; Krey, Viktoria M.; Jeßberger, Nadja; Frenzel, Elrike; Scherer, Siegfried

    2016-01-01

    Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5′ intergenic regions (5′ IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5′ IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5′ untranslated regions (5′ UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5′ UTR in B. cereus INRA C3 showed that the entire 331 bp 5′ UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5′ UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5′ IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5′ UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. Plc

  12. Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz

    PubMed Central

    Moghassem Hamidi, R; Hosseinzadeh, S; Shekarforoush, S. S.; Poormontaseri, M; Derakhshandeh, A

    2015-01-01

    The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended. PMID:27175208

  13. Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma.

    PubMed

    Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise M; Litvinov, Ivan V; Fredholm, Simon; Petersen, David L; Nastasi, Claudia; Gniadecki, Robert; Mongan, Nigel P; Sasseville, Denis; Wasik, Mariusz A; Bonefeld, Charlotte M; Geisler, Carsten; Woetmann, Anders; Iversen, Lars; Kilian, Mogens; Koralov, Sergei B; Odum, Niels

    2016-03-10

    Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient-derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk-dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis. PMID:26738536

  14. Staphylococcal enterotoxin A (SEA) stimulates STAT3 activation and IL-17 expression in cutaneous T-cell lymphoma

    PubMed Central

    Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise M.; Litvinov, Ivan V.; Fredholm, Simon; Petersen, David L.; Nastasi, Claudia; Gniadecki, Robert; Mongan, Nigel P.; Sasseville, Denis; Wasik, Mariusz A.; Bonefeld, Charlotte M.; Geisler, Carsten; Woetmann, Anders; Iversen, Lars; Kilian, Mogens; Koralov, Sergei B.

    2016-01-01

    Cutaneous T-cell lymphoma (CTCL) is characterized by proliferation of malignant T cells in a chronic inflammatory environment. With disease progression, bacteria colonize the compromised skin barrier and half of CTCL patients die of infection rather than from direct organ involvement by the malignancy. Clinical data indicate that bacteria play a direct role in disease progression, but little is known about the mechanisms involved. Here, we demonstrate that bacterial isolates containing staphylococcal enterotoxin A (SEA) from the affected skin of CTCL patients, as well as recombinant SEA, stimulate activation of signal transducer and activator of transcription 3 (STAT3) and upregulation of interleukin (IL)-17 in immortalized and primary patient–derived malignant and nonmalignant T cells. Importantly, SEA induces STAT3 activation and IL-17 expression in malignant T cells when cocultured with nonmalignant T cells, indicating an indirect mode of action. In accordance, malignant T cells expressing an SEA-nonresponsive T-cell receptor variable region β chain are nonresponsive to SEA in monoculture but display strong STAT3 activation and IL-17 expression in cocultures with SEA-responsive nonmalignant T cells. The response is induced via IL-2 receptor common γ chain cytokines and a Janus kinase 3 (JAK3)-dependent pathway in malignant T cells, and blocked by tofacitinib, a clinical-grade JAK3 inhibitor. In conclusion, we demonstrate that SEA induces cell cross talk–dependent activation of STAT3 and expression of IL-17 in malignant T cells, suggesting a mechanism whereby SEA-producing bacteria promote activation of an established oncogenic pathway previously implicated in carcinogenesis. PMID:26738536

  15. Integrins alpha1beta1 and alpha2beta1 are receptors for the rotavirus enterotoxin.

    PubMed

    Seo, Neung-Seon; Zeng, Carl Q-Y; Hyser, Joseph M; Utama, Budi; Crawford, Sue E; Kim, Kate J; Höök, Magnus; Estes, Mary K

    2008-07-01

    Rotavirus NSP4 is a viral enterotoxin capable of causing diarrhea in neonatal mice. This process is initiated by the binding of extracellular NSP4 to target molecule(s) on the cell surface that triggers a signaling cascade leading to diarrhea. We now report that the integrins alpha1beta1 and alpha2beta1 are receptors for NSP4. NSP4 specifically binds to the alpha1 and alpha2 I domains with apparent K(d) = 1-2.7 muM. Binding is mediated by the I domain metal ion-dependent adhesion site motif, requires Mg(2+) or Mn(2+), is abolished with EDTA, and an NSP4 point mutant, E(120)A, fails to bind alpha2 integrin I domain. NSP4 has two distinct integrin interaction domains. NSP4 amino acids 114-130 are essential for binding to the I domain, and NSP4 peptide 114-135 blocks binding of the natural ligand, collagen I, to integrin alpha2. NSP4 amino acids 131-140 are not associated with the initial binding to the I domain, but elicit signaling that leads to the spreading of attached C2C12-alpha2 cells, mouse myoblast cells stably expressing the human alpha2 integrin. NSP4 colocalizes with integrin alpha2 on the basolateral surface of rotavirus-infected polarized intestinal epithelial (Caco-2) cells as well as surrounding noninfected cells. NSP4 mutants that fail to bind or signal through integrin alpha2 were attenuated in diarrhea induction in neonatal mice. These results indicate that NSP4 interaction with integrin alpha1 and alpha2 is an important component of enterotoxin function and rotavirus pathogenesis, further distinguishing this viral virulence factor from other microbial enterotoxins. PMID:18587047

  16. Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus aureus Enterotoxin B

    PubMed Central

    2011-01-01

    Background Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays. Results Here we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL. Conclusion The anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies. PMID:21933444

  17. Assay of Blood and Synovial Fluid of Patients With Rheumatoid Arthritis for Staphylococcus aureus Enterotoxin D: Absence of Bacteria But Presence of Its Toxin

    PubMed Central

    Ataee, Ramezan Ali; Kashefi, Reyhane; Alishiri, Gholam Hossein; Esmaieli, Davoud

    2015-01-01

    Background: Rheumatoid arthritis (RA) is the most common chronic inflammatory disease. The staphylococcal superantigens are considered as the causative agent of RA disease. Objectives: This study aimed to assess the presence of staphylococcal enterotoxin D in synovial fluid and blood of patients with RA. Patients and Methods: A total of 120 blood and SF samples of patients with RA were studied. Bacterial culture, primer pairs design, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) methods have been used to assess of the staphylococcal enterotoxin D. The data were analyzed through descriptive statistics. Results: During this study and after sequential subcultures, only 5 bacterial strains were isolated. The results of PCR showed the presence of staphylococcal enterotoxin D gene in almost 50% of SF and also in 48.4% of blood samples of patients with RA. Similarly, the ELISA method detected staphylococcal enterotoxin D in 36.16% of SF and in 33.33% of blood of patients with RA. Conclusions: The result of this study showed that a high percentage of patients with RA have shown staphylococcal enterotoxin D (superantigen D) or entD gene in SF and in blood. However, the origin of this superantigen was not clarified and no Staphylococcus aureus enterotoxin D producer was isolated. This finding indicates other role of this superantigen besides its intoxication. Therefore, staphylococcal enterotoxin D as a biomarker may provide a good model for the diagnosis and treatment of patients with RA. PMID:26870313

  18. Staphylococcal enterotoxin induced mitogenesis: toxin binding and cell-cell interactions.

    PubMed

    Buxser, E S; Bonventre, P F; Archer, D L

    1983-07-01

    The binding characteristics of 125I-labelled staphylococcal enterotoxin A (125I-SEA), a T-cell mitogen, to murine lymphoid cell subpopulations were analyzed. Both T- and B-lymphocytes from murine spleens possess specific binding sites for SEA, as do T-lymphocytes from thymus. B-lymphocytes appear to have a greater capacity for binding of 125-SEA than do T-lymphocytes from either thymus or spleen. Enterotoxin did not specifically bind to thioglycollate-induced peritoneal exudate cells (PECs), used as a source of macrophages. Adherent PECs however, incorporated 125-ISEA by fluid phase endocytosis. When exposed to SEA and thoroughly washed, macrophages stimulate lymphocyte mitogenesis in spleen or thymus cell cultures not directly exposed to toxin. Maximum mitogenic stimulation took place only when both PECs and lymphocytes were exposed to SEA. The presence of splenic B-lymphocytes enhanced the mitogenic response of thymus derived T-cells to SEA. Thus, B-lymphocytes appear to contribute to SEA mitogenesis. These data suggest that mitogenic stimulation and possibly other immunological phenomena associated with SEA occur as a result of complex interactions between cellular components of the immune system. PMID:6605472

  19. Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

    NASA Astrophysics Data System (ADS)

    Gordon, Geoffrey; Lo, Chun-Min

    2007-03-01

    Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

  20. Impacts of enterotoxin gene cluster-encoded superantigens on local and systemic experimental Staphylococcus aureus infections.

    PubMed

    Nowrouzian, F L; Ali, A; Badiou, C; Dauwalder, O; Lina, G; Josefsson, E

    2015-07-01

    Staphylococcus aureus is both a component of the normal skin flora and an important pathogen. It expresses a range of recognized and putative virulence factors, such as enterotoxins with superantigenic properties. Several superantigen genes, i.e., seg, sei, selm, seln, and selo, are encoded by the enterotoxin gene cluster (egc), which is found in the majority of S. aureus isolates. Carriage of egc is associated with fitness of S. aureus in the gut microbiota, but it is not known if it contributes to pathogenicity. We constructed egc+ (functional for the seg, selm, and selo genes) and isogenic egc- S. aureus mutants, and investigated their virulence profiles in murine infection models. No effect of egc was seen in a local skin and soft tissue infection model, but in an invasive infection model, increased weight loss was observed after infection with the egc+ as compared to the egc- mutant. Mortality and arthritis were not affected by egc status. Our data suggest that egc has limited effects on the virulence of S. aureus. It may primarily function as a colonization factor increasing commensal fitness, although it might have some aggravating effects on the infection when the bacteria reach the blood. PMID:25864191

  1. Staphylococcal Enterotoxin B Primes Cytokine Secretion and Lytic Activity in Response to Native Bacterial Antigens

    PubMed Central

    Mason, Kevin M.; Dryden, Tricia D.; Bigley, Nancy J.; Fink, Pamela S.

    1998-01-01

    Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-γ) and interleukin-12 than did sham-injected controls. The majority of IFN-γ production appeared to be CD8+ T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-γ. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to “bystander” fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-γ decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo. PMID:9784507

  2. Staphylococcal Enterotoxin O Exhibits Cell Cycle Modulating Activity

    PubMed Central

    Hodille, Elisabeth; Alekseeva, Ludmila; Berkova, Nadia; Serrier, Asma; Badiou, Cedric; Gilquin, Benoit; Brun, Virginie; Vandenesch, François; Terman, David S.; Lina, Gerard

    2016-01-01

    Maintenance of an intact epithelial barrier constitutes a pivotal defense mechanism against infections. Staphylococcus aureus is a versatile pathogen that produces multiple factors including exotoxins that promote tissue alterations. The aim of the present study is to investigate the cytopathic effect of staphylococcal exotoxins SEA, SEG, SEI, SElM, SElN and SElO on the cell cycle of various human cell lines. Among all tested exotoxins only SEIO inhibited the proliferation of a broad panel of human tumor cell lines in vitro. Evaluation of a LDH release and a DNA fragmentation of host cells exposed to SEIO revealed that the toxin does not induce necrosis or apoptosis. Analysis of the DNA content of tumor cells synchronized by serum starvation after exposure to SEIO showed G0/G1 cell cycle delay. The cell cycle modulating feature of SEIO was confirmed by the flow cytometry analysis of synchronized cells exposed to supernatants of isogenic S. aureus strains wherein only supernatant of the SElO producing strain induced G0/G1 phase delay. The results of yeast-two-hybrid analysis indicated that SEIO’s potential partner is cullin-3, involved in the transition from G1 to S phase. In conclusion, we provide evidence that SEIO inhibits cell proliferation without inducing cell death, by delaying host cell entry into the G0/G1 phase of the cell cycle. We speculate that this unique cell cycle modulating feature allows SEIO producing bacteria to gain advantage by arresting the cell cycle of target cells as part of a broader invasive strategy. PMID:27148168

  3. Staphylococcal Enterotoxin O Exhibits Cell Cycle Modulating Activity.

    PubMed

    Hodille, Elisabeth; Alekseeva, Ludmila; Berkova, Nadia; Serrier, Asma; Badiou, Cedric; Gilquin, Benoit; Brun, Virginie; Vandenesch, François; Terman, David S; Lina, Gerard

    2016-01-01

    Maintenance of an intact epithelial barrier constitutes a pivotal defense mechanism against infections. Staphylococcus aureus is a versatile pathogen that produces multiple factors including exotoxins that promote tissue alterations. The aim of the present study is to investigate the cytopathic effect of staphylococcal exotoxins SEA, SEG, SEI, SElM, SElN and SElO on the cell cycle of various human cell lines. Among all tested exotoxins only SEIO inhibited the proliferation of a broad panel of human tumor cell lines in vitro. Evaluation of a LDH release and a DNA fragmentation of host cells exposed to SEIO revealed that the toxin does not induce necrosis or apoptosis. Analysis of the DNA content of tumor cells synchronized by serum starvation after exposure to SEIO showed G0/G1 cell cycle delay. The cell cycle modulating feature of SEIO was confirmed by the flow cytometry analysis of synchronized cells exposed to supernatants of isogenic S. aureus strains wherein only supernatant of the SElO producing strain induced G0/G1 phase delay. The results of yeast-two-hybrid analysis indicated that SEIO's potential partner is cullin-3, involved in the transition from G1 to S phase. In conclusion, we provide evidence that SEIO inhibits cell proliferation without inducing cell death, by delaying host cell entry into the G0/G1 phase of the cell cycle. We speculate that this unique cell cycle modulating feature allows SEIO producing bacteria to gain advantage by arresting the cell cycle of target cells as part of a broader invasive strategy. PMID:27148168

  4. A probability model for enterotoxin production of Bacillus cereus as a function of pH and temperature

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacillus cereus is frequently isolated from a variety of foods including vegetables, dairy products, meat, and other raw and processed foods. The bacterium is capable of producing enterotoxin and emetic toxin that can cause severe nausea, vomiting and diarrhea. The objectives of this study were to a...

  5. Ocurrence of Staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products

    PubMed Central

    Pelisser, Marcia Regina; Klein, Cátia Silene; Ascoli, Kelen Regina; Zotti, Thaís Regina; Arisi, Ana Carolina Maisonnave

    2009-01-01

    Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products. From 102 CPS isolates, 91 were positive for femA, 10 for sea, 12 for sed and four for see. PMID:24031334

  6. DEVELOPING A FLUORESCENT LATEX MICRO-PARTICLE IMMUNOASSAY USING ALEXA FLUOR 568 FOR DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN A (SEA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcus aureus produces heat stable toxins that cause major foodborne gastroenteritis. Staphylococcal enterotoxin A (SEA) is the most recovered in staphylococcal food poisoning outbreaks. A sensitive method for detection of SEA is needed for food safety and food defense monitoring. Our resea...

  7. AN EVALUATION OF ASCORBIC ACID AS A QUORUM SENSING ANALOGUE TO CONTROL GROWTH, SPORULATION, AND ENTEROTOXIN PRODUCTION IN CLOSTRIDIUM PERFRINGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inhibition of quorum sensing by enterotoxin-producing strains of Clostridium perfringens was investigated. Autoinducer-2 (AI-2) activity was measured in the presence and absence of ascorbic acid (vitamin C; concentrations ranging from 10 to 300 mM), an AI-2 analogue. Subsequent effects on AI-2 pro...

  8. Necrotizing enterocolitis and death in a goat kid associated with enterotoxin (CPE)-producing Clostridium perfringens type A

    PubMed Central

    Fernandez Miyakawa, Mariano E.; Saputo, Julian; St. Leger, Judy; Puschner, Birgit; Fisher, Derek J.; McClane, Bruce A.; Uzal, Francisco A.

    2007-01-01

    A goat kid died after being depressed for several days. No significant gross abnormalities were observed at postmortem examination, while histopathological analysis revealed diffuse necrotizing enterocolitis. Isolation of Clostridium perfringens type A secreting enterotoxin (CPE) and presence of CPE in the small intestine suggest that CPE contributed to the death of this kid. PMID:18189049

  9. Enterotoxin-producing bacteria and parasites in stools of Ethiopian children with diarrhoeal disease.

    PubMed Central

    Wadström, T; Aust-Kettis, A; Habte, D; Holmgren, J; Meeuwisse, G; Möllby, R; Söderlind, O

    1976-01-01

    Enterotoxinogenic bacteria were isolated from 131 (37%) of 354 Ethiopian infants and children with acute gastrointestinal symptoms. Only one of these isolates belonged to the classical enteropathogenic serotypes of Esch. coli. Two colonies from each patient were isolated and tested for production of enterotoxin by the rabbit ileal loop test, the rabbit skin test, and an adrenal cell assay. However, only 38% of the isolated enterotoxinogenic strains were Esch. coli; the others belonged to Klebsiella, Enterobacter, Proteus, Citrobacter, Serratia, and Aeromonas. In 18 patients both isolates were toxinogenic and belonged to different species. The incidence of intestinal parasites was 35% with no apparent correlation to the occurrence of toxinogenic bacteria in the stools. PMID:1008593

  10. Enterotoxin-producing bacteria and parasites in stools of Ethiopian children with diarrhoeal disease.

    PubMed

    Wadström, T; Aust-Kettis, A; Habte, D; Holmgren, J; Meeuwisse, G; Möllby, R; Söderlind, O

    1976-11-01

    Enterotoxinogenic bacteria were isolated from 131 (37%) of 354 Ethiopian infants and children with acute gastrointestinal symptoms. Only one of these isolates belonged to the classical enteropathogenic serotypes of Esch. coli. Two colonies from each patient were isolated and tested for production of enterotoxin by the rabbit ileal loop test, the rabbit skin test, and an adrenal cell assay. However, only 38% of the isolated enterotoxinogenic strains were Esch. coli; the others belonged to Klebsiella, Enterobacter, Proteus, Citrobacter, Serratia, and Aeromonas. In 18 patients both isolates were toxinogenic and belonged to different species. The incidence of intestinal parasites was 35% with no apparent correlation to the occurrence of toxinogenic bacteria in the stools. PMID:1008593