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Sample records for cholera toxin entry

  1. Radiolabelling of cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Santos, R.G.; Neves, Nicoli M.J. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Abdalla, L.F.; Brandao, R.L.; Etchehebehere, L. [Ouro Preto Univ., MG (Brazil). Escola de Farmacia. Lab. de Fisiologia e Bioquimica de Microorganismos; Lima, M.E. de [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Dept. de Bioquimica e Imunologia; Nicoli, J.R. [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Dept. de Microbiologia

    1999-11-01

    Binding of cholera toxin to ganglioside receptors of enterocyte microvilli catalyzes the activation of adenylate cyclase causing a rise in cAMP which final result is a copious diarrhea. Saccharomyces boulardii, a nonpathogenic yeast has been used to prevent diarrhea. Although the antidiarrheic properties of S. boulardii are widely recognized, this yeast has been used on empirical basis, and the mechanism of this protective effect is unknown. The addition of cholera toxin to S. boulardii induces the raising of cAMP that triggers the activation of neutral trehalase. This suggests that toxin specifically binding to cells, is internalized and active the protein phosphorylation cascade. Our objective is labeling the cholera toxin to verify the presence of binding sites on yeast cell surfaces for the cholera toxin. Cholera toxin was radiolabelled with Na {sup 125} I by a chloramine-T method modified from Cuatrecasas and Griffiths et alii. The {sup 125} I-Cholera toxin showed a specific radioactivity at about 1000 cpm/fmol toxin. Biological activity of labeled cholera toxin measured by trehalase activation was similar to the native toxin. (author) 5 refs., 3 figs.; e-mail: nevesmj at urano.cdtn.br

  2. Cholera toxin structure, gene regulation and pathophysiological and immunological aspects.

    Science.gov (United States)

    Sánchez, J; Holmgren, J

    2008-05-01

    Many notions regarding the function, structure and regulation of cholera toxin expression have remained essentially unaltered in the last 15 years. At the same time, recent findings have generated additional perspectives. For example, the cholera toxin genes are now known to be carried by a non-lytic bacteriophage, a previously unsuspected condition. Understanding of how the expression of cholera toxin genes is controlled by the bacterium at the molecular level has advanced significantly and relationships with cell-density-associated (quorum-sensing) responses have recently been discovered. Regarding the cell intoxication process, the mode of entry and intracellular transport of cholera toxin are becoming clearer. In the immunological field, the strong oral immunogenicity of the non-toxic B subunit of cholera toxin (CTB) has been exploited in the development of a now widely licensed oral cholera vaccine. Additionally, CTB has been shown to induce tolerance against co-administered (linked) foreign antigens in some autoimmune and allergic diseases.

  3. Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism

    DEFF Research Database (Denmark)

    Hansen, Gert H; Dalskov, Stine-Mathilde; Rasmussen, Christina Rehné;

    2005-01-01

    accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating...

  4. Binding of cholera toxin to Giardia lamblia.

    OpenAIRE

    McCardell, B. A.; Madden, J M; Stanfield, J T; Tall, B D; Stephens, M. J.

    1987-01-01

    Binding of cholera toxin to Giardia lamblia was demonstrated by two slightly different methods: an immunofluorescence technique using antibody to cholera toxin and anti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate, and a one-step fluorescence method in which G. lamblia was incubated with the B subunit of cholera toxin conjugated to fluorescein isothiocyanate.

  5. Evaluation of Cholera Toxin Expression in Different Populations of Vibrio cholera

    OpenAIRE

    Sedigheh Ebrahimi Kasgari; Mahnaz Nourani; Yousef Yahyapour; Seyed Ehsanollah Mousavi; Enayatollah Kalantar; Hami Kaboosi (PhD); Seyed Mahmoud Amin Marashi

    2015-01-01

    Background: Cholera is one of the most diseases of human. Cholera toxin is the most important pathogenic factor in humans that causes diarrhea. The cholera toxin is produced by V. cholerae and CTXфPhage. Objectives: In this study, we have investigated the production cholera toxin with different density of Vibrio cholerae. Materials and Methods: With this propose we inoculated classical strain O1 of Vibrio cholerae ATCC 14035 and Vibrio cholerae O1biovar El Tor N16961 into th...

  6. Tetra- versus Pentavalent Inhibitors of Cholera Toxin

    NARCIS (Netherlands)

    Fu, Ou; Pukin, Aliaksei V.; Quarles Van Ufford, Linda; Branson, Thomas R.; Thies-Weesie, Dominique M E; Turnbull, W. Bruce; Visser, Gerben M.; Pieters, Roland J.

    2015-01-01

    The five B-subunits (CTB5) of the Vibrio cholerae (cholera) toxin can bind to the intestinal cell surface so the entire AB5 toxin can enter the cell. Simultaneous binding can occur on more than one of the monosialotetrahexosylganglioside (GM1) units present on the cell surface.

  7. Tetra- versus Pentavalent Inhibitors of Cholera Toxin

    NARCIS (Netherlands)

    Fu, Ou; Pukin, Aliaksei V.; Quarles Van Ufford, Linda; Branson, Thomas R.; Thies-Weesie, Dominique M E; Turnbull, W. Bruce; Visser, Gerben M.; Pieters, Roland J.

    2015-01-01

    The five B-subunits (CTB5) of the Vibrio cholerae (cholera) toxin can bind to the intestinal cell surface so the entire AB5 toxin can enter the cell. Simultaneous binding can occur on more than one of the monosialotetrahexosylganglioside (GM1) units present on the cell surface.

  8. Non-O1 Vibrio cholerae in Thailand: homology with cloned cholera toxin genes.

    OpenAIRE

    Hanchalay, S; Seriwatana, J; Echeverria, P.; Holmgren, J.; Tirapat, C.; Moseley, S L; Taylor, D N

    1985-01-01

    We examined 281 non-O1 Vibrio cholerae isolates from Thailand for homology with genes coding for cholera toxin. Five isolates from environmental sources were homologous with the cholera toxin gene probe and produced both the A and B subunits of cholera toxin.

  9. Microbes and microbial Toxins: paradigms for microbial-mucosal toxins. V. Cholera: invasion of the intestinal epithelial barrier by a stably folded protein toxin.

    Science.gov (United States)

    Lencer, W I

    2001-05-01

    Cholera toxin (CT) produced by Vibrio cholerae is the virulence factor responsible for the massive secretory diarrhea seen in Asiatic cholera. To cause disease, CT enters the intestinal epithelial cell as a stably folded protein by co-opting a lipid-based membrane receptor, ganglioside G(M1). G(M1) sorts the toxin into lipid rafts and a retrograde trafficking pathway to the endoplasmic reticulum, where the toxin unfolds and transfers its enzymatic subunit to the cytosol, probably by dislocation through the translocon sec61p. The molecular determinants that drive entry of CT into this pathway are encoded entirely within the structure of the protein toxin itself.

  10. Impact of solar irradiation on cholera toxin secretion by different strains of Vibrio cholerae

    CSIR Research Space (South Africa)

    Ssemakalu, CC

    2013-09-01

    Full Text Available Cholera toxin is the aetiological agent of cholera – a deadly waterborne disease acquired through the consumption of untreated water contaminated with CTX bacteriophage harbouring strains of V. cholerae. Solar disinfection is a re...

  11. 5-hydroxytryptamine release into human jejunum by cholera toxin.

    OpenAIRE

    Bearcroft, C P; Perrett, D.; Farthing, M J

    1996-01-01

    BACKGROUND: Cholera toxin produces intestinal secretion by activation of the adenylate cyclase complex. However animal studies have shown 5-hydroxytryptamine may be released after exposure to cholera toxin, and thereby contribute to the secretory state. AIM: To determine whether cholera toxin releases 5-hydroxytryptamine in human jejunum. SUBJECTS: Seven male subjects were given a subclinical dose of cholera toxin in a paired, controlled, randomised, double blind study. METHODS: A closed 10 c...

  12. Molecular mechanism of acquisition of the cholera toxin genes.

    Science.gov (United States)

    Das, Bhabatosh; Bischerour, Julien; Barre, Francois-Xavier

    2011-02-01

    One of the major pathogenic determinants of Vibrio cholerae, the cholera toxin, is encoded in the genome of a filamentous phage, CTXφ. CTXφ makes use of the chromosome dimer resolution system of V. cholerae to integrate its single stranded genome into one, the other, or both V. cholerae chromosomes. Here, we review current knowledge about this smart integration process.

  13. Characterisation of cholera toxin by liquid chromatography - Electrospray mass spectrometry

    NARCIS (Netherlands)

    Baar, B.L.M. van; Hulst, A.G.; Wils, E.R.J.

    1999-01-01

    Cholera toxin, one of the toxins that may be generated by various strains of the bacterium Vibrio cholerae, can be considered as a substance possibly used in biological warfare. The possibilities of characterising the toxin by liquid chromatography electrospray mass spectrometry (LC-ES-MS) were inve

  14. Characterisation of cholera toxin by liquid chromatography - Electrospray mass spectrometry

    NARCIS (Netherlands)

    Baar, B.L.M. van; Hulst, A.G.; Wils, E.R.J.

    1999-01-01

    Cholera toxin, one of the toxins that may be generated by various strains of the bacterium Vibrio cholerae, can be considered as a substance possibly used in biological warfare. The possibilities of characterising the toxin by liquid chromatography electrospray mass spectrometry (LC-ES-MS) were

  15. Antitoxic immunity to cholera in dogs immunized orally with cholera toxin.

    Science.gov (United States)

    Pierce, N F; Cray, W C; Engel, P F

    1980-02-01

    Colera toxin was evaluated as an oral immunogen against experimental canine cholera. Dogs were immunized orally with 100-microgram doses of purified cholera toxin or comparable doses of crude toxin. Both doses caused moderate diarrhea in most nonimmune dogs. Repeated oral doses (12 doses in 54 days) gave marked protection against the diarrheal effect of oral toxin, provoked a vigorous antitoxic response in jejunal mucosa, and gave nearly complete protection against subsequent oral challenge with living virulent Vibrio cholerae. Protection appeared to be due largely to the antitoxic response in intestinal mucosa. The effectiveness of cholera toxin as an oral vaccine contrasts with the previously described ineffectiveness of toxoid given orally. This study provides an example of mucosal immunity due to a nonreplicating vaccine given orally and suggests that cholera toxin may be useful as a component of an oral vaccine for cholera.

  16. Actions of cholera toxin and the prevention and treatment of cholera

    Science.gov (United States)

    Holmgren, Jan

    1981-07-01

    The drastic intestinal secretion of fluid and electrolytes that is characteristic of cholera is the result of reasonably well understood cellular and biochemical actions of the toxin secreted by Vibrio cholerae. Based on this understanding it is possible to devise new techniques for the treatment and prophylaxis of cholera to complement those based on fluid replacement therapy and sanitation.

  17. Improved purification process for cholera toxin and its application to the quantification of residual toxin in cholera vaccines.

    Science.gov (United States)

    Jang, Hyun; Kim, Hyo Seung; Kim, Jeong Ah; Seo, Jin Ho; Carbis, Rodney

    2009-01-01

    A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-microm crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-microm permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH7.0, containing 1.0M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of 3.1 EU/microg of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a G(M1) ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The G(M1) ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.

  18. Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli.

    OpenAIRE

    Jacob, C O; Leitner, M.; Zamir, A.; Salomon, D.; Arnon, R

    1985-01-01

    A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of ...

  19. Expression and secretion of cholera toxin B subunit in lactobacilli.

    Science.gov (United States)

    Okuno, Takahiro; Kashige, Nobuhiro; Satho, Tomomitsu; Irie, Keiichi; Hiramatsu, Yukihiro; Sharmin, Tanjina; Fukumitsu, Yuki; Uyeda, Saori; Yamada, Seitaro; Harakuni, Tetsuya; Miyata, Takeshi; Arakawa, Takeshi; Imoto, Masumi; Toda, Akihisa; Nakashima, Yukihiko; Miake, Fumio

    2013-01-01

    Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

  20. Internalization of cholera toxin by different endocytic mechanisms

    National Research Council Canada - National Science Library

    Torgersen, M L; Skretting, G; van Deurs, B; Sandvig, K

    2001-01-01

    The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A...

  1. Removal of Cholera Toxin from Aqueous Solution by Probiotic Bacteria

    Directory of Open Access Journals (Sweden)

    Jussi A. O. Meriluoto

    2012-06-01

    Full Text Available Cholera remains a serious health problem, especially in developing countries where basic hygiene standards are not met. The symptoms of cholera are caused by cholera toxin, an enterotoxin, which is produced by the bacterium Vibrio cholerae. We have recently shown that human probiotic bacteria are capable of removing cyanobacterial toxins from aqueous solutions. In the present study we investigate the ability of the human probiotic bacteria, Lactobacillus rhamnosus strain GG (ATCC 53103 and Bifidobacterium longum 46 (DSM 14583, to remove cholera toxin from solution in vitro. Lactobacillus rhamnosus strain GG and Bifidobacterium longum 46 were able to remove 68% and 59% of cholera toxin from aqueous solutions during 18 h of incubation at 37 °C, respectively. The effect was dependent on bacterial concentration and L. rhamnosus GG was more effective at lower bacterial concentrations. No significant effect on cholera toxin concentration was observed when nonviable bacteria or bacterial supernatant was used.

  2. Cholera- and anthrax-like toxins are among several new ADP-ribosyltransferases.

    Directory of Open Access Journals (Sweden)

    Robert J Fieldhouse

    Full Text Available Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins we identified and characterized using in silico and cell-based techniques. We also uncovered medically relevant toxins from Mycobacterium avium and Enterococcus faecalis. We found agriculturally relevant toxins in Photorhabdus luminescens and Vibrio splendidus. These toxins belong to the ADP-ribosyltransferase family that has conserved structure despite low sequence identity. Therefore, our search for new toxins combined fold recognition with rules for filtering sequences--including a primary sequence pattern--to reduce reliance on sequence identity and identify toxins using structure. We used computers to build models and analyzed each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. We confirmed activity using a yeast growth test. In this era where an expanding protein structure library complements abundant protein sequence data--and we need high-throughput validation--our approach provides insight into the newest toxin ADP-ribosyltransferases.

  3. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    Science.gov (United States)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E; Cervin, Jakob; Dedic, Benjamin; Rodriguez, Andrea C; Nischan, Nicole; Bond, Michelle R; Mettlen, Marcel; Trudgian, David C; Lemoff, Andrew; Quiding-Järbrink, Marianne; Gustavsson, Bengt; Steentoft, Catharina; Clausen, Henrik; Mirzaei, Hamid; Teneberg, Susann; Yrlid, Ulf; Kohler, Jennifer J

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera. DOI: http://dx.doi.org/10.7554/eLife.09545.001 PMID:26512888

  4. Toxin(s), Other Than Cholera Toxin, Produced by Environmental Non O1 Non O139 Vibrio cholerae

    Institute of Scientific and Technical Information of China (English)

    Kohinur Begum; Chowdhury R. Ahsan; Mohammad Ansaruzzaman; Dilip K. Dutta; Qazi S.Ahmad; Kaisar A. Talukder

    2006-01-01

    A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis.Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin (ST), as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary (CHO) cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin (CT). Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen (CAMP) method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

  5. Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.

    OpenAIRE

    Sandvig, K.; Garred, O; van Deurs, B.

    1996-01-01

    Cholera toxin is normally observed only in the Golgi apparatus and not in the endoplasmic reticulum (ER) although the enzymatically active A subunit of cholera toxin has a KDEL sequence. Here we demonstrate transport of horseradish peroxidase-labeled cholera toxin to the ER by electron microscopy in thapsigargin-treated A431 cells. Thapsigargin treatment strongly increased cholera toxin-induced cAMP production, and the formation of the catalytically active A1 fragment was somewhat increased. ...

  6. Isolation of isoelectrically pure cholera toxin for crystallization

    Science.gov (United States)

    Spangler, Brenda D.; Westbrook, Edwin M.

    1991-03-01

    We have determined that the failure of cholera toxin to crystallize well results from its isoelectric heterogeneity, which is probably due to a post-translational process such as deamidation of its B subunit. Every sample of cholera toxin we have examined from commercial or academic suppliers has been heterogeneous; heterogeneous cholera toxin does not crystallize satisfactorily. We have overcome this problem by using ion-exchange fast protein liquid chromatography (FPLC) to obtain an isoelectrically homogeneous species of cholera toxin. Homogeneous cholera toxin crystallizes readily, forming single, nonmosaic crystals suitable for X-ray diffraction studies. For this process, protein was applied to a MonoQ ion-exchange column, then eluted with an isocratic low salt buffer followed by a linear salt gradient (0-100 mM NaCl). Column fractions were analyzed on isoelectric focusing gels, and those fractions containing the desired homogeneous species were pooled and concentrated. Crystals formed within 24 to 48 h in a MOPS/PEG buffer, which made use of slow isoelectric precipitation to induce crystallization.

  7. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    Directory of Open Access Journals (Sweden)

    Keegan J. Baldauf

    2015-03-01

    Full Text Available Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT, which consists of two subunits: the A subunit (CTA and the B subunit (CTB. CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  8. Cholera toxin B: one subunit with many pharmaceutical applications.

    Science.gov (United States)

    Baldauf, Keegan J; Royal, Joshua M; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2015-03-20

    Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  9. Functional characterization of cholera toxin inhibitors using human intestinal organoids

    NARCIS (Netherlands)

    Zomer-van Ommen, Domenique D.; Pukin, Aliaksei V.; Fu, Ou; Quarles Van Ufford, Linda H C; Janssens, Hettie M.; Beekman, Jeffrey M.; Pieters, Roland J.

    2016-01-01

    Preclinical drug testing in primary human cell models that recapitulate disease can significantly reduce animal experimentation and time-to-the-clinic. We used intestinal organoids to quantitatively study the potency of multivalent cholera toxin inhibitors. The method enabled the determination of IC

  10. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    DEFF Research Database (Denmark)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we...

  11. Functional characterization of cholera toxin inhibitors using human intestinal organoids

    NARCIS (Netherlands)

    Zomer-van Ommen, Domenique D.; Pukin, Aliaksei V.; Fu, Ou; Quarles Van Ufford, Linda H C; Janssens, Hettie M.; Beekman, Jeffrey M.; Pieters, Roland J.

    2016-01-01

    Preclinical drug testing in primary human cell models that recapitulate disease can significantly reduce animal experimentation and time-to-the-clinic. We used intestinal organoids to quantitatively study the potency of multivalent cholera toxin inhibitors. The method enabled the determination of

  12. Strong Inhibition of Cholera Toxin by Multivalent GM1 Derivatives

    NARCIS (Netherlands)

    Pukin, A.V.; Branderhorst, H.M.; Sisu, C.; Weijers, C.A.G.M.; Gilbert, M.; Liskamp, R.M.J.; Visser, G.M.; Zuilhof, H.; Pieters, R.J.

    2007-01-01

    (Chemical Equation Presented) Sticky fingers. The optimal ligand for the cholera toxin (CT), GM1-oligosaccharide (GM1os), was linked to dendritic structures that contained long spacer arms by using highly efficient "click" chemistry coupling. In the inhibition studies very large multivalency effects

  13. Molecular Dynamics Simulation of Cholera Toxin A-1 Polypeptide

    Directory of Open Access Journals (Sweden)

    Badshah Syed Lal

    2016-01-01

    Full Text Available A molecular dynamics (MD simulation study of the enzymatic portion of cholera toxin; cholera toxin A-1 polypeptide (CTA1 was performed at 283, 310 and 323 K. From total energy analysis it was observed that this toxin is stable thermodynamically and these outcomes were likewise confirmed by root mean square deviations (RMSD investigations. The Cα root mean square fluctuation (RMSF examinations revealed that there are a number of residues inside CTA1, which can be used as target for designing and synthesizing inhibitory drugs, in order to inactivate cholera toxin inside the human body. The fluctuations in the radius of gyration and hydrogen bonding in CTA1 proved that protein unfolding and refolding were normal routine phenomena in its structure at all temperatures. Solvent accessible surface area study identified the hydrophilic nature of the CTA1, and due to this property it can be a potential biological weapon. The structural identification (STRIDE algorithm for proteins was successfully used to determine the partially disordered secondary structure of CTA1. On account of this partially disordered secondary structure, it can easily deceive the proteolytic enzymes of the endoplasmic reticulum of host cells.

  14. Synthetic multivalent ligands for cholera & cholera-like toxins: Protected cyclic neoglycopeptides.

    Science.gov (United States)

    Kumar, Vajinder; Yadav, Narender; Kartha, K P Ravindranathan

    2016-08-05

    Synthesis of a set of novel glycopeptide analogues as potential cholera/cholera-like toxin inhibitors in their protected form is described. They include di-, tri-, tetra- and pentavalent scaffolds. The synthetic steps were achieved using a combination of solvent-free mechanochemical as well as the conventional solution-phase reactions. During the conventional DIC-HOBt-mediated peptide coupling followed for the preparation of certain glycopeptide analogues an interesting in situ Fmoc deprotection was observed which has been demonstrated to hold potential for synthesiszing glycopeptides/neoglycopeptides with extended polyamide chains.

  15. Cholera Toxin Production Induced upon Anaerobic Respiration is Suppressed by Glucose Fermentation in Vibrio cholerae.

    Science.gov (United States)

    Oh, Young Taek; Lee, Kang-Mu; Bari, Wasimul; Kim, Hwa Young; Kim, Hye Jin; Yoon, Sang Sun

    2016-03-01

    The causative agent of pandemic cholera, Vibrio cholerae, infects the anaerobic environment of the human intestine. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly induced during anaerobic respiration with trimethylamine N-oxide (TMAO) as an alternative electron acceptor. However, the molecular mechanism of TMAO-stimulated CT production is not fully understood. Herein, we reveal that CT production during anaerobic TMAO respiration is affected by glucose fermentation. When the seventh pandemic V. cholerae O1 strain N16961 was grown with TMAO and additional glucose, CT production was markedly reduced. Furthermore, an N16961 Δcrp mutant, devoid of cyclic AMP receptor protein (CRP), was defective in CT production during growth by anaerobic TMAO respiration, further suggesting a role of glucose metabolism in regulating TMAO-mediated CT production. TMAO reductase activity was noticeably decreased when grown together with glucose or by mutation of the crp gene. A CRP binding region was identified in the promoter region of the torD gene, which encodes a structural subunit of the TMAO reductase. Gel shift assays further confirmed the binding of purified CRP to the torD promoter sequence. Together, our results suggest that the bacterial ability to respire using TMAO is controlled by CRP, whose activity is dependent on glucose availability. Our results reveal a novel mechanism for the regulation of major virulence factor production by V. cholerae under anaerobic growth conditions.

  16. Gas phase characterization of the noncovalent quaternary structure of cholera toxin and the cholera toxin B subunit pentamer.

    Science.gov (United States)

    Williams, Jonathan P; Smith, Daniel C; Green, Brian N; Marsden, Brian D; Jennings, Keith R; Roberts, Lynne M; Scrivens, James H

    2006-05-01

    Cholera toxin (CTx) is an AB5 cytotonic protein that has medical relevance in cholera and as a novel mucosal adjuvant. Here, we report an analysis of the noncovalent homopentameric complex of CTx B chain (CTx B5) using electrospray ionization triple quadrupole mass spectrometry and tandem mass spectrometry and the analysis of the noncovalent hexameric holotoxin usingelectrospray ionization time-of-flight mass spectrometry over a range of pH values that correlate with those encountered by this toxin after cellular uptake. We show that noncovalent interactions within the toxin assemblies were maintained under both acidic and neutral conditions in the gas phase. However, unlike the related Escherichia coli Shiga-like toxin B5 pentamer (SLTx B), the CTx B5 pentamer was stable at low pH, indicating that additional interactions must be present within the latter. Structural comparison of the CTx B monomer interface reveals an additional alpha-helix that is absent in the SLTx B monomer. In silico energy calculations support interactions between this helix and the adjacent monomer. These data provide insight into the apparent stabilization of CTx B relative to SLTx B.

  17. Activation of cholera toxin production by anaerobic respiration of trimethylamine N-oxide in Vibrio cholerae.

    Science.gov (United States)

    Lee, Kang-Mu; Park, Yongjin; Bari, Wasimul; Yoon, Mi Young; Go, Junhyeok; Kim, Sang Cheol; Lee, Hyung-Il; Yoon, Sang Sun

    2012-11-16

    Vibrio cholerae is a gram-negative bacterium that causes cholera. Although the pathogenesis caused by this deadly pathogen takes place in the intestine, commonly thought to be anaerobic, anaerobiosis-induced virulence regulations are not fully elucidated. Anerobic growth of the V. cholerae strain, N16961, was promoted when trimethylamine N-oxide (TMAO) was used as an alternative electron acceptor. Strikingly, cholera toxin (CT) production was markedly induced during anaerobic TMAO respiration. N16961 mutants unable to metabolize TMAO were incapable of producing CT, suggesting a mechanistic link between anaerobic TMAO respiration and CT production. TMAO reductase is transported to the periplasm via the twin arginine transport (TAT) system. A similar defect in both anaerobic TMAO respiration and CT production was also observed in a N16961 TAT mutant. In contrast, the abilities to grow on TMAO and to produce CT were not affected in a mutant of the general secretion pathway. This suggests that V. cholerae may utilize the TAT system to secrete CT during TMAO respiration. During anaerobic growth with TMAO, N16961 cells exhibit green fluorescence when stained with 2',7'-dichlorofluorescein diacetate, a specific dye for reactive oxygen species (ROS). Furthermore, CT production was decreased in the presence of an ROS scavenger suggesting a positive role of ROS in regulating CT production. When TMAO was co-administered to infant mice infected with N16961, the mice exhibited more severe pathogenic symptoms. Together, our results reveal a novel anaerobic growth condition that stimulates V. cholerae to produce its major virulence factor.

  18. Cross-reactivity of outer membrane proteins of Campylobacter species with cholera toxin

    OpenAIRE

    Albert, M. John

    2011-01-01

    Campylobacter jejuni is a foodborne pathogen and a leading cause of diarrhoea worldwide. It is believed that a cholera toxin-like toxin (CTLT) produced by C. jejuni may mediate watery diarrhoea. However, the production of a CTLT by C. jejuni is controversial. A cholera toxin gene (ctx) homologue has not been identified in Campylobacter species. We investigated the identity of the CT cross-reactive antigen from Campylobacter species previously and the results are reviewed here. Filtrates of C....

  19. Hybrid microarray based on double biomolecular markers of DNA and carbohydrate for simultaneous genotypic and phenotypic detection of cholera toxin-producing Vibrio cholerae.

    Science.gov (United States)

    Shin, Hwa Hui; Seo, Jeong Hyun; Kim, Chang Sup; Hwang, Byeong Hee; Cha, Hyung Joon

    2016-05-15

    Life-threatening diarrheal cholera is usually caused by water or food contaminated with cholera toxin-producing Vibrio cholerae. For the prevention and surveillance of cholera, it is crucial to rapidly and precisely detect and identify the etiological causes, such as V. cholerae and/or its toxin. In the present work, we propose the use of a hybrid double biomolecular marker (DBM) microarray containing 16S rRNA-based DNA capture probe to genotypically identify V. cholerae and GM1 pentasaccharide capture probe to phenotypically detect cholera toxin. We employed a simple sample preparation method to directly obtain genomic DNA and secreted cholera toxin as target materials from bacterial cells. By utilizing the constructed DBM microarray and prepared samples, V. cholerae and cholera toxin were detected successfully, selectively, and simultaneously; the DBM microarray was able to analyze the pathogenicity of the identified V. cholerae regardless of whether the bacteria produces toxin. Therefore, our proposed DBM microarray is a new effective platform for identifying bacteria and analyzing bacterial pathogenicity simultaneously. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Microfluidic biosensor for cholera toxin detection in fecal samples.

    Science.gov (United States)

    Bunyakul, Natinan; Promptmas, Chamras; Baeumner, Antje J

    2015-01-01

    Sample preparation and processing steps are the most critical assay aspects that require our attention in the development of diagnostic devices for analytes present in complex matrices. In the best scenarios, diagnostic devices should use only simple sample processing. We have therefore investigated minimal preparation of stool samples and their effect on our sensitive microfluidic immunosensor for the detection of cholera toxin. This biosensor was previously developed and tested in buffer solutions only, using either fluorescence or electrochemical detection strategies. The microfluidic devices were made from polydimethylsiloxane using soft lithography and silicon templates. Cholera toxin subunit B (CTB)-specific antibodies immobilized onto superparamagnetic beads and ganglioside GM1-containing liposomes were used for CTB recognition in the detection system. Quantification of CTB was tested by spiking it in human stool samples. Here, optimal minimal sample processing steps, including filtration and centrifugation, were optimized using a microtiter plate assay owing to its high-throughput capabilities. Subsequently, it was transferred to the microfluidic systems, enhancing the diagnostic characteristic of the biosensor. It was found that the debris removal obtained through simple centrifugation resulted in an acceptable removal of matrix effects for the fluorescence format, reaching a limit of detection of only 9.0 ng/mL. However, the electron transfer in the electrochemical format was slightly negatively affected (limit of detection of 31.7 ng/mL). Subsequently, cross-reactivity using the heat-labile Escherichia coli toxin was investigated using the electrochemical microfluidic immunosensors and was determined to be negligible. With minimal sample preparation required, these microfluidic liposome-based systems have demonstrated excellent analytical performance in a complex matrix and will thus be applicable to other sample matrices.

  1. A Protein-Based Pentavalent Inhibitor of the Cholera Toxin B-Subunit

    NARCIS (Netherlands)

    Branson, T.R.; McAllister, T.E.; Garcia Hartjes, J.; Fascione, M.A.; Ross, J.F.; Warriner, S.L.; Wennekes, T.; Zuilhof, H.; Turnbull, W.B.

    2014-01-01

    Protein toxins produced by bacteria are the cause of many life-threatening diarrheal diseases. Many of these toxins, including cholera toxin (CT), enter the cell by first binding to glycolipids in the cell membrane. Inhibiting these multivalent protein/carbohydrate interactions would prevent the

  2. Cholera toxin expression by El Tor Vibrio cholerae in shallow culture growth conditions.

    Science.gov (United States)

    Cobaxin, Mayra; Martínez, Haydee; Ayala, Guadalupe; Holmgren, Jan; Sjöling, Asa; Sánchez, Joaquín

    2014-01-01

    Vibrio cholerae O1 classical, El Tor and O139 are the primary biotypes that cause epidemic cholera, and they also express cholera toxin (CT). Although classical V. cholerae produces CT in various settings, the El Tor and O139 strains require specific growth conditions for CT induction, such as the so-called AKI conditions, which consist of growth in static conditions followed by growth under aerobic shaking conditions. However, our group has demonstrated that CT production may also take place in shallow static cultures. How these type of cultures induce CT production has been unclear, but we now report that in shallow culture growth conditions, there is virtual depletion of dissolved oxygen after 2.5 h of growth. Concurrently, during the first three to 4 h, endogenous CO2 accumulates in the media and the pH decreases. These findings may explain CT expression at the molecular level because CT production relies on a regulatory cascade, in which the key regulator AphB may be activated by anaerobiosis and by low pH. AphB activation stimulates TcpP synthesis, which induces ToxT production, and ToxT directly stimulates ctxAB expression, which encodes CT. Importantly, ToxT activity is enhanced by bicarbonate. Therefore, we suggest that in shallow cultures, AphB is activated by initial decreases in oxygen and pH, and subsequently, ToxT is activated by intracellular bicarbonate that has been generated from endogenous CO2. This working model would explain CT production in shallow cultures and, possibly, also in other growth conditions.

  3. Picomolar inhibition of cholera toxin by a pentavalent ganglioside GM1os-calix[5]arene

    NARCIS (Netherlands)

    Garcia-Hartjes, J.; Bernardi, S.; Weijers, C.A.G.M.; Wennekes, T.; Gilbert, M.; Sansone, F.; Casnati, A.; Zuilhof, H.

    2013-01-01

    Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells. Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding do

  4. Reinitiation of Growth in Senescent Mouse Mammary Epithelium in Response to Cholera Toxin

    Science.gov (United States)

    Daniel, Charles W.; Silberstein, Gary B.; Strickland, Phyllis

    1984-06-01

    Several lines of mouse mammary tissue that had been serially transplanted until mitotic senescence was reached were exposed in vivo to plastic implants that slowly released cholera toxin. Gland tissue surrounding the implants displayed new end buds, indicating reinitiation of growth and morphogenesis. The ability of cholera toxin, which elevates intracellular adenosine 3',5'-monophosphate, to temporarily reverse the senescent phenotype suggests that this mitotic dysfunction results not from generalized cellular deterioration but from specific changes in cell regulation.

  5. Promotion of colonization and virulence by cholera toxin is dependent on neutrophils.

    Science.gov (United States)

    Queen, Jessica; Satchell, Karla J F

    2013-09-01

    The innate immune response to Vibrio cholerae infection is poorly understood, but this knowledge is critical for the design of safe, effective vaccines. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host immunity, as well as the effect of cholera toxin and other secreted factors on this response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to similar survival rates of mice infected with low or moderate doses of toxigenic V. cholerae El Tor O1. At a high dose, neutropenic mice showed increased rates of survival compared to neutrophil-replete animals. Expression of cholera toxin was found to be protective to the neutropenic host, and this phenotype can be replicated by the administration of purified toxin. Neutrophils do not effectively clear colonizing bacteria from the small intestine, nor do they alter induction of early immune-modulating signals. In both neutropenic and neutrophil-replete animals, the local response to infection is characterized by expression of interleukin 6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha (MIP-2). Overall, these data indicate that the innate immune response to toxigenic V. cholerae infection differs dramatically from the host response to nontoxigenic infection or vaccination, where neutrophils are protective to the host. In the absence of neutrophils, cholera toxin induces immunomodulatory effects that increase host survival. In cholera toxin-producing strains, similar to nontoxigenic infection, accessory toxins are critical to virulence, indicating that cholera toxin and the other secreted toxins modulate the host response by different mechanisms, with both contributing to bacterial persistence and virulence.

  6. OBTAINING OF MONOCLONAL ANTIBODIES AGAINST CHOLERA TOXIN AND HEAT LABILE ENTEROTOXIN OF E. coli FOR DEVELOPMENT OF THE TOXINS DIPLEX ANALYSIS IN ENVIRONMENTAL SPECIMENS

    OpenAIRE

    Eu. V. Grishin; T. I. Valiakina

    2013-01-01

    The present study focuses on development of monoclonal antibodies (MAbs) which specifically interact with cholera toxin or heat labile enterotoxin of E. coli. Such monoclonal antibodies MAbs are possessed of ability to identify cholera toxin or heat labile enterotoxin in different immunochemical assays. We obtained hybridoma clones which produced monoclonal antibodies of IgG isotypes to cholera toxin and heat labile enterotoxin. On application of the method of serial dilutions we selected the...

  7. Intranasal immunization with recombinant toxin-coregulated pilus and cholera toxin B subunit protects rabbits against Vibrio cholerae O1 challenge.

    Science.gov (United States)

    Kundu, Juthika; Mazumder, Rupa; Srivastava, Ranjana; Srivastava, Brahm S

    2009-07-01

    Intranasal immunization, a noninvasive method of vaccination, has been found to be effective in inducing systemic and mucosal immune responses. The present study was aimed at investigating the efficacy of intranasal immunization in inducing mucosal immunity in experimental cholera by subunit recombinant protein vaccines from Vibrio cholerae O1. The structural genes encoding toxin-coregulated pilus A (TcpA) and B subunit of cholera toxin (CtxB) from V. cholerae O1 were cloned and expressed in Escherichia coli. Rabbits were immunized intranasally with purified TcpA and CtxB alone or a mixture of TcpA and CtxB. Immunization with TcpA and CtxB alone conferred, respectively, 41.1% and 70.5% protection against V. cholerae challenge, whereas immunization with a mixture of both antigens conferred complete (100%) protection, as assayed in the rabbit ileal loop model. Serum titers of immunoglobulin G (IgG) antibodies to TcpA and CtxB, and anti-TcpA- and anti-CtxB-specific sIgA in intestinal lavage of vaccinated animals were found to be significantly elevated compared with unimmunized controls. Vibriocidal antibodies were detected at remarkable levels in rabbits receiving TcpA antigen and their titers correlated with protection. Thus, mucosal codelivery of pertinent cholera toxoids provides enhanced protection against experimental cholera.

  8. Entry of Shiga toxin into cells

    DEFF Research Database (Denmark)

    Sandvig, Kirsten; van Deurs, Bo

    1994-01-01

    Cellebiologi, Shiga toxin, receptors, glycolipids, endocytosis, trans-Golgi network, endoplasmic reticulum, retrograde transport......Cellebiologi, Shiga toxin, receptors, glycolipids, endocytosis, trans-Golgi network, endoplasmic reticulum, retrograde transport...

  9. Role of receptor-mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity.

    Science.gov (United States)

    El Hage, Tatiana; Merlen, Clémence; Fabrega, Sylvie; Authier, François

    2007-05-01

    Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment, and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin A, Authier F (2005) FEBS J272, 4385-4397]. Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma. Following administration of a saturating dose of cholera toxin to rats, rapid endocytosis of both cholera toxin subunits was observed, coincident with massive internalization of both the 45 kDa and 47 kDa Gsalpha proteins. These events coincided with the endosomal recruitment of ADP-ribosylation factor proteins, especially ADP-ribosylation factor-6, with a time course identical to that of toxin and the A subunit of the stimulatory G protein (Gsalpha) translocation. After an initial lag phase of 30 min, these constituents were linked to NAD-dependent ADP-ribosylation of endogenous Gsalpha, with maximum accumulation observed at 30-60 min postinjection. Assessment of the subsequent postendosomal fate of internalized Gsalpha revealed sustained endolysosomal transfer of the two Gsalpha isoforms. Concomitantly, cholera toxin increased in vivo endosome acidification rates driven by the ATP-dependent H(+)-ATPase pump and in vitro vacuolar acidification in hepatoma HepG2 cells. The vacuolar H(+)-ATPase inhibitor bafilomycin and the cathepsin D inhibitor pepstatin A partially inhibited, both in vivo and in vitro, the cAMP response to cholera toxin. This cathepsin D-dependent action of cholera toxin under the control of endosomal acidity was confirmed using cellular systems in which modification of the expression levels of cathepsin D, either by transfection of the cathepsin D gene or small interfering RNA, was followed by parallel changes in the cytotoxic

  10. Immunization with cholera toxin B subunit induces high-level protection in the suckling mouse model of cholera.

    Directory of Open Access Journals (Sweden)

    Gregory A Price

    Full Text Available Cholera toxin (CT is the primary virulence factor responsible for severe cholera. Vibrio cholerae strains unable to produce CT show severe attenuation of virulence in animals and humans. The pentameric B subunit of CT (CTB contains the immunodominant epitopes recognized by antibodies that neutralize CT. Although CTB is a potent immunogen and a promising protective vaccine antigen in animal models, immunization of humans with detoxified CT failed to protect against cholera. We recently demonstrated however that pups reared from mice immunized intraperitoneally (IP with 3 doses of recombinant CTB were well protected against a highly lethal challenge dose of V. cholerae N16961. The present study investigated how the route and number of immunizations with CTB could influence protective efficacy in the suckling mouse model of cholera. To this end female mice were immunized with CTB intranasally (IN, IP, and subcutaneously (SC. Serum and fecal extracts were analyzed for anti-CTB antibodies by quantitative ELISA, and pups born to immunized mothers were challenged orogastrically with a lethal dose of V. cholerae. Pups from all immunized groups were highly protected from death by 48 hours (64-100% survival. Cox regression showed that percent body weight loss at 24 hours predicted death by 48 hours, but we were unable to validate a specific amount of weight loss as a surrogate marker for protection. Although CTB was highly protective in all regimens, three parenteral immunizations showed trends toward higher survival and less weight loss at 24 hours post infection. These results demonstrate that immunization with CTB by any of several routes and dosing regimens can provide protection against live V. cholerae challenge in the suckling mouse model of cholera. Our data extend the results of previous studies and provide additional support for the inclusion of CTB in the development of a subunit vaccine against V. cholerae.

  11. Immunization with cholera toxin B subunit induces high-level protection in the suckling mouse model of cholera.

    Science.gov (United States)

    Price, Gregory A; McFann, Kim; Holmes, Randall K

    2013-01-01

    Cholera toxin (CT) is the primary virulence factor responsible for severe cholera. Vibrio cholerae strains unable to produce CT show severe attenuation of virulence in animals and humans. The pentameric B subunit of CT (CTB) contains the immunodominant epitopes recognized by antibodies that neutralize CT. Although CTB is a potent immunogen and a promising protective vaccine antigen in animal models, immunization of humans with detoxified CT failed to protect against cholera. We recently demonstrated however that pups reared from mice immunized intraperitoneally (IP) with 3 doses of recombinant CTB were well protected against a highly lethal challenge dose of V. cholerae N16961. The present study investigated how the route and number of immunizations with CTB could influence protective efficacy in the suckling mouse model of cholera. To this end female mice were immunized with CTB intranasally (IN), IP, and subcutaneously (SC). Serum and fecal extracts were analyzed for anti-CTB antibodies by quantitative ELISA, and pups born to immunized mothers were challenged orogastrically with a lethal dose of V. cholerae. Pups from all immunized groups were highly protected from death by 48 hours (64-100% survival). Cox regression showed that percent body weight loss at 24 hours predicted death by 48 hours, but we were unable to validate a specific amount of weight loss as a surrogate marker for protection. Although CTB was highly protective in all regimens, three parenteral immunizations showed trends toward higher survival and less weight loss at 24 hours post infection. These results demonstrate that immunization with CTB by any of several routes and dosing regimens can provide protection against live V. cholerae challenge in the suckling mouse model of cholera. Our data extend the results of previous studies and provide additional support for the inclusion of CTB in the development of a subunit vaccine against V. cholerae.

  12. Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds

    Science.gov (United States)

    Cherubin, Patrick; Garcia, Maria Camila; Curtis, David; Britt, Christopher B. T.; Craft, John W.; Burress, Helen; Berndt, Chris; Reddy, Srikar; Guyette, Jessica; Zheng, Tianyu; Huo, Qun; Quiñones, Beatriz; Briggs, James M.

    2016-01-01

    Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT. PMID:27829022

  13. Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds.

    Science.gov (United States)

    Cherubin, Patrick; Garcia, Maria Camila; Curtis, David; Britt, Christopher B T; Craft, John W; Burress, Helen; Berndt, Chris; Reddy, Srikar; Guyette, Jessica; Zheng, Tianyu; Huo, Qun; Quiñones, Beatriz; Briggs, James M; Teter, Ken

    2016-01-01

    Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.

  14. In Vitro Inhibition of Cholera Toxin Production in Vibrio cholerae by Methanol Extract of Sweet Fennel Seeds and Its Components.

    Science.gov (United States)

    Chatterjee, Shruti; Zahid, M Shamim Hasan; Awasthi, Sharda Prasad; Chowdhury, Nityananda; Asakura, Masahiro; Hinenoya, Atsushi; Ramamurthy, T; Iwaoka, Emiko; Aoki, Shunji; Yamasaki, Shinji

    2016-09-21

    A newly emerged Vibrio cholerae O1 El Tor variant strain with multidrug resistance is considered a threat to public health. Recent strategies to suppress virulence factors production instead of bacterial growth may lead to less selective pressure for the emergence of resistant strains. The use of spices and their active constituents as the inhibitory agents against cholera toxin (CT) production in V. cholerae may be an alternative approach to treat cholera. In this study, we examined the potential of sweet fennel seed (Foeniculum vulgare Miller var. dulce) methanol extract to inhibit CT production in V. cholerae without affecting viability. The methanol extract of sweet fennel seeds significantly inhibited CT production in various V. cholerae strains, regardless of serogroup or biotype. Interestingly, trans-anethole and 4-allylanisole, essential oil components of sweet fennel seeds, also demonstrated similar effects. Here, we report that sub-bactericidal concentrations of sweet fennel seed methanol extract and its major components can drastically inhibit CT production in various V. cholerae strains.

  15. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    Science.gov (United States)

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  16. Prolonged colonization of mice by Vibrio cholerae El Tor O1 depends on accessory toxins.

    Science.gov (United States)

    Olivier, Verena; Salzman, Nita H; Satchell, Karla J Fullner

    2007-10-01

    Cholera epidemics caused by Vibrio cholerae El Tor O1 strains are typified by a large number of asymptomatic carriers who excrete vibrios but do not develop diarrhea. This carriage state was important for the spread of the seventh cholera pandemic as the bacterium was mobilized geographically, allowing the global dispersion of this less virulent strain. Virulence factors associated with the development of the carriage state have not been previously identified. We have developed an animal model of cholera in adult C57BL/6 mice wherein V. cholerae colonizes the mucus layer and forms microcolonies in the crypts of the distal small bowel. Colonization occurred 1 to 3 h after oral inoculation and peaked at 10 to 12 h, when bacterial loads exceeded the inoculum by 10- to 200-fold, indicating bacterial growth within the small intestine. After a clearance phase, the number of bacteria within the small intestine, but not those in the cecum or colon, stabilized and persisted for at least 72 h. The ability of V. cholerae to prevent clearance and establish this prolonged colonization was associated with the accessory toxins hemolysin, the multifunctional autoprocessing RTX toxin, and hemagglutinin/protease and did not require cholera toxin or toxin-coregulated pili. The defect in colonization attributed to the loss of the accessory toxins may be extracellularly complemented by inoculation of the defective strain with an isogenic colonization-proficient V. cholerae strain. This work thus demonstrates that secreted accessory toxins modify the host environment to enable prolonged colonization of the small intestine in the absence of overt disease symptoms and thereby contribute to disease dissemination via asymptomatic carriers.

  17. Conjugated Linoleic Acid Reduces Cholera Toxin Production In Vitro and In Vivo by Inhibiting Vibrio cholerae ToxT Activity.

    Science.gov (United States)

    Withey, Jeffrey H; Nag, Drubhajyoti; Plecha, Sarah C; Sinha, Ritam; Koley, Hemanta

    2015-12-01

    The severe diarrheal disease cholera is endemic in over 50 countries. Current therapies for cholera patients involve oral and/or intravenous rehydration, often combined with the use of antibiotics to shorten the duration and intensity of the disease. However, as antibiotic resistance increases, treatment options will become limited. Linoleic acid has been shown to be a potent negative effector of V. cholerae virulence that acts on the major virulence transcription regulator protein, ToxT, to inhibit virulence gene expression. ToxT activates transcription of the two major virulence factors required for disease, cholera toxin (CT) and toxin-coregulated pilus (TCP). A conjugated form of linoleic acid (CLA) is currently sold over the counter as a dietary supplement and is generally recognized as safe by the U.S. Food and Drug Administration. This study examined whether CLA could be used as a new therapy to reduce CT production, which, in turn, would decrease disease duration and intensity in cholera patients. CLA could be used in place of traditional antibiotics and would be very unlikely to generate resistance, as it affects only virulence factor production and not bacterial growth or survival. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Changing genotypes of cholera toxin (CT) of Vibrio cholerae O139 in Bangladesh and description of three new CT genotypes.

    Science.gov (United States)

    Bhuiyan, Nurul A; Nusrin, Suraia; Alam, Munirul; Morita, Masatomo; Watanabe, Haruo; Ramamurthy, Thandavarayan; Cravioto, Alejandro; Nair, Gopinath Balakrish

    2009-11-01

    We determined the genotype of cholera toxin by amplifying and sequencing the B-subunit in a sequential collection of 90 strains of Vibrio cholerae O139 isolated over the past 13 years since its first description in 1992. Representative strains isolated during 1993-1997 harboured ctxB of El Tor type (genotype 3). Twenty-six strains isolated during 1999, 2001, 2005 and three strains isolated in 1998, 2000 and 2002 were identified to belong to new ctxB genotypes 4 and 5, respectively. Genotype 5 was similar to genotype 1 except at position 28 (D-->A). The genotype 6 was similar to genotype 4 except at position 34 (H-->P). The implication of switch in terms of function of the toxin and its impact on human disease is unclear. How this change has influenced their prevalence relative to that of V. cholerae O1 in human infection is also not clear. The other common virulence gene clusters including the Vibrio pathogenicity island-1, Vibrio seventh pandemic island (VSP)-I and VSP-II of V. cholerae O139 did not show any remarkable difference from that of the O1 El Tor strains. Overall, the majority of the O139 strains tested in this study were similar to the El Tor strains but had altered ctxB genotype. This change and the impact that it causes to the epidemiology of cholera caused by O139 should be closely monitored.

  19. Conjugated Linoleic Acid Reduces Cholera Toxin Production In Vitro and In Vivo by Inhibiting Vibrio cholerae ToxT Activity

    Science.gov (United States)

    Nag, Drubhajyoti; Plecha, Sarah C.; Sinha, Ritam; Koley, Hemanta

    2015-01-01

    The severe diarrheal disease cholera is endemic in over 50 countries. Current therapies for cholera patients involve oral and/or intravenous rehydration, often combined with the use of antibiotics to shorten the duration and intensity of the disease. However, as antibiotic resistance increases, treatment options will become limited. Linoleic acid has been shown to be a potent negative effector of V. cholerae virulence that acts on the major virulence transcription regulator protein, ToxT, to inhibit virulence gene expression. ToxT activates transcription of the two major virulence factors required for disease, cholera toxin (CT) and toxin-coregulated pilus (TCP). A conjugated form of linoleic acid (CLA) is currently sold over the counter as a dietary supplement and is generally recognized as safe by the U.S. Food and Drug Administration. This study examined whether CLA could be used as a new therapy to reduce CT production, which, in turn, would decrease disease duration and intensity in cholera patients. CLA could be used in place of traditional antibiotics and would be very unlikely to generate resistance, as it affects only virulence factor production and not bacterial growth or survival. PMID:26392502

  20. Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay.

    Science.gov (United States)

    Khan, Sadia Afrin; DeGrasse, Jeffrey A; Yakes, Betsy Jean; Croley, Timothy R

    2015-09-10

    Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV-Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.

  1. A large cholera outbreak due to a new cholera toxin variant of the Vibrio cholerae O1 El Tor biotype in Orissa, Eastern India.

    Science.gov (United States)

    Kumar, P; Jain, M; Goel, A K; Bhadauria, S; Sharma, S K; Kamboj, D V; Singh, L; Ramamurthy, T; Nair, G B

    2009-02-01

    A total of 32 Vibrio cholerae isolates were collected during a recent large cholera outbreak in Eastern India. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. Two multiplex PCR assays confirmed the presence of various toxigenic and pathogenic genes - ace, ctxAB, hlyA, ompU, ompW, rfbO1, rtx, tcp, toxR and zot - in all of the isolates. Sequencing of the ctxB gene from the isolates revealed a novel mutation in the gene. Sequencing also confirmed the presence of altered cholera toxin B of the classical biotype in all of the El Tor isolates, suggesting infection of isolates by classical CTXPhi. The molecular diversity of V. cholerae isolates studied by enterobacterial repetitive intergenic consensus sequence PCR, BOX-PCR and randomly amplified polymorphic DNA analysis uniformly showed the clonal relationship among the outbreak V. cholerae O1 isolates. The results of this study suggest that cholera-causing V. cholerae strains are constantly evolving in epidemic areas, highlighting the potential of the emergence of more virulent strains.

  2. Expression of toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

    Science.gov (United States)

    Sharma, Manoj Kumar; Singh, Nirmal Kumar; Jani, Dewal; Sisodia, Rama; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

    2008-02-01

    For protection against cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio cholerae infections. Earlier, expression of cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and G(M1)-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against cholera infection.

  3. Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae

    Directory of Open Access Journals (Sweden)

    M Boustanshenas

    2013-01-01

    Full Text Available Purpose: The aim of this study was to assess the production of recombinant cholera toxin B subunit (rCTB protein in two different expression systems (pAE_ctxB and pQE_ctxB constructs in Escherichia coli BL21 (DE3. Materials and Methods: The ctxB fragment was amplified from Vibrio cholerae O 1 ATCC14035 and cloned in pGETM-T easy vector after which it was transformed to E. coli Top 10F′ and grown on LB-ampicillin agar medium. Sequence analysis confirmed the complete ctxB gene sequence in the construct which was further subcloned to pQE-30 vector. The construct was subsequently transformed to E. coli M15 (pREP4. The recombinant pAE_ctxB and pQE_ctxB were transformed to competent E. coli BL21 (DE3 cells to express CTB protein. Result: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE analysis showed the maximum expression of rCTB in both systems at 5 h after induction and western blot analysis confirmed the presence of recombinant CTB in blotting membranes. Conclusion: Expression of rCTB in pAE_ctxB construct was more efficient (15-fold than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3 is more compatible with the former construct. This expression system can be used to produce recombinant CTB in high yield which may enable us to study the oral tolerance or mucosal adjuvant properties of rCTB using animal models.

  4. Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

    Energy Technology Data Exchange (ETDEWEB)

    Jorgensen, Rene; Purdy, Alexandra E.; Fieldhouse, Robert J.; Kimber, Matthew S.; Bartlett, Douglas H.; Merrill, A. Rod (Guelph); (NIH); (UCSD)

    2008-07-15

    The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 {+-} 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 {+-} 2 {mu}g/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-{angstrom} resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.

  5. Nanomechanical detection of cholera toxin using microcantilevers functionalized with ganglioside nanodiscs

    Energy Technology Data Exchange (ETDEWEB)

    Tark, Soo-Hyun; Dravid, Vinayak P [Department of Materials Science and Engineering, Northwestern University, Evanston, IL 60208 (United States); Das, Aditi; Sligar, Stephen, E-mail: s-sligar@illinois.edu, E-mail: v-dravid@northwestern.edu [Department of Biochemistry and Chemistry, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States)

    2010-10-29

    The label-free detection of cholera toxin is demonstrated using microcantilevers functionalized with ganglioside nanodiscs. The cholera toxin molecules bind specifically to the active membrane protein encased in nanodiscs, nanoscale lipid bilayers surrounded by an amphipathic protein belt, immobilized on the cantilever surface. The specific molecular binding results in cantilever deflection via the formation of a surface stress-induced bending moment. The nanomechanical cantilever response is quantitatively monitored by optical interference. The consistent and reproducible nanomechanical detection of cholera toxin in nanomolar range concentrations is demonstrated. The results validated with such a model system suggest that the combination of a microcantilever platform with receptor nanodiscs is a promising approach for monitoring invasive pathogens and other types of biomolecular detection relevant to drug discovery.

  6. Single-mode tapered optical fiber loop immunosensor II: assay of anti-cholera toxin immunoglobulins

    Science.gov (United States)

    Marks, Robert S.; Hale, Zoe M.; Levine, Myron M.; Lowe, C. R.; Payne, Frank P.

    1994-07-01

    An evanescent wave immunoassay for cholera antitoxin immunoglobulins was performed using a single mode tapered optical fiber loop sensor. The transducer was silanized with 3- glycidoxypropyltrimethoxysilane and chemically modified to link covalently either cholera toxin B subunit or a synthetic peptide derived from it, CTP3. The sensor was exposed to seral fluids, obtained from human volunteers having been exposed to live virulent Vibrio cholerae 01 and shown to produce rice-water stools. Other toxins of interest, such as Clostridium botulinum toxin A, have been tested on similar systems. The bound unlabelled immunoglobulins were then exposed to a mixture of FITC-anti-IgG and TRITC-anti-IgA, without requirement for a separation step. The emanating fluorescent emissions of fluorescein and rhodamine, excited by the input laser light, were coupled back into the guided mode of the tapered fiber, and used to determine the concentrations of the complementary antigens.

  7. Nanomechanical detection of cholera toxin using microcantilevers functionalized with ganglioside nanodiscs

    Science.gov (United States)

    Tark, Soo-Hyun; Das, Aditi; Sligar, Stephen; Dravid, Vinayak P.

    2010-10-01

    The label-free detection of cholera toxin is demonstrated using microcantilevers functionalized with ganglioside nanodiscs. The cholera toxin molecules bind specifically to the active membrane protein encased in nanodiscs, nanoscale lipid bilayers surrounded by an amphipathic protein belt, immobilized on the cantilever surface. The specific molecular binding results in cantilever deflection via the formation of a surface stress-induced bending moment. The nanomechanical cantilever response is quantitatively monitored by optical interference. The consistent and reproducible nanomechanical detection of cholera toxin in nanomolar range concentrations is demonstrated. The results validated with such a model system suggest that the combination of a microcantilever platform with receptor nanodiscs is a promising approach for monitoring invasive pathogens and other types of biomolecular detection relevant to drug discovery.

  8. Blood Group O-Dependent Cellular Responses to Cholera Toxin: Parallel Clinical and Epidemiological Links to Severe Cholera.

    Science.gov (United States)

    Kuhlmann, F Matthew; Santhanam, Srikanth; Kumar, Pardeep; Luo, Qingwei; Ciorba, Matthew A; Fleckenstein, James M

    2016-08-03

    Because O blood group has been associated with more severe cholera infections, it has been hypothesized that cholera toxin (CT) may bind non-O blood group antigens of the intestinal mucosae, thereby preventing efficient interaction with target GM1 gangliosides required for uptake of the toxin and activation of cyclic adenosine monophosphate (cAMP) signaling in target epithelia. Herein, we show that after exposure to CT, human enteroids expressing O blood group exhibited marked increase in cAMP relative to cells derived from blood group A individuals. Likewise, using CRISPR/Cas9 engineering, a functional group O line (HT-29-A(-/-)) was generated from a parent group A HT-29 line. CT stimulated robust cAMP responses in HT-29-A(-/-) cells relative to HT-29 cells. These findings provide a direct molecular link between blood group O expression and differential cellular responses to CT, recapitulating clinical and epidemiologic observations.

  9. Lipid requirements for entry of protein toxins into cells.

    Science.gov (United States)

    Sandvig, Kirsten; Bergan, Jonas; Kavaliauskiene, Simona; Skotland, Tore

    2014-04-01

    The plant toxin ricin and the bacterial toxin Shiga toxin both belong to a group of protein toxins having one moiety that binds to the cell surface, and another, enzymatically active moiety, that enters the cytosol and inhibits protein synthesis by inactivating ribosomes. Both toxins travel all the way from the cell surface to endosomes, the Golgi apparatus and the ER before the ribosome-inactivating moiety enters the cytosol. Shiga toxin binds to the neutral glycosphingolipid Gb3 at the cell surface and is therefore dependent on this lipid for transport into the cells, whereas ricin binds both glycoproteins and glycolipids with terminal galactose. The different steps of transport used by these toxins have specific requirements for lipid species, and with the recent developments in mass spectrometry analysis of lipids and microscopical and biochemical dissection of transport in cells, we are starting to see the complexity of endocytosis and intracellular transport. In this article we describe lipid requirements and the consequences of lipid changes for the entry and intoxication with ricin and Shiga toxin. These toxins can be a threat to human health, but can also be exploited for diagnosis and therapy, and have proven valuable as tools to study intracellular transport. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Lack of immunological analogy between the beta-subunits of cholera toxin and human choriogonadotropin.

    Science.gov (United States)

    Acevedo, H F; Kellen, J A

    1986-01-01

    A chemical relatedness has been described between the beta-subunit of cholera toxin and that of the four dimeric glycoprotein hormones (hCG, hLH, hFSH and hTSH). However, antibodies induced by cholera toxin did not crossreact, when tested by labeled hCG binding and immunocytochemistry, with the beta-subunit of hCG. It appears that differences in the tertiary structures, as shown in this study, account for distinct epitopes. Similarities in biological activity between these two compounds, such as induction of adenyl cyclase or a protective effect against some tumors, are not based on immunological mechanisms.

  11. Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

    Science.gov (United States)

    Sharma, Manoj Kumar; Jani, Dewal; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

    2008-05-20

    In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

  12. OBTAINING OF MONOCLONAL ANTIBODIES AGAINST CHOLERA TOXIN AND HEAT LABILE ENTEROTOXIN OF E. coli FOR DEVELOPMENT OF THE TOXINS DIPLEX ANALYSIS IN ENVIRONMENTAL SPECIMENS

    Directory of Open Access Journals (Sweden)

    Eu. V. Grishin

    2013-08-01

    Full Text Available The present study focuses on development of monoclonal antibodies (MAbs which specifically interact with cholera toxin or heat labile enterotoxin of E. coli. Such monoclonal antibodies MAbs are possessed of ability to identify cholera toxin or heat labile enterotoxin in different immunochemical assays. We obtained hybridoma clones which produced monoclonal antibodies of IgG isotypes to cholera toxin and heat labile enterotoxin. On application of the method of serial dilutions we selected the clones which produced monoclonal antibodies with specific activity against only one of the toxins. We found the 16 pairs of monoclonal antibodies to cholera toxin and 28 ones to heat labile enterotoxin. By means of these monoclonal antibodies it was possible to realize the quantitative analysis of theses toxins in sandwich immunoassay ELISA and diplex sandwich xMAP-assay. The limits of detection of cholera toxin and heat labile enterotoxin in ELISA in control buffer were 0.2 and 0.4 ng/ml, respectively, and in xMAP assay — 0.01 and 0.08 ng/ml, respectively. In probes of cow milk, meat soup, pond water and nasopharyngeal washes cholera toxin was detected in the both assays with the same limits of detections, but heat labile enterotoxin limits of detections were above the ones in control buffers.

  13. A therapeutic chemical chaperone inhibits cholera intoxication and unfolding/translocation of the cholera toxin A1 subunit.

    Science.gov (United States)

    Taylor, Michael; Banerjee, Tuhina; Navarro-Garcia, Fernando; Huerta, Jazmin; Massey, Shane; Burlingame, Mansfield; Pande, Abhay H; Tatulian, Suren A; Teter, Ken

    2011-04-19

    Cholera toxin (CT) travels as an intact AB(5) protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera.

  14. Radioimmune assay of ganglioside GM/sub 1/ synthase using cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Honke, K.; Taniguchi, N.; Makita, A.

    1986-01-01

    A radioimmune assay for uridine 5'-diphosphate-galactose (UDP-Gal):GM/sub 2/ galactosyltransferase, which synthesizes GM/sub 1/, has been developed utilizing cholera toxin. This assay is more sensitive and simpler than previously used assays. Radioactive nucleotide substrate and GM/sub 2/ were incubated with an enzyme sample, and a radiolabeled product, GM/sub 1/, was reacted with cholera toxin. The GM/sub 1/-cholera toxin complex was further reacted with anti-cholera toxin and Staphylococcus aureus cell suspension. The resulting complex was transferred onto a nitrocellulose membrane and quantitated by liquid scintillation counting. This assay was found to be sensitive for the detection of 100 pmol of the reaction product, GM/sub 1/. With this assay method, some properties of the crude enzyme extracts from rat liver were studied. The enzyme had a pH optimum of 6.5-7.0 and required Mn/sup 2 +/. The K/sub m/ values for UDP-Gal and GM/sub 2/ were 0.12 mM and 6 ..mu..M, respectively.

  15. Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization

    NARCIS (Netherlands)

    Slos, P.; Speck, D.; Accart, N.; Kolbe, H.V.; Schubnel, D.; Bouchon, B.; Bischoff, Rainer; Kieny, M.P.

    1994-01-01

    The gene coding for cholera toxin subunit B (CT-B) was fused to a modified ompA signal sequence and subsequently cloned into a high expression vector based on the regulatory signals of the arabinose operon of Salmonella typhimurium. Upon induction of gene expression in Escherichia coli, a product of

  16. Crystallization of the HigBA2 toxin-antitoxin complex from Vibrio cholerae

    DEFF Research Database (Denmark)

    Hadǽi, San; Garcia-Pino, Abel; Martinez-Rodriguez, Sergio

    2013-01-01

    The genome of Vibrio cholerae encodes two higBA toxin-antitoxin (TA) modules that are activated by amino-acid starvation. Here, the TA complex of the second module, higBA2, as well as the C-terminal domain of the corresponding HigA2 antitoxin, have been purified and crystallized. The HigBA2 complex...

  17. Recombinant cholera toxin B subunit in Escherichia coli: high-level secretion, purification, and characterization

    NARCIS (Netherlands)

    Slos, P.; Speck, D.; Accart, N.; Kolbe, H.V.; Schubnel, D.; Bouchon, B.; Bischoff, Rainer; Kieny, M.P.

    1994-01-01

    The gene coding for cholera toxin subunit B (CT-B) was fused to a modified ompA signal sequence and subsequently cloned into a high expression vector based on the regulatory signals of the arabinose operon of Salmonella typhimurium. Upon induction of gene expression in Escherichia coli, a product of

  18. Multivalent drug design and inhibition of cholera toxin by specific and transient protein-ligand interactions.

    Science.gov (United States)

    Liu, Jiyun; Begley, Darren; Mitchell, Daniel D; Verlinde, Christophe L M J; Varani, Gabriele; Fan, Erkang

    2008-05-01

    Multivalent inhibitors of the cholera toxin B pentamer are potential therapeutic drugs for treating cholera and serve as models for demonstrating multivalent ligand effects through a structure-based approach. A crucial yet often overlooked aspect of multivalent drug design is the length, rigidity and chemical composition of the linker used to connect multiple binding moieties. To specifically study the role of chemical linkers in multivalent ligand design, we have synthesized a series of compounds with one and two binding motifs connected by several different linkers. These compounds have affinity for and potency against the cholera toxin B pentamer despite the fact that none can simultaneously bind two toxin receptor sites. Results from saturation transfer difference NMR reveal transient, non-specific interactions between the cholera toxin and linker groups contribute significantly to overall binding affinity of monovalent compounds. However, the same random protein-ligand interactions do not appear to affect binding of bivalent molecules. Moreover, the binding affinities and potencies of these 'non-spanning' bivalent ligands appear to be wholly independent of linker length. Our detailed analysis identifies multiple effects that account for the improved inhibitory potencies of bivalent ligands and suggest approaches to further improve the activity of this class of compounds.

  19. Cholera toxin production during anaerobic trimethylamine N-oxide respiration is mediated by stringent response in Vibrio cholerae.

    Science.gov (United States)

    Oh, Young Taek; Park, Yongjin; Yoon, Mi Young; Bari, Wasimul; Go, Junhyeok; Min, Kyung Bae; Raskin, David M; Lee, Kang-Mu; Yoon, Sang Sun

    2014-05-09

    As a facultative anaerobe, Vibrio cholerae can grow by anaerobic respiration. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly promoted during anaerobic growth using trimethylamine N-oxide (TMAO) as an alternative electron acceptor. Here, we investigated the molecular mechanisms of TMAO-stimulated CT production and uncovered the crucial involvement of stringent response in this process. V. cholerae 7th pandemic strain N16961 produced a significantly elevated level of ppGpp, the bacterial stringent response alarmone, during anaerobic TMAO respiration. Bacterial viability was impaired, and DNA replication was also affected under the same growth condition, further suggesting that stringent response is induced. A ΔrelA ΔspoT ppGpp overproducer strain produced an enhanced level of CT, whereas anaerobic growth via TMAO respiration was severely inhibited. In contrast, a ppGpp-null strain (ΔrelA ΔspoT ΔrelV) grew substantially better, but produced no CT, suggesting that CT production and bacterial growth are inversely regulated in response to ppGpp accumulation. Bacterial capability to produce CT was completely lost when the dksA gene, which encodes a protein that works cooperatively with ppGpp, was deleted. In the ΔdksA mutant, stringent response growth inhibition was alleviated, further supporting the inverse regulation of CT production and anaerobic growth. In vivo virulence of ΔrelA ΔspoT ΔrelV or ΔdksA mutants was significantly attenuated. The ΔrelA ΔspoT mutant maintained virulence when infected with exogenous TMAO despite its defective growth. Together, our results reveal that stringent response is activated under TMAO-stimulated anaerobic growth, and it regulates CT production in a growth-dependent manner in V. cholerae.

  20. Development of an Immunochromatographic Test Strip for Detection of Cholera Toxin

    Directory of Open Access Journals (Sweden)

    Eiki Yamasaki

    2013-01-01

    Full Text Available Because cholera toxin (CT is responsible for most of the symptoms induced by Vibrio cholerae infection, detection of CT is critical for diagnosis of the disease. In this study, we constructed an immunochromatographic test strip for detection of CT (CT-IC with polyclonal antibodies developed against purified recombinant whole CT protein. The detection limit of the CT-IC was 10 ng/mL of purified recombinant CT, and it could detect the CT in culture supernatant of all 15 toxigenic V. cholerae isolates examined, whereas no false-positive signal was detected in all 5 nontoxigenic V. cholerae isolates examined. The specificity of the CT-IC was examined with recombinant heat-labile toxin (LT, which shares high homology with CT, and it was revealed that the minimum detection limit for LT was 100 times higher than that for CT. In addition, lt gene-positive enterotoxigenic Escherichia coli (ETEC was examined by CT-IC. The false-positive signals were observed in 3 out of 12 ETEC isolates, but these signals were considerably faint. The CT-IC did not develop false-positive signals with all 7 V. parahaemolyticus isolates. These results showed the high specificity of CT-IC and the feasible use of it for the detection and surveillance of toxigenic V. cholerae.

  1. Neoglycolipid analogues of ganglioside G sub M1 as functional receptors of cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Pacuszka, T.; Bradley, R.M.; Fishman, P.H. (National Inst. of Health, Bethesda, MD (United States))

    1991-03-12

    The authors synthesized several lipid analogues of ganglioside G{sub M1} by attaching its oligosaccharide moiety (G{sub M1}OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. They incubated G{sub M1}-deficient rat glioma C6 cells with each of the derivatives as well as native G{sub M1} and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of {sup 125}I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C{sub 10} derivative was ineffective, C{sub 12} and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native G{sub M1}, whereas the phospholipid derivatives were less effective. The distance between G{sub M1}OS and the phospholipid also appeared to influence its functional activity. The results indicate that although G{sub M1}OS provides the recognition site for the binding of cholera toxin, the nature of the lipid moiety plays an important role in the action of the toxin.

  2. Multiple domains of tetanus toxin direct entry into primary neurons

    OpenAIRE

    Blum, Faith C.; Tepp, William H.; Eric A. Johnson; Barbieri, Joseph T

    2014-01-01

    Tetanus toxin elicits spastic paralysis by cleaving VAMP-2 to inhibit neurotransmitter release in inhibitory neurons of the central nervous system. While the retrograde transport of TeNT from endosomes has been described, the initial steps that define how TeNT initiates trafficking to the retrograde system are undefined. The current study examines TeNT entry into primary cultured cortical neurons by TIRF microscopy. The initial association of TeNT with the plasma membrane was dependent upon g...

  3. Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium

    Science.gov (United States)

    Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.

    1986-08-01

    The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

  4. Orphan nuclear receptor Nur77 is required for the differentiation of C6 glioma cells induced by cholera toxin

    Institute of Scientific and Technical Information of China (English)

    Dong XU; Yi-jun HUANG; Yan LI; Wei YIN; Guang-mei YAN

    2009-01-01

    Aim: To investigate a possible regulator gene involved in the cholera toxin-induced differentiation of rat C6 glioma cells. Methods: The global changes in the mRNA expression pattern induced by cholera toxin were analyzed using gene chip microarray. The selected gene was then silenced by RNA interference or overexpressed with an ORF plasmid to determine its necessity in this process. Results: Nur77, a member of the orphan nuclear receptor family (NR4A), was markedly up-regulated during the process of differentiation. Furthermore, RNAi of nur77 attenuated the induction effect of cholera toxin on C6 cells, whereas overexpression of nur77 led to similarly differentiated behavior, including morphologic and biomarker changes, as well as cell cycle arrest. Conclusion: Nur77 participated actively and essentially as an important regulator in the cholera toxin-induced differentiation of C6 cells.

  5. Determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and BALB/c mouse macrophages.

    Science.gov (United States)

    Tamari, Farshad; Tychowski, Joanna; Lorentzen, Laura

    2013-05-30

    The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be cholera infections.

  6. GALACTOSE-BINDING SITE IN ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN (LT) AND CHOLERA-TOXIN (CT)

    NARCIS (Netherlands)

    MERRITT, EA; SIXMA, TK; KALK, KH; VANZANTEN, BAM; HOL, WGJ

    The galactose-binding site in cholera toxin and the closely related heat-labile enterotoxin (LT) from Escherichia coil is an attractive target for the rational design of potential anti-cholera drugs. In this paper we analyse the molecular structure of this binding site as seen in several crystal

  7. GALACTOSE-BINDING SITE IN ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN (LT) AND CHOLERA-TOXIN (CT)

    NARCIS (Netherlands)

    MERRITT, EA; SIXMA, TK; KALK, KH; VANZANTEN, BAM; HOL, WGJ

    1994-01-01

    The galactose-binding site in cholera toxin and the closely related heat-labile enterotoxin (LT) from Escherichia coil is an attractive target for the rational design of potential anti-cholera drugs. In this paper we analyse the molecular structure of this binding site as seen in several crystal str

  8. GALACTOSE-BINDING SITE IN ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN (LT) AND CHOLERA-TOXIN (CT)

    NARCIS (Netherlands)

    MERRITT, EA; SIXMA, TK; KALK, KH; VANZANTEN, BAM; HOL, WGJ

    1994-01-01

    The galactose-binding site in cholera toxin and the closely related heat-labile enterotoxin (LT) from Escherichia coil is an attractive target for the rational design of potential anti-cholera drugs. In this paper we analyse the molecular structure of this binding site as seen in several crystal str

  9. [Evaluation of toxin producing abilities of non-O1/non-O139 serogroup Vibrio cholerae isolated from humans].

    Science.gov (United States)

    Telesmanich, N R; Akulova, M V; Lomov, Iu M; Alekseeva, L P; Kruglikova, V D; Agafonova, V V; Tatarenko, O A; Evdokimova, V V

    2011-01-01

    Determination of non-O1/non-O139 Vibrio cholerae toxin (CT) gene expression by using EIA, and biological effect of non-O1/non-O139 V. cholerae supernatant on cell cultures evaluation. 39 V. cholerae strains from various serological groups were studied. Hemolytic activity of strains was determined by using Greig test, and cholera toxin production--in GM1-EIA and in continuous cell lines by registering cytotonic, cytotoxic and proteolitic effect. GM1-EIA method does not detect CT production in 29 museum strains of non-O1/non-O139 V. cholerae in vitro. CT was detected only in 1 non-O1/non-O139 V. cholerae strain supernatant with OD = 0.577 that is substantially lower than in O1 V. cholerae strains (OD = 2.176). In cell cultures non-O1/non-O139 V. cholerae supernatants diluted to 1:100 caused elongation only in single cells. Cytological model is a more sensitive technique to evaluate toxin producing abilities of non-O1/non-O139 V. cholerae strains and is appropriate for use.

  10. Cystic Fibrosis Heterozygote Resistance to Cholera Toxin in the Cystic Fibrosis Mouse Model

    Science.gov (United States)

    Gabriel, Sherif E.; Brigman, Kristen N.; Koller, Beverly H.; Boucher, Richard C.; Stutts, M. Jackson

    1994-10-01

    The effect of the number of cystic fibrosis (CF) alleles on cholera toxin (CT)-induced intestinal secretion was examined in the CF mouse model. CF mice that expressed no CF transmembrane conductance regulator (CFTR) protein did not secrete fluid in response to CT. Heterozygotes expressed 50 percent of the normal amount of CFTR protein in the intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion in response to CT. This correlation between CFTR protein and CT-induced chloride ion and fluid secretion suggests that CF heterozygotes might possess a selective advantage of resistance to cholera.

  11. Role of 6-gingerol in reduction of cholera toxin activity in vitro and in vivo.

    Science.gov (United States)

    Saha, Pallashri; Das, Bornita; Chaudhuri, Keya

    2013-09-01

    Vibrio cholerae is one of the major bacterial pathogens responsible for the devastating diarrheal disease called cholera. Chemotherapy is often used against V. cholerae infections; however, the emergence of V. cholerae with multidrug resistance (MDR) toward the chemotherapeutic agents is a serious clinical problem. This scenario has provided us with the impetus to look into herbal remediation, especially toward blocking the action of cholera toxin (CT). Our studies were undertaken to determine the antidiarrheal potential of 6-gingerol (6G) on the basis of its effect on CT, the virulence factor secreted by V. cholerae. We report here that 6G binds to CT, hindering its interaction with the GM1 receptor present on the intestinal epithelial cells. The 50% inhibitory concentration (IC50) was determined to be 10 μg/ml. The detailed mechanistic study was conducted by enzyme-linked immunosorbent assay (ELISA), fluorescence spectroscopy, and isoelectric focusing. These results were validated with in vitro studies performed with the CHO, HeLa, and HT-29 cell lines, whereas a rabbit ileal loop assay was done to estimate the in vivo action, which confirms the efficacy of 6G in remediation of the choleragenic effects of CT. Thus, 6G can be an effective adjunctive therapy with oral rehydration solution for severe CT-mediated diarrhea.

  12. Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins.

    Science.gov (United States)

    Markov, Alexander G; Falchuk, Evgeny L; Kruglova, Natalia M; Rybalchenko, Oksana V; Fromm, Michael; Amasheh, Salah

    2014-11-01

    Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.

  13. Ganglioside embedded in reconstituted lipoprotein binds cholera toxin with elevated affinity.

    Science.gov (United States)

    Bricarello, Daniel A; Mills, Emily J; Petrlova, Jitka; Voss, John C; Parikh, Atul N

    2010-09-01

    The ability to exogenously present cell-surface receptors in high-affinity conformations in a synthetic system offers an opportunity to provide host cells with protection from pathogenic toxins. This strategy requires improvement of the synthetic receptor binding affinity against its native counterpart, particularly with polyvalent toxins where clustering of membrane receptors can hinder binding. Here we demonstrate that reconstituted lipoprotein, nanometer-sized discoidal lipid bilayers bounded by apolipoprotein and functionalized by incorporation of pathogen receptors, provides a means to enhance toxin-receptor binding through molecular-level control over the receptor microenvironment (specifically, its rigidity, composition, and heterogeneity). Using a Foerster Resonance Energy Transfer (FRET)-based assay, we found that reconstituted lipoprotein incorporating low concentrations of ganglioside monosialotetrahexosylganglioside (GM1) binds polymeric cholera toxin with significantly higher affinity than liposomes or supported lipid bilayers, most likely a result of the enhanced control over receptor clustering provided by the lipoprotein platform. Using wide-area epifluorescence, we found that this enhanced binding capacity can be effectively utilized to divert cholera toxin away from populations of healthy mammalian cells. In summary, we found that reconstitutions of high-density lipoprotein can be engineered to include specific pathogen receptors; that their pathogen binding affinity is altered, presumably due to attenuation of receptor aggregation; and that these assemblies are effective at protecting cells from biological toxins.

  14. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø;

    1997-01-01

    The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions...... in the fimA gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fimA in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted...... with respect to host background in three different Escherichia coli strains, i.e. an isogenic set of K-12 strains, differing in the presence of an indigenous fim gene cluster, as well as a wild-type isolate. Immunization of rabbits with purified chimeric fimbriae resulted in serum which specifically recognized...

  15. Toxin-coregulated pilus-loaded microparticles as a vaccine against Vibrio cholerae O139

    Institute of Scientific and Technical Information of China (English)

    杜艳; 贾文祥; 刘莉

    2004-01-01

    @@ The cholera epidemics is an important public health problem in many developing countries. Highly effective and preventive vaccines against cholera are under investigation as alternatives to the one available presently. Much of the vaccine research focuses on colonization factors. Colonization of a human by the Vibrio cholerae (V. cholerae Ol strain is mediated by toxin-coregulated pilus (TCP), 1 which was shown to play a role in the infant mouse cholera model and subsequently in human volunteers. 2 TCP-loaded vaccines could potentially provide cross-protection among experimental strains. Data have indicated that poly (D,L-lactide)-polyethylene glycol copolymer (PELA)microparticles loaded antigens were strongly immunogenic, 3 and that these microparticles served as an effective delivery system for a single dose of vaccine. 4Microparticle formulation could represent the next generation of vaccines, as they are highly effective at delivery of vaccines, thus requiring fewer doses. 5 We prepared PELA microparticles loaded with TCP for testing as a vaccine; their targeting distributions were identified and related immune responses were analyzed.

  16. Prolonged pretreatment of mice with cholera toxin, but not isoproterenol, alleviates acute lethal systemic inflammatory response.

    Science.gov (United States)

    Wang, Jingyang; Guo, Xiangrui; Cao, Junxia; Zhang, Xueying; Zhang, Jiyan; Sun, Dejun; Wang, Qingyang

    2014-11-01

    Isoproterenol, a synthetic non-selective β-adrenergic agonist, is often used during the immediate postoperative period after open heart surgery for its chronotropic and vasodilatory effects. It has been demonstrated that isoproterenol pretreatment followed by immediate LPS administration leads to reduced tumor necrosis factor-α (TNF-α) response in vivo. However, sepsis never happens immediately after the surgery, but rather severe immune dysfunction occurs at least 24h later. It remains elusive what effects isoproterenol might exert to innate immunity during the period. In this scenario, we investigated the effects of 24-h isoproterenol pretreatment on septic shock induced by experimental endotoxemia and bacterial peritonitis, with cholera toxin as another cAMP elevator. Unexpectedly, we found that isoproterenol and cholera toxin exhibited distinct effects in acute lethal systemic inflammatory response. Isoproterenol worsened liver injury without enhancing NK/NKT activity. Meanwhile, cholera toxin but not isoproterenol showed dramatically reduced TNF-α response in LPS induced septic shock. Our data provide a caution for the clinical use of isoproterenol and suggest that isoproterenol has cAMP-independent functions.

  17. Cholera Toxin Suppresses Expression of Ubiquitin Editing Enzyme A20 and Enhances Transcytosis

    Directory of Open Access Journals (Sweden)

    Ming-Yang Li

    2013-03-01

    Full Text Available Background: The ubiquitin editing enzyme A20 plays an important role in maintaining the homeostasis in the body Microbe-derived adjuvants are commonly used in animal models of intestinal allergy. Objective: This study aims to investigate the role of cholera toxin-induced A20 suppression in compromising intestinal barrier function. Methods: Human intestinal epithelial cells were cultured into monolayers as an in vitro epithelial barrier model. Ovalbumin (OVA was used as a specific allergen to test the degrading capability of intestinal epithelial cells for the endocytic allergens. The fusion of endosomes and lysosomes in epithelial cells was observed by immunocytochemistry. The antigenicity of OVA was tested by T cell proliferation assay. Results: A20 was detectable in the intestinal cell lines and mouse intestinal epithelialum. A20 was required in the degradation of endocytic allergens in HT-29 cells. The allergen, OVA, could pass through A20-deficient HT-29 monolayer barrier. Exposure to microbial adjuvant, cholera toxin, suppressed the expression of A20 in HT-29 cells, which compromised the epithelial barrier function. Conclusion: The microbial product, cholera toxin, interferes with the expression of A20 in intestinal epithelial cells, which compromises the intestinal epithelial barrier function.

  18. Inhibition of cholera toxin and other AB toxins by polyphenolic compounds

    Science.gov (United States)

    All AB-type protein toxins have intracellular targets despite an initial extracellular location. These toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against AB toxins are therefore hard to develop because the toxins use dif...

  19. Cholera

    OpenAIRE

    Harris, Jason B.; LaRocque, Regina C; Qadri, Firdausi; Edward T. Ryan; Calderwood, Stephen B.

    2012-01-01

    Cholera is an acute, secretory diarrhea caused by infection with Vibrio cholerae of the O1 and O139 serogroups. Cholera is endemic in over 50 countries and also causes large epidemics. Since 1817, seven cholera pandemics have spread from Asia to much of the world. The 7th pandemic began in 1961 and affects 3–5 million people each year, killing 120,000. Although mild cholera may be indistinguishable from other diarrheal illnesses, the presentation of severe cholera is distinct, with dramatic d...

  20. The three-dimensional crystal structure of cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Rong-Guang; Westbrook, M.L.; Nance, S.; Spangler, B.D. [Argonne National Lab., IL (United States); Scott, D.L. [Yale Univ., New Haven, CT (United States). Dept. of Molecular Biophysics and Biochemistry; Westbrook, E.M. [Northwestern Univ., Evanston, IL (United States)

    1996-02-01

    The clinical manifestations of cholera are largely attributable to the actions of a secreted hexameric AB{sub 5} enterotoxin (choleragen). We have solved the three-dimensional structure of choleragen at 2.5 {Angstrom} resolution and compared the refined coordinates with those of choleragenoid (isolated B pentamer) and the heat-labile enterotoxin from Escherichia coli (LT). The crystalline coordinates provide a detailed view of the stereochemistry implicated in binding to GM1 gangliosides and in carrying out ADP-ribosylation. The A2 chain of choleragen, in contrast to that of LT, is a nearly continuous {alpha}-helix with an interpretable carboxyl tail.

  1. A Conformational Shift in the Dissociated Cholera Toxin A1 Subunit Prevents Reassembly of the Cholera Holotoxin.

    Science.gov (United States)

    Taylor, Michael; Curtis, David; Teter, Ken

    2015-07-20

    Cholera toxin (CT) consists of a catalytic A1 subunit, an A2 linker, and a homopentameric cell-binding B subunit. The intact holotoxin moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) where CTA1 is released from the rest of the toxin. The dissociated CTA1 subunit then shifts to an unfolded conformation, which triggers its export to the cytosol by a process involving the quality control system of ER-associated degradation (ERAD). We hypothesized that the unfolding of dissociated CTA1 would prevent its non-productive reassociation with CTA2/CTB5. To test this prediction, we monitored the real-time reassociation of CTA1 with CTA2/CTB5 by surface plasmon resonance. Folded but not disordered CTA1 could interact with CTA2/CTB5 to form a stable, functional holotoxin. Our data, thus, identified another role for the intrinsic instability of the isolated CTA1 polypeptide in host-toxin interactions: in addition to activating the ERAD translocation mechanism, the spontaneous unfolding of free CTA1 at 37 °C prevents the non-productive reassembly of a CT holotoxin in the ER.

  2. A Conformational Shift in the Dissociated Cholera Toxin A1 Subunit Prevents Reassembly of the Cholera Holotoxin

    Directory of Open Access Journals (Sweden)

    Michael Taylor

    2015-07-01

    Full Text Available Cholera toxin (CT consists of a catalytic A1 subunit, an A2 linker, and a homopentameric cell-binding B subunit. The intact holotoxin moves by vesicle carriers from the cell surface to the endoplasmic reticulum (ER where CTA1 is released from the rest of the toxin. The dissociated CTA1 subunit then shifts to an unfolded conformation, which triggers its export to the cytosol by a process involving the quality control system of ER-associated degradation (ERAD. We hypothesized that the unfolding of dissociated CTA1 would prevent its non-productive reassociation with CTA2/CTB5. To test this prediction, we monitored the real-time reassociation of CTA1 with CTA2/CTB5 by surface plasmon resonance. Folded but not disordered CTA1 could interact with CTA2/CTB5 to form a stable, functional holotoxin. Our data, thus, identified another role for the intrinsic instability of the isolated CTA1 polypeptide in host-toxin interactions: in addition to activating the ERAD translocation mechanism, the spontaneous unfolding of free CTA1 at 37 °C prevents the non-productive reassembly of a CT holotoxin in the ER.

  3. Inhibition of the sodium-translocating NADH-ubiquinone oxidoreductase [Na+-NQR] decreases cholera toxin production in Vibrio cholerae O1 at the late exponential growth phase.

    Science.gov (United States)

    Minato, Yusuke; Fassio, Sara R; Reddekopp, Rylan L; Häse, Claudia C

    2014-01-01

    Two virulence factors produced by Vibrio cholerae, cholera toxin (CT) and toxin-corregulated pilus (TCP), are indispensable for cholera infection. ToxT is the central regulatory protein involved in activation of CT and TCP expression. We previously reported that lack of a respiration-linked sodium-translocating NADH-ubiquinone oxidoreductase (Na(+)-NQR) significantly increases toxT transcription. In this study, we further characterized this link and found that Na(+)-NQR affects toxT expression only at the early-log growth phase, whereas lack of Na(+)-NQR decreases CT production after the mid-log growth phase. Such decreased CT production was independent of toxT and ctxB transcription. Supplementing a respiratory substrate, l-lactate, into the growth media restored CT production in the nqrA-F mutant, suggesting that decreased CT production in the Na(+)-NQR mutant is dependent on electron transport chain (ETC) activity. This notion was supported by the observations that two chemical inhibitors, a Na(+)-NQR specific inhibitor 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO) and a succinate dehydrogenase (SDH) inhibitor, thenoyltrifluoroacetone (TTFA), strongly inhibited CT production in both classical and El Tor biotype strains of V. cholerae. Accordingly, we propose the main respiratory enzyme of V. cholerae, as a potential drug target to treat cholera because human mitochondria do not contain Na(+)-NQR orthologs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Comprehensive analysis of blood group antigen binding to classical and El Tor cholera toxin B-pentamers by NMR.

    Science.gov (United States)

    Vasile, Francesca; Reina, José J; Potenza, Donatella; Heggelund, Julie E; Mackenzie, Alasdair; Krengel, Ute; Bernardi, Anna

    2014-08-01

    Cholera is a diarrheal disease responsible for the deaths of thousands, possibly even hundreds of thousands of people every year, and its impact is predicted to further increase with climate change. It has been known for decades that blood group O individuals suffer more severe symptoms of cholera compared with individuals with other blood groups (A, B and AB). The observed blood group dependence is likely to be caused by the major virulence factor of Vibrio cholerae, the cholera toxin (CT). Here, we investigate the binding of ABH blood group determinants to both classical and El Tor CTB-pentamers using saturation transfer difference NMR and show that all three blood group determinants bind to both toxin variants. Although the details of the interactions differ, we see no large differences between the two toxin genotypes and observe very similar binding constants. We also show that the blood group determinants bind to a site distinct from that of the primary receptor, GM1. Transferred NOESY data confirm that the conformations of the blood group determinants in complex with both toxin variants are similar to those of reported X-ray and solution structures. Taken together, this detailed analysis provides a framework for the interpretation of the epidemiological data linking the severity of cholera infection and an individual's blood group, and brings us one step closer to understanding the molecular basis of cholera blood group dependence.

  5. Functions of cholera toxin B-subunit as a raft cross-linker.

    Science.gov (United States)

    Day, Charles A; Kenworthy, Anne K

    2015-01-01

    Lipid rafts are putative complexes of lipids and proteins in cellular membranes that are proposed to function in trafficking and signalling events. CTxB (cholera toxin B-subunit) has emerged as one of the most studied examples of a raft-associated protein. Consisting of the membrane-binding domain of cholera toxin, CTxB binds up to five copies of its lipid receptor on the plasma membrane of the host cell. This multivalency of binding gives the toxin the ability to reorganize underlying membrane structure by cross-linking otherwise small and transient lipid rafts. CTxB thus serves as a useful model for understanding the properties and functions of protein-stabilized domains. In the present chapter, we summarize current evidence that CTxB associates with and cross-links lipid rafts, discuss how CTxB binding modulates the architecture and dynamics of membrane domains, and describe the functional consequences of this cross-linking behaviour on toxin uptake into cells via endocytosis.

  6. Quantitative detection of Vibrio cholera toxin by real-time and dynamic cytotoxicity monitoring.

    Science.gov (United States)

    Jin, Dazhi; Luo, Yun; Zheng, Min; Li, Haijing; Zhang, Jing; Stampfl, Melinda; Xu, Xiao; Ding, Gangqiang; Zhang, Yanjun; Tang, Yi-Wei

    2013-12-01

    We report here the quantitative detection of Vibrio cholerae toxin (CT) in isolates and stool specimens by dynamic monitoring of the full course of CT-mediated cytotoxicity in a real-time cell analysis (RTCA) system. Four cell lines, including Y-1 mouse adrenal tumor cells, Chinese hamster ovary (CHO) cells, small intestine epithelial (FHs74Int) cells, and mouse adrenal gland (PC12-Adh) cells, were evaluated for their suitability for CT-induced cytotoxicity testing. Among them, the Y-1 line was demonstrated to be the most sensitive for CT-mediated cytotoxicity, with limits of detection of 7.0 pg/ml for purified CT and 0.11 ng/ml for spiked CT in pooled negative stool specimens. No CT-mediated cytotoxicity was observed for nontoxigenic V. cholerae, non-V. cholerae species, or non-V. cholerae enterotoxins. The CT-RTCA assay was further validated with 100 stool specimens consecutively collected from patients with diarrhea and 200 V. cholerae isolates recovered from patients and the environment, in comparison to a reference using three detection methods. The CT-RTCA assay had sensitivities and specificities of 97.5% and 100.0%, respectively, for V. cholerae isolates and 90.0% and 97.2% for stool specimens. For stool specimens spiked with CT concentrations ranging from 3.5 pg/ml to 1.8 ng/ml, the inoculation-to-detection time was 1.12 ± 0.38 h, and the values were inversely correlated with CT concentrations (ρ = -1; P = 0.01). The results indicate that the CT-RTCA assay with the Y-1 cell line provides a rapid and sensitive tool for the quantitative detection of CT activities in clinical specimens.

  7. CHOLERA

    African Journals Online (AJOL)

    a cholera-like disease in the United Arab Republic, Ethio- pia, Malawi, Senegal, Sudan ... spread dissemination and sharp outbreaks of cholera. This is particularly so in ... Given early and adequate treatment cholera is no longer a higWy lethal ...

  8. The 2.3 {angstrom} crystal structure of cholera toxin B subunit pentamer: Choleragenoid

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Rong-Guang; Westbrook, M.L. [Argonne National Lab., IL (United States); Maulik, P.R.; Reed, R.A.; Shipley, G. [Boston Univ., MA (United States). School of Medicine; Westbrook, E.M. [Argonne National Lab., IL (United States)]|[Northwestern Univ., Evanston, IL (United States); Scott, D.L.; Otwinowski, Z. [Yale Univ., New Haven, CT (United States)

    1996-02-01

    Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{sub 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.

  9. Insights into binding of cholera toxin to GM1 containing membrane.

    Science.gov (United States)

    Basu, Ipsita; Mukhopadhyay, Chaitali

    2014-12-23

    Interactions of cholera toxin (CT) with membrane are associated with the massive secretory diarrhea seen in Asiatic cholera. Ganglioside GM1 has been shown to be responsible for the binding of the B subunit of cholera toxin (CT-B), which then helps CT to pass through the membrane, but the exact mechanism remains to be explored. In this work, we have carried out atomistic scale molecular dynamics simulation to investigate the structural changes of CT upon membrane binding and alteration in membrane structure and dynamics. Starting from the initial structure where the five units of B subunit bind with five GM1, only three of five units remain bound and the whole CT is tilted such that the three binding units are deeper in the membrane. The lipids that are in contact with those units of the CT-B behave differently from the rest of the lipids. Altogether, our results demonstrate the atomistic interaction of CT with GM1 containing lipid membrane and provide a probable mechanism of the early stage alteration of lipid structure and dynamics, which can make a passage for penetration of CT on membrane surface.

  10. Successful small intestine colonization of adult mice by Vibrio cholerae requires ketamine anesthesia and accessory toxins.

    Directory of Open Access Journals (Sweden)

    Verena Olivier

    Full Text Available Vibrio cholerae colonizes the small intestine of adult C57BL/6 mice. In this study, the physical and genetic parameters that facilitate this colonization were investigated. Successful colonization was found to depend upon anesthesia with ketamine-xylazine and neutralization of stomach acid with sodium bicarbonate, but not streptomycin treatment. A variety of common mouse strains were colonized by O1, O139, and non-O1/non-O139 strains. All combinations of mutants in the genes for hemolysin, the multifunctional, autoprocessing RTX toxin (MARTX, and hemagglutinin/protease were assessed, and it was found that hemolysin and MARTX are each sufficient for colonization after a low dose infection. Overall, this study suggests that, after intragastric inoculation, V. cholerae encounters barriers to infection including an acidic environment and an immediate immune response that is circumvented by sodium bicarbonate and the anti-inflammatory effects of ketamine-xylazine. After initial adherence in the small intestine, the bacteria are subjected to additional clearance mechanisms that are evaded by the independent toxic action of hemolysin or MARTX. Once colonization is established, it is suggested that, in humans, these now persisting bacteria initiate synthesis of the major virulence factors to cause cholera disease. This adult mouse model of intestinal V. cholerae infection, now well-characterized and fully optimized, should serve as a valuable tool for studies of pathogenesis and testing vaccine efficacy.

  11. Cholera.

    OpenAIRE

    Kaper, J B; Morris, J G; Levine, M M

    1995-01-01

    Despite more than a century of study, cholera still presents challenges and surprises to us. Throughout most of the 20th century, cholera was caused by Vibrio cholerae of the O1 serogroup and the disease was largely confined to Asia and Africa. However, the last decade of the 20th century has witnessed two major developments in the history of this disease. In 1991, a massive outbreak of cholera started in South America, the one continent previously untouched by cholera in this century. In 199...

  12. A comparative structure-function analysis of active-site inhibitors of Vibrio cholerae cholix toxin.

    Science.gov (United States)

    Lugo, Miguel R; Merrill, A Rod

    2015-09-01

    Cholix toxin from Vibrio cholerae is a novel mono-ADP-ribosyltransferase (mART) toxin that shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae diphtheria toxin. Herein, we have used the high-resolution X-ray structure of full-length cholix toxin in the apo form, NAD(+) bound, and 10 structures of the cholix catalytic domain (C-domain) complexed with several strong inhibitors of toxin enzyme activity (NAP, PJ34, and the P-series) to study the binding mode of the ligands. A pharmacophore model based on the active pose of NAD(+) was compared with the active conformation of the inhibitors, which revealed a cationic feature in the side chain of the inhibitors that may determine the active pose. Moreover, a conformational search was conducted for the missing coordinates of one of the main active-site loops (R-loop). The resulting structural models were used to evaluate the interaction energies and for 3D-QSAR modeling. Implications for a rational drug design approach for mART toxins were derived.

  13. Detection of cholera toxin in seafood using a ganglioside-liposome immunoassay.

    Science.gov (United States)

    Ahn, Soohyoun; Durst, Richard A

    2008-05-01

    Microbiological contamination of foods continues to be a major concern in public health. Biological toxins are one class of important contaminants that can cause various human diseases. Outbreaks related to contamination by biological toxins or toxin-producing microorganisms have made it extremely important to develop rapid (approximately 20 min), sensitive and cost-effective analytical methods. This paper describes the development of a sensitive bioassay for the detection of cholera toxin (CT) in selected seafood samples, using ganglioside-incorporated liposomes. In this study, the assays were run with food samples spiked with various concentrations of CT. The limit of detection (LOD) increased by a factor of about 10-20 in most food samples, compared with the LOD in the buffer system previously reported. However, the LOD of toxins in food samples (8 × 10-3 × 10(3) fg/mL for CT) was still comparable to, or lower than, that previously reported for other assays. The results from this study demonstrate that the bioassays using ganglioside-liposomes can detect the toxin directly in the field screening of food samples rapidly, simply and reliably, without the need for complex instrumentation.

  14. Anti-bacterial and anti-toxic immunity induced by a killed whole-cell-cholera toxin B subunit cholera vaccine is essential for protection against lethal bacterial infection in mouse pulmonary cholera model.

    Science.gov (United States)

    Kang, S-S; Yang, J S; Kim, K W; Yun, C-H; Holmgren, J; Czerkinsky, C; Han, S H

    2013-07-01

    The lack of appropriate animal model for studying protective immunity has limited vaccine development against cholera. Here, we demonstrate a pulmonary cholera model conferred by intranasal administration of mice with live Vibrio cholerae. The bacterial components, but not cholera toxin, caused lethal and acute pneumonia by inducing massive inflammation. Intranasal immunization with Dukoral, comprising killed whole bacteria and recombinant cholera toxin B subunit (rCTB), developed both mucosal and systemic antibody responses with protection against the lethal challenge. Either rCTB-free Dukoral or rCTB alone partially protected the mice against the challenge. However, reconstitution of rCTB-free Dukoral with rCTB restored full protection. Parenteral immunization with Dukoral evoked strong systemic immunity without induction of mucosal immunity or protection from the challenge. These results suggest that both anti-bacterial and anti-toxic immunity are required for protection against V. cholerae-induced pneumonia, and this animal model is useful for pre-clinical evaluation of candidate cholera vaccines.

  15. Efficacy of a food plant-based oral cholera toxin B subunit vaccine.

    Science.gov (United States)

    Arakawa, T; Chong, D K; Langridge, W H

    1998-03-01

    Transgenic potatoes were engineered to synthesize a cholera toxin B subunit (CTB) pentamer with affinity for GMI-ganglioside. Both serum and intestinal CTB-specific antibodies were induced in orally immunized mice. Mucosal antibody titers declined gradually after the last immunization but were restored following an oral booster of transgenic potato. The cytopathic effect of cholera holotoxin (CT) on Vero cells was neutralized by serum from mice immunized with transgenic potato tissues. Following intraileal injection with CT, the plant-immunized mice showed up to a 60% reduction in diarrheal fluid accumulation in the small intestine. Protection against CT was based on inhibition of enterotoxin binding to the cell-surface receptor GMI-ganglioside. These results demonstrate the ability of transgenic food plants to generate protective immunity in mice against a bacterial enterotoxin.

  16. Picomolar inhibition of cholera toxin by a pentavalent ganglioside GM1os-calix[5]arene.

    Science.gov (United States)

    Garcia-Hartjes, Jaime; Bernardi, Silvia; Weijers, Carel A G M; Wennekes, Tom; Gilbert, Michel; Sansone, Francesco; Casnati, Alessandro; Zuilhof, Han

    2013-07-14

    Cholera toxin (CT), the causative agent of cholera, displays a pentavalent binding domain that targets the oligosaccharide of ganglioside GM1 (GM1os) on the periphery of human abdominal epithelial cells. Here, we report the first GM1os-based CT inhibitor that matches the valency of the CT binding domain (CTB). This pentavalent inhibitor contains five GM1os moieties linked to a calix[5]arene scaffold. When evaluated by an inhibition assay, it achieved a picomolar inhibition potency (IC50 = 450 pM) for CTB. This represents a significant multivalency effect, with a relative inhibitory potency of 100,000 compared to a monovalent GM1os derivative, making GM1os-calix[5]arene one of the most potent known CTB inhibitors.

  17. Novel GM1 ganglioside-like peptide mimics prevent the association of cholera toxin to human intestinal epithelial cells in vitro

    OpenAIRE

    Robert K Yu; Usuki, Seigo; Itokazu, Yutaka; Wu, Han-Chung

    2015-01-01

    Cholera is an acute diarrheal disease caused by infection in the gastrointestinal tract by the gram-negative bacterium, Vibrio cholerae, and is a serious public health threat worldwide. There has not been any effective treatment for this infectious disease. Cholera toxin (CT), which is secreted by V. cholerae, can enter host cells by binding to GM1, a monosialoganglioside widely distributed on the plasma membrane surface of various animal epithelial cells. The present study was undertaken to ...

  18. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, B; Pallesen, L; Jensen, LB

    1997-01-01

    . Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested......The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions...... in the fimA gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fimA in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted...

  19. Nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor

    DEFF Research Database (Denmark)

    Borch, Jonas; Torta, Federico; Sligar, Stephen G;

    2008-01-01

    nanodiscs and their incorporated membrane receptors can be attached to surface plasmon resonance sensorchips and used to measure the kinetics of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. Cholera toxin and its glycolipid receptor G(M1) constitute...... partner cholera toxin B subunit to the receptor with the sensorchip-based surface plasmon resonance (SPR) technology. The measured stoichiometric and kinetic values of the interaction are in agreement with those reported by previous studies, thus providing proof-of-principle that nanodiscs can be employed...

  20. Experimental parameters differentially affect the humoral response of the cholera-toxin-based murine model of food allergy

    DEFF Research Database (Denmark)

    Kroghsbo, S.; Christensen, Hanne Risager; Frøkiær, Hanne

    2003-01-01

    Background: Recent studies have developed a murine model of IgE-mediated food allergy based on oral coadministration of antigen and cholera toxin (CT) to establish a maximal response for studying immunopathogenic mechanisms and immunotherapeutic strategies. However, for studying subtle immunomodu......Background: Recent studies have developed a murine model of IgE-mediated food allergy based on oral coadministration of antigen and cholera toxin (CT) to establish a maximal response for studying immunopathogenic mechanisms and immunotherapeutic strategies. However, for studying subtle...

  1. Infrared and visible ellipsometric studies of cholera toxin in ELISA structures

    Science.gov (United States)

    Thompson, Daniel W.; Pfeiffer, Galen L.; Berberov, Emil; Castro, Leon; Woollam, John A.

    2003-07-01

    Ellipsometry is well known for its extreme sensitivity to the presence and properties of ultra-thin films. In the infrared, resonance response to chemical bonds allows chemical identification in monolayer-thick biological films. In this paper we show results of attachment repeatability for successive layers of monosialoganglioside, cholera toxin, and related antibodies using in situ visible spectroscopic ellipsometry. Several factors contributing to difficulty in obtaining reproducible results are discussed. Soecifically, these include freshness of reagents; surface type, cleaning, and preparation; temperature; birefringence of liquid cell windows; and cell design. Sensitivity and signal noise considerations for infrared spectra of molecular monolayers are discussed.

  2. Molecular Characterization of High-Level-Cholera-Toxin-Producing El Tor Variant Vibrio cholerae Strains in the Zanzibar Archipelago of Tanzania

    Science.gov (United States)

    Naha, A.; Chowdhury, G.; Ghosh-Banerjee, J.; Senoh, M.; Takahashi, T.; Ley, B.; Thriemer, K.; Deen, J.; Seidlein, L. V.; Ali, S. M.; Khatib, A.; Ramamurthy, T.; Nandy, R. K.; Nair, G. B.; Takeda, Y.

    2013-01-01

    Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis. PMID:23325815

  3. Molecular characterization of high-level-cholera-toxin-producing El Tor variant Vibrio cholerae strains in the Zanzibar Archipelago of Tanzania.

    Science.gov (United States)

    Naha, A; Chowdhury, G; Ghosh-Banerjee, J; Senoh, M; Takahashi, T; Ley, B; Thriemer, K; Deen, J; Seidlein, L V; Ali, S M; Khatib, A; Ramamurthy, T; Nandy, R K; Nair, G B; Takeda, Y; Mukhopadhyay, A K

    2013-03-01

    Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.

  4. Cholera toxin B subunits assemble into pentamers--proposition of a fly-casting mechanism.

    Directory of Open Access Journals (Sweden)

    Jihad Zrimi

    Full Text Available The cholera toxin B pentamer (CtxB(5, which belongs to the AB(5 toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i acquisition of a fully assembly-competent fold by the CtxB monomer, (ii association of CtxB monomer into oligomers, (iii acquisition of the native fold by the CtxB pentamer. The results show that CtxB(5 and the related heat labile enterotoxin LTB(5 have distinct mechanisms of assembly despite sharing high sequence identity (84% and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them.

  5. The Ero1alpha-PDI redox cycle regulates retro-translocation of cholera toxin.

    Science.gov (United States)

    Moore, Paul; Bernardi, Kaleena M; Tsai, Billy

    2010-04-01

    Cholera toxin (CT) is transported from the plasma membrane of host cells to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit retro-translocates to the cytosol to induce toxicity. Our previous analyses demonstrated that the ER oxidoreductase protein disulfide isomerase (PDI) acts as a redox-dependent chaperone to unfold CTA1, a reaction postulated to initiate toxin retro-translocation. In its reduced state, PDI binds and unfolds CTA1; subsequent oxidation of PDI by Ero1alpha enables toxin release. Whether this in vitro model describes events in cells that control CTA1 retro-translocation is unknown. Here we show that down-regulation of Ero1alpha decreases retro-translocation of CTA1 by increasing reduced PDI and blocking efficient toxin release. Overexpression of Ero1alpha also attenuates CTA1 retro-translocation, an effect due to increased PDI oxidation, which prevents PDI from engaging the toxin effectively. Interestingly, Ero1alpha down-regulation increases interaction between PDI and Derlin-1, an ER membrane protein that is a component of the retro-translocation complex. These findings demonstrate that an appropriate Ero1alpha-PDI ratio is critical for regulating the binding-release cycle of CTA1 by PDI during retro-translocation, and implicate PDI's redox state in targeting it to the retro-translocon.

  6. Statistical pattern matching facilitates the design of polyvalent inhibitors of anthrax and cholera toxins.

    Science.gov (United States)

    Rai, Prakash; Padala, Chakradhar; Poon, Vincent; Saraph, Arundhati; Basha, Saleem; Kate, Sandesh; Tao, Kevin; Mogridge, Jeremy; Kane, Ravi S

    2006-05-01

    Numerous biological processes involve the recognition of a specific pattern of binding sites on a target protein or surface. Although ligands displayed by disordered scaffolds form stochastic rather than specific patterns, theoretical models predict that recognition will occur between patterns that are characterized by similar or "matched" statistics. Endowing synthetic biomimetic structures with statistical pattern matching capabilities may improve the specificity of sensors and resolution of separation processes. We demonstrate that statistical pattern matching enhances the potency of polyvalent therapeutics. We functionalized liposomes with an inhibitory peptide at different densities and observed a transition in potency at an interpeptide separation that matches the distance between ligand-binding sites on the heptameric component of anthrax toxin. Pattern-matched polyvalent liposomes inhibited anthrax toxin in vitro at concentrations four orders of magnitude lower than the corresponding monovalent peptide, and neutralized this toxin in vivo. Statistical pattern matching also enhanced the potency of polyvalent inhibitors of cholera toxin. This facile strategy should be broadly applicable to the detection and neutralization of toxins and pathogens.

  7. Fighting Cholera One-on-One: The Development and Efficacy of Multivalent Cholera-Toxin-Binding Molecules.

    Science.gov (United States)

    Zuilhof, Han

    2016-02-16

    A series of diseases, ranging from cholera via travelers' diarrhea to hamburger disease, are caused by bacterially produced toxic proteins. In particular, a toxic protein unit is brought into the host cell upon binding to specific membrane-bound oligosaccharides on the host cell membrane. For example, the protein that causes cholera, cholera toxin (CT), has five identical, symmetrically placed binding pockets (B proteins), on top of which the toxic A protein resides. A promising strategy to counteract the devastating biological effects of this AB5 protein involves the development of inhibitors that can act as mimics of membrane-bound GM1 molecules, i.e., that can bind CT strongly and selectively. To reach this goal, two features are essential: First of all, the inhibitor should display oligosaccharides that resemble as much as possible the naturally occurring cell-surface pentasaccharide onto which CT normally binds, the so-called GM1 sugar (the oligosaccharide part of which is then labeled GM1os). Second, the inhibitor should be able to bind CT via multivalent interactions so as to bind CT as strongly as possible to allow for a real competition with the cell-membrane-bound GM1 molecules. In this Account, we present elements of the path that leads to strong CT inhibition by outlining the roles of multivalency and the development and use of GM1 mimics. First, multivalency effects were investigated using "sugar-coated" platforms, ranging from dendritic structures with up to eight oligosaccharides to platforms that mimicked the fivefold symmetry of CT itself. The latter goal was reached either via synthetic scaffolds like corannulene or calix[5]arene or via the development of a neolectin CT mimic that itself carries five GM1os groups. Second, the effect of the nature of the oligosaccharide appended to this platform was investigated via the use of oligosaccharides of increasing complexity, from galactose and lactose to the tetrasaccharide GM2os and eventually to GM1os

  8. Cholera.

    Science.gov (United States)

    Kaper, J B; Morris, J G; Levine, M M

    1995-01-01

    Despite more than a century of study, cholera still presents challenges and surprises to us. Throughout most of the 20th century, cholera was caused by Vibrio cholerae of the O1 serogroup and the disease was largely confined to Asia and Africa. However, the last decade of the 20th century has witnessed two major developments in the history of this disease. In 1991, a massive outbreak of cholera started in South America, the one continent previously untouched by cholera in this century. In 1992, an apparently new pandemic caused by a previously unknown serogroup of V. cholerae (O139) began in India and Bangladesh. The O139 epidemic has been occurring in populations assumed to be largely immune to V. cholerae O1 and has rapidly spread to many countries including the United States. In this review, we discuss all aspects of cholera, including the clinical microbiology, epidemiology, pathogenesis, and clinical features of the disease. Special attention will be paid to the extraordinary advances that have been made in recent years in unravelling the molecular pathogenesis of this infection and in the development of new generations of vaccines to prevent it.

  9. Cross-reactivity of outer membrane proteins of Campylobacter species with cholera toxin.

    Science.gov (United States)

    Albert, M John

    2011-02-01

    Campylobacter jejuni is a foodborne pathogen and a leading cause of diarrhoea worldwide. It is believed that a cholera toxin-like toxin (CTLT) produced by C. jejuni may mediate watery diarrhoea. However, the production of a CTLT by C. jejuni is controversial. A cholera toxin gene (ctx) homologue has not been identified in Campylobacter species. We investigated the identity of the CT cross-reactive antigen from Campylobacter species previously and the results are reviewed here. Filtrates of C. jejuni grown in four different liquid media, reported to promote CTLT production, were tested by Chinese hamster ovary (CHO) cell elongation assay for functional toxin and for reactivity with CT antibody using GM1 ganglioside ELISA (GM1 ELISA) and immunoblotting. Protein sequence of the CT antibody-reactive band was determined by matrix-assisted laser desorption ionization-time of flight (MALDI TOF-TOF). Non-jejuni species (C. coli, C. lari, C. foetus, C. hyointestinalis and C. upsaliensis) were investigated by CHO cell assay and immunoblotting. Filtrates from seven C. jejuni reference strains reported to produce CTLT and from 80 clinical strains were negative in the CHO cell assay. However, filtrates from three reference strains and 16 clinical strains were positive by GM1 ELISA. All strains irrespective of GM1 ELISA reactivity, possessed a 53-kDa protein which reacted with CT antibody by immunoblotting. This band was identified as the major outer membrane protein (PorA) of C. jejuni. CT antibody reacted with a C. jejuni recombinant PorA on immunoblotting. All non-C. jejuni strains were negative by CHO cell assay, but the common 53-kDa proteins reacted with CT antibody on immunoblots. The cross-reactivity of PorAs of Campylobacter species with CT may lead to the erroneous conclusion that Campylobacter species produce a functional CTLT.

  10. Sensitization of spinal cord nociceptive neurons with a conjugate of substance P and cholera toxin

    Directory of Open Access Journals (Sweden)

    Perez Federico M

    2007-05-01

    Full Text Available Abstract Background Several investigators have coupled toxins to neuropeptides for the purpose of lesioning specific neurons in the central nervous system. By producing deficits in function these toxin conjugates have yielded valuable information about the role of these cells. In an effort to specifically stimulate cells rather than kill them we have conjugated the neuropeptide substance P to the catalytic subunit of cholera toxin (SP-CTA. This conjugate should be taken up selectively by neurokinin receptor expressing neurons resulting in enhanced adenylate cyclase activity and neuronal firing. Results The conjugate SP-CTA stimulates adenylate cyclase in cultured cells that are transfected with either the NK1 or NK2 receptor, but not the NK3 receptor. We further demonstrate that intrathecal injection of SP-CTA in rats induces the phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB and also enhances the expression of the immediate early gene c-Fos. Behaviorally, low doses of SP-CTA (1 μg injected intrathecally produce thermal hyperalgesia. At higher doses (10 μg peripheral sensitivity is suppressed suggesting that descending inhibitory pathways may be activated by the SP-CTA induced sensitization of spinal cord neurons. Conclusion The finding that stimulation of adenylate cyclase in neurokinin receptor expressing neurons in the spinal cord produces thermal hyperalgesia is consistent with the known actions of these neurons. These data demonstrate that cholera toxin can be targeted to specific cell types by coupling the catalytic subunit to a peptide agonist for a g-protein coupled receptor. Furthermore, these results demonstrate that SP-CTA can be used as a tool to study sensitization of central neurons in vivo in the absence of an injury.

  11. Oral administration of a recombinant cholera toxin B subunit promotes mucosal healing in the colon.

    Science.gov (United States)

    Baldauf, K J; Royal, J M; Kouokam, J C; Haribabu, B; Jala, V R; Yaddanapudi, K; Hamorsky, K T; Dryden, G W; Matoba, N

    2016-11-02

    Cholera toxin B subunit (CTB) is a component of a licensed oral cholera vaccine. However, CTB has pleiotropic immunomodulatory effects whose impacts on the gut are not fully understood. Here, we found that oral administration in mice of a plant-made recombinant CTB (CTBp) significantly increased several immune cell populations in the colon lamina propria. Global gene expression analysis revealed that CTBp had more pronounced impacts on the colon than the small intestine, with significant activation of TGFβ-mediated pathways in the colon epithelium. The clinical relevance of CTBp-induced impacts on colonic mucosa was examined. In a human colon epithelial model using Caco2 cells, CTBp, but not the non-GM1-binding mutant G33D-CTBp, induced TGFβ-mediated wound healing. In a dextran sodium sulfate (DSS) acute colitis mouse model, oral administration of CTBp protected against colon mucosal damage as manifested by mitigated body weight loss, decreased histopathological scores, and blunted escalation of inflammatory cytokine levels while inducing wound healing-related genes. Furthermore, biweekly oral administration of CTBp significantly reduced disease severity and tumorigenesis in the azoxymethane/DSS model of ulcerative colitis and colon cancer. Altogether, these results demonstrate CTBp's ability to enhance mucosal healing in the colon, highlighting its potential application in ulcerative colitis therapy besides cholera vaccination.Mucosal Immunology advance online publication 2 November 2016. doi:10.1038/mi.2016.95.

  12. Zinc oxide nanoparticles provide anti-cholera activity by disrupting the interaction of cholera toxin with the human GM1 receptor.

    Science.gov (United States)

    Sarwar, Shamila; Ali, Asif; Pal, Mahadeb; Chakrabarti, Pinak

    2017-09-07

    Vibrio cholerae causes cholera and is the leading cause of diarrhea in the developing countries, highlighting the need for the development of new treatment strategies to combat this disease agent. While exploring the possibility of using zinc oxide (ZnO) nanoparticles (NPs) in cholera treatment, we previously found that ZnO NPs reduce fluid accumulation in mouse ileum induced by the cholera toxin (CT) protein. To uncover the mechanism of action of ZnO NPs on CT activity, here we used classical (O395) and El Tor (C6706) V. cholerae biotypes in growth and biochemical assays. We found that a ZnO NP concentration of 10 µg/ml did not affect the growth rates of these two strains, nor did we observe that ZnO NPs reduce the expression levels of CT mRNA and protein. It was observed that ZnO NPs form a complex with CT, appear to disrupt the CT secondary structure, and block its interaction with the GM1 ganglioside receptor in the outer leaflet of the plasma membrane in intestinal (HT-29) cells and thereby reduce CT uptake into the cells. In the range of 2.5-10 µg/ml, ZnO NPs exhibited no cytotoxicity on kidney (HEK293) and HT-29 cells. We conclude that ZnO NPs prevent the first step in the translocation of cholera toxin into intestinal epithelial cells without exerting measurable toxic effects on HEK293 and HT-29 cells. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  13. Intracellular Signal Triggered by Cholera Toxin in Saccharomyces boulardii and Saccharomyces cerevisiae

    Science.gov (United States)

    Brandão, Rogelio L.; Castro, Ieso M.; Bambirra, Eduardo A.; Amaral, Sheila C.; Fietto, Luciano G.; Tropia, Maria José M.; Neves, Maria José; Dos Santos, Raquel G.; Gomes, Newton C. M.; Nicoli, Jacques R.

    1998-01-01

    As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts. PMID:9464394

  14. Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites

    Energy Technology Data Exchange (ETDEWEB)

    Prochazkova, Katerina; Shuvalova, Ludmilla A.; Minasov, George; Voburka, Zden& #283; k; Anderson, Wayne F.; Satchell, Karla J.F.; (NWU); (Czech Academy)

    2009-10-05

    The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP{sub 6}). In this study, we demonstrated that InsP{sub 6} is not simply an allosteric cofactor, but rather binding of InsP{sub 6} stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-{angstrom} crystal structure of this InsP{sub 6}-bound unprocessed form of CPD was determined and revealed the scissile bond Leu{sup 3428}-Ala{sup 3429} captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP{sub 6}, but was reactivated for high affinity binding of InsP{sub 6} by cooperative binding of both a new substrate and InsP{sub 6}. Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.

  15. Identification of host cell factors required for intoxication through use of modified cholera toxin.

    Science.gov (United States)

    Guimaraes, Carla P; Carette, Jan E; Varadarajan, Malini; Antos, John; Popp, Maximilian W; Spooner, Eric; Brummelkamp, Thijn R; Ploegh, Hidde L

    2011-11-28

    We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ~12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.

  16. Revisiting the oligomerization mechanism of Vibrio cholerae cytolysin, a beta-barrel pore-forming toxin.

    Science.gov (United States)

    Rai, Anand Kumar; Chattopadhyay, Kausik

    2016-06-03

    Vibrio cholerae cytolysin (VCC) is a membrane-damaging beta-barrel pore-forming toxin (beta-PFT). VCC causes permeabilization of the target membranes by forming transmembrane oligomeric beta-barrel pores. Oligomerization is a key step in the mode of action of any beta-PFT, including that of VCC. Earlier studies have identified some of the key residues in VCC that are directly involved in the generation of the inter-protomer contacts, thus playing critical roles in the oligomerization of the membrane-bound toxin. Analysis of the VCC oligomeric pore structure reveals a potential hydrogen-bond network that appears to connect the sidechain of an asparagine residue (Asn582; located within an inter-domain linker sequence) from one protomer to the backbone CO- and NH-groups of the neighbouring protomer, indirectly through water molecules at most of the inter-protomer interfaces. In the present study, we show that the mutation of Asn582Ala affects the oligomerization and the pore-forming activity of VCC in the membrane lipid bilayer of the synthetic lipid vesicles, while the replacement of Asn582Gln results into the restoration of the oligomeric pore-forming ability of the toxin. Using a number of truncated variants of VCC, having deletion in the C-terminal region of the toxin starting from the Asn582 residue or beyond, we also show that the presence of Asn582 is critically required for the oligomerization of the truncated form of the protein.

  17. Cholera.

    Science.gov (United States)

    Lippi, Donatella; Gotuzzo, Eduardo; Caini, Saverio

    2016-08-01

    Cholera is an acute disease of the gastrointestinal tract caused by Vibrio cholerae. Cholera was localized in Asia until 1817, when a first pandemic spread from India to several other regions of the world. After this appearance, six additional major pandemics occurred during the 19th and 20th centuries, the latest of which originated in Indonesia in the 1960s and is still ongoing. In 1854, a cholera outbreak in Soho, London, was investigated by the English physician John Snow (1813 to 1858). He described the time course of the outbreak, managed to understand its routes of transmission, and suggested effective measures to stop its spread, giving rise to modern infectious disease epidemiology. The germ responsible for cholera was discovered twice: first by the Italian physician Filippo Pacini during an outbreak in Florence, Italy, in 1854, and then independently by Robert Koch in India in 1883, thus favoring the germ theory over the miasma theory of disease. Unlike many other infectious diseases, such as plague, smallpox, and poliomyelitis, cholera persists as a huge public health problem worldwide, even though there are effective methods for its prevention and treatment. The main reasons for its persistence are socioeconomic rather than purely biological; cholera flourishes where there are unsatisfactory hygienic conditions and where a breakdown of already fragile sanitation and health infrastructure occurs because of natural disasters or humanitarian crises.

  18. Phase Partitioning of GM1 and Its Bodipy-Labeled Analog Determine Their Different Binding to Cholera Toxin

    DEFF Research Database (Denmark)

    Rissanen, Sami; Grzybek, Michal; Orłowski, Adam

    2017-01-01

    membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane...

  19. Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes.

    Science.gov (United States)

    Angström, J; Teneberg, S; Karlsson, K A

    1994-12-06

    Binding studies of various glycolipids, mainly belonging to the ganglio series, to the toxins isolated from Vibrio cholerae, Escherichia coli, and Clostridium tetani have been performed, using the microtiter well assay. By using the found binding preferences in conjunction with minimum-energy conformations obtained from molecular modeling of the various ligands, binding epitopes on the natural receptor glycolipids for the toxins have been defined. The binding preferences for the cholera toxin and the heat-labile E. coli toxin are very similar, with the ganglioside GM1 being the most efficient ligand. The tetanus toxin binds strongly to gangliosides of the G1b series, with GT1b as the most efficient ligand. It is found that the binding epitope on GM1 for the cholera and heat-labile toxins to a large extent overlaps with the epitope on GQ1b for the tetanus toxin.

  20. Cholera toxin B subunit-binding and ganglioside GM1 immuno-expression are not necessarily correlated in human salivary glands

    DEFF Research Database (Denmark)

    Kirkeby, Svend

    2014-01-01

    OBJECTIVE: To determine and compare the presence and in situ localization of the glycosphingolipid ganglioside GM1 in human salivary glands using the biomarkers for GM1: cholera toxin and antibodies against GM1. MATERIALS AND METHODS: Immunohistochemical analyses were performed on sections of adult...... human submandibular, parotid and palatinal glands using cholera toxin sub-unit B and two polyclonal antibodies against ganglioside GM1 as biomarkers. RESULTS: Immunofluorescence microscopy showed that the toxin and antibodies were co-localized in some acini but not in others. The cholera toxin mainly...... reacted with the cell membranes of the mucous acini in the submandibular gland, while incubation with the antibody against GM1 gave rise to a staining of the cytoplasm. The cytoplasm in some secretory acinar cells in the parotid gland was stained by the cholera toxin, whereas only small spots...

  1. Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose

    DEFF Research Database (Denmark)

    Kirkeby, S

    2016-01-01

    FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini...... to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human...

  2. Cholera toxin subunit B-mediated intracellular trafficking of mesoporous silica nanoparticles toward the endoplasmic reticulum

    Science.gov (United States)

    Walker, William Andrew

    In recent decades, pharmaceutical research has led to the development of numerous treatments for human disease. Nanoscale delivery systems have the potential to maximize therapeutic outcomes by enabling target specific delivery of these therapeutics. The intracellular localization of many of these materials however, is poorly controlled, leading to sequestration in degradative cellular pathways and limiting the efficacy of their payloads. Numerous proteins, particularly bacterial toxins, have evolved mechanisms to subvert the degradative mechanisms of the cell. Here, we have investigated a possible strategy for shunting intracellular delivery of encapsulated cargoes from these pathways by modifying mesoporous silica nanoparticles (MSNs) with the well-characterized bacterial toxin Cholera toxin subunit B (CTxB). Using established optical imaging methods we investigated the internalization, trafficking, and subcellular localization of our modified MSNs in an in vitro animal cell model. We then attempted to demonstrate the practical utility of this approach by using CTxB-modified mesoporous silica nanoparticles to deliver propidium iodide, a membrane-impermeant fluorophore.

  3. Inhibition of Binding of the AB5-Type Enterotoxins LT-I and Cholera Toxin to Ganglioside GM1 by Galactose-Rich Dietary Components

    NARCIS (Netherlands)

    Becker, P.M.; Widjaja-Greefkes, H.C.A.; Wikselaar, van P.G.

    2010-01-01

    Cholera, travelers' diarrhea, or colibacillosis in pigs can possibly be prevented or attenuated by dietary provision of competitive inhibitors that react with the GM1-binding sites of the enterotoxins cholera toxin (CT), human Escherichia coli heat-labile enterotoxin of serogroup I (LTh-I), and porc

  4. Molecular modeling of methyl-α-Neu5Ac analogues docked against cholera toxin--a molecular dynamics study.

    Science.gov (United States)

    Blessy, J Jino; Sharmila, D Jeya Sundara

    2015-02-01

    Molecular modeling of synthetic methyl-α-Neu5Ac analogues modified in C-9 position was investigated by molecular docking and molecular dynamics (MD) simulation methods. Methyl-α-Neu5Ac analogues were docked against cholera toxin (CT) B subunit protein and MD simulations were carried out for three Methyl-α-Neu5Ac analogue-CT complexes (30, 10 and 10 ns) to estimate the binding activity of cholera toxin-Methyl-α-Neu5Ac analogues using OPLS_2005 force field. In this study, direct and water mediated hydrogen bonds play a vital role that exist between the methyl-α-9-N-benzoyl-amino-9-deoxy-Neu5Ac (BENZ)-cholera toxin active site residues. The Energy plot, RMSD and RMSF explain that the simulation was stable throughout the simulation run. Transition of phi, psi and omega angle for the complex was calculated. Molecular docking studies could be able to identify the binding mode of methyl-α-Neu5Ac analogues in the binding site of cholera toxin B subunit protein. MD simulation for Methyl-α-9-N-benzoyl-amino-9-deoxy-Neu5Ac (BENZ), Methyl-α-9-N-acetyl-9-deoxy-9-amino-Neu5Ac and Methyl-α-9-N-biphenyl-4-acetyl-deoxy-amino-Neu5Ac complex with CT B subunit protein was carried out, which explains the stable nature of interaction. These methyl-α-Neu5Ac analogues that have computationally acceptable pharmacological properties may be used as novel candidates for drug design for cholera disease.

  5. Protein deprivation causes reversible impariment of mucosal immune response to cholera toxoid/toxin in rat gut.

    Science.gov (United States)

    Barry, W S; Pierce, N F

    1979-09-06

    Scretory antibodies may be the major defence against mucosal infections, especially those due to viruses and non-invasive pathogens such as Vibrio cholerae and toxinogenic Escherichia coli. The high incidence of mucosal infections in malnourished protein-deficient children may result from defective antibody production, but evidence for this is conflicting. We report here that protein deficiency markedly impairs the mucosal immune reponse to cholera toxiod/toxin (CT), a protein antigen, in rats and that this impairment is rapidly reversed by refeeding.

  6. N-Glycosylation of cholera toxin B subunit: serendipity for novel plant-made vaccines?

    Directory of Open Access Journals (Sweden)

    Nobuyuki eMatoba

    2015-12-01

    Full Text Available The non-toxic B subunit of cholera toxin (CTB has attracted considerable interests from vaccinologists due to strong mucosal immunomodulatory effects and potential utility as a vaccine scaffold for heterologous antigens. Along with other conventional protein expression systems, various plant species have been used as recombinant production hosts for CTB and its fusion proteins. However, it has recently become clear that the protein is N-glycosylated within the endoplasmic reticulum of plant cells – a eukaryotic post-translational modification that is not present in native CTB. While functionally active aglycosylated variants have been successfully engineered to circumvent potential safety and regulatory issues related to glycosylation, this modification may actually provide advantageous characteristics to the protein as a vaccine platform. Based on data from our recent studies, I discuss the unique features of N-glycosylated CTB produced in plants for the development of novel vaccines.

  7. N-Glycosylation of Cholera Toxin B Subunit: Serendipity for Novel Plant-Made Vaccines?

    Science.gov (United States)

    Matoba, Nobuyuki

    2015-01-01

    The non-toxic B subunit of cholera toxin (CTB) has attracted considerable interests from vaccinologists due to strong mucosal immunomodulatory effects and potential utility as a vaccine scaffold for heterologous antigens. Along with other conventional protein expression systems, various plant species have been used as production hosts for CTB and its fusion proteins. However, it has recently become clear that the protein is N-glycosylated within the endoplasmic reticulum of plant cells-a eukaryotic post-translational modification that is not present in native CTB. While functionally active aglycosylated variants have been successfully engineered to circumvent potential safety and regulatory issues related to glycosylation, this modification may actually provide advantageous characteristics to the protein as a vaccine platform. Based on data from our recent studies, I discuss the unique features of N-glycosylated CTB produced in plants for the development of novel vaccines.

  8. Cellular Endocytosis and Trafficking of Cholera Toxin B-Modified Mesoporous Silica Nanoparticles

    Science.gov (United States)

    Walker, William A.; Tarannum, Mubin; Vivero-Escoto, Juan L.

    2016-01-01

    In this study, mesoporous silica nanoparticles (MSNs) were functionalized with Cholera toxin subunit B (CTxB) protein to influence their intracellular trafficking pathways. The CTxB-MSN carrier was synthesized, and its chemical and structural properties were characterized. Endocytic pathway inhibition assays showed that the uptake of CTxB-MSNs in human cervical cancer (HeLa) cells was partially facilitated by both chlatrin- and caveolae-mediated endocytosis mechanisms. Laser scanning confocal microscopy (LSCM) experiments demonstrated that CTxB-MSNs were taken up by the cells and partially trafficked through the trans-Golgi network into to the endoplasmic reticulum in a retrograde fashion. The delivery abilities of CTxB-MSNs were evaluated using propidium iodide, an impermeable cell membrane dye. LSCM images depicted the release of propidium iodide in the endoplasmic reticulum and cell nucleus of HeLa cells. PMID:27134749

  9. Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector.

    Science.gov (United States)

    Moore, Lauren; Hamorsky, Krystal; Matoba, Nobuyuki

    2016-01-01

    Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE(®) tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapatite resins. This procedure yields on average 400 mg of low-endotoxin CTBp from 1 kg of fresh leaf material.

  10. Facile Method for the Production of Recombinant Cholera Toxin B Subunit in E. coli.

    Science.gov (United States)

    Hamorsky, Krystal; Matoba, Nobuyuki

    2016-01-01

    Herein, we report an Escherichia coli-based expression and purification method of recombinant cholera toxin B subunit (CTB). The CTB gene (E. coli codon optimized) is cloned into commercial pET-22b(+) vector using standard molecular biology techniques and the resulting vector is transformed into BL21(DE3) electrocompetent cells. The bacterial cells are grown and induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) results in accumulation of CTB in the culture medium. CTB is purified from the culture medium using a simple two-step chromatography process: immobilized metal affinity chromatography (IMAC) followed by ceramic hydroxyapatite (CHT). CTB is purified to >95 % homogeneity with a yield of over 10 mg per liter of culture. Depending on the application, endotoxin is removed using a commercially available endotoxin removal resin to <1 EU/mg.

  11. N-Glycosylation of Cholera Toxin B Subunit: Serendipity for Novel Plant-Made Vaccines?

    Science.gov (United States)

    Matoba, Nobuyuki

    2015-01-01

    The non-toxic B subunit of cholera toxin (CTB) has attracted considerable interests from vaccinologists due to strong mucosal immunomodulatory effects and potential utility as a vaccine scaffold for heterologous antigens. Along with other conventional protein expression systems, various plant species have been used as production hosts for CTB and its fusion proteins. However, it has recently become clear that the protein is N-glycosylated within the endoplasmic reticulum of plant cells—a eukaryotic post-translational modification that is not present in native CTB. While functionally active aglycosylated variants have been successfully engineered to circumvent potential safety and regulatory issues related to glycosylation, this modification may actually provide advantageous characteristics to the protein as a vaccine platform. Based on data from our recent studies, I discuss the unique features of N-glycosylated CTB produced in plants for the development of novel vaccines. PMID:26732492

  12. Antigen-Independent Restriction of Pneumococcal Density by Mucosal Adjuvant Cholera Toxin Subunit B.

    Science.gov (United States)

    Kuipers, Kirsten; Diavatopoulos, Dimitri A; van Opzeeland, Fred; Simonetti, Elles; van den Kieboom, Corné H; Kerstholt, Mariska; Borczyk, Malgorzata; van IngenSchenau, D; Brandsma, Eelke T; Netea, Mihai G; de Jonge, Marien I

    2016-11-15

    For many bacterial respiratory infections, development of (severe) disease is preceded by asymptomatic colonization of the upper airways. For Streptococcus pneumoniae, the transition to severe lower respiratory tract infection is associated with an increase in nasopharyngeal colonization density. Insight into how the mucosal immune system restricts colonization may provide new strategies to prevent clinical symptoms. Several studies have provided indirect evidence that the mucosal adjuvant cholera toxin subunit B (CTB) may confer nonspecific protection against respiratory infections. Here, we show that CTB reduces the pneumococcal load in the nasopharynx, which required activation of the caspase-1/11 inflammasome, mucosal T cells, and macrophages. Our findings suggest that CTB-dependent activation of the local innate response synergizes with noncognate T cells to restrict bacterial load. Our study not only provides insight into the immunological components required for containment and clearance of pneumococcal carriage, but also highlights an important yet often understudied aspect of adjuvants.

  13. Bovine lactoferrin decreases cholera-toxin-induced intestinal fluid accumulation in mice by ganglioside interaction.

    Directory of Open Access Journals (Sweden)

    Fulton P Rivera

    Full Text Available Secretory diarrhea caused by cholera toxin (CT is initiated by binding of CT's B subunit (CTB to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01. We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea.

  14. Bovine Lactoferrin Decreases Cholera-Toxin-Induced Intestinal Fluid Accumulation in Mice by Ganglioside Interaction

    Science.gov (United States)

    Rivera, Fulton P.; Medina, Anicia M.; Bezada, Sandra; Valencia, Roberto; Bernal, María; Meza, Rina; Maves, Ryan C.; Ochoa, Theresa J.

    2013-01-01

    Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT’s B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea. PMID:23580005

  15. Plasmon Coupling Enhanced Raman Scattering Nanobeacon for Single-Step, Ultrasensitive Detection of Cholera Toxin.

    Science.gov (United States)

    Zhang, Chong-Hua; Liu, Ling-Wei; Liang, Ping; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

    2016-08-02

    We report the development of a novel plasmon coupling enhanced Raman scattering (PCERS) method, PCERS nanobeacon, for ultrasensitive, single-step, homogeneous detection of cholera toxin (CT). This method relies on our design of the plasmonic nanoparticles, which have a bilayer phospholipid coating with embedded Raman indicators and CT-binding ligands of monosialoganglioside (GM1). This design allows a facile synthesis of the plasmonic nanoparticle via two-step self-assembly without any specific modification or chemical immobilization. The realization of tethering GM1 on the surface imparts the plasmonic nanoparticles with high affinity, excellent specificity, and multivalence for interaction with CT. The unique lipid-based bilayer coated structure also affords excellent biocompatibility and stability for the plasmonic nanoparticles. The plasmonic nanoparticles are able to show substantial enhancement of the surface-enhanced Raman scattering (SERS) signals in a single-step interaction with CT, because of their assembly into aggregates in response to the CT-sandwiched interactions. The results reveal that the developed nanobeacon provides a simple but ultrasensitive sensor for rapid detection of CT with a large signal-to-background ratio and excellent reproducibility in a wide dynamic range, implying its potential for point-of-care applications in preventive and diagnostic monitoring of cholera.

  16. Construction and preclinical evaluation of mmCT, a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae.

    Science.gov (United States)

    Lebens, Michael; Terrinoni, Manuela; Karlsson, Stefan L; Larena, Maximilian; Gustafsson-Hedberg, Tobias; Källgård, Susanne; Nygren, Erik; Holmgren, Jan

    2016-04-19

    There is an urgent need for new adjuvants that are effective with mucosally administered vaccines. Cholera toxin (CT) is the most powerful known mucosal adjuvant but is much too toxic for human use. In an effort to develop a useful mucosal adjuvant we have generated a novel non-toxic mutant CT molecule that retains much of the adjuvant activity of native CT. This was achieved by making the enzymatically active A subunit (CTA) recalcitrant to the site-specific proteolytic cleavage ("nicking") required for toxicity, which was found to require mutations not only in the two residues rendering the molecule resistant to trypsin but also in neighboring sites protecting against cleavage by Vibrio cholerae proteases. This multiple-mutated CT (mmCT) adjuvant protein could be efficiently produced in and purified from the extracellular medium of CT-deleted V. cholerae. The mmCT completely lacked detectable enterotoxicity in an infant mouse model and had >1000-fold reduced cAMP inducing activity compared to native CT in a sensitive mammalian target cell system. It nonetheless proved to have potent adjuvant activity on mucosal and systemic antibody as well as cellular immune responses to mucosally co-administered antigens including oral cholera and intranasal influenza vaccines. We conclude that mmCT is an attractive novel non-toxic mucosal adjuvant for enhancing immune responses to co-administered mucosal vaccines. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Purification and characterization of Yersinia enterocolitica and Yersinia pestis LcrV-cholera toxin A(2)/B chimeras.

    Science.gov (United States)

    Tinker, Juliette K; Davis, Chadwick T; Arlian, Britni M

    2010-11-01

    Yersinia pestis is a virulent human pathogen and potential biological weapon. Despite a long history of research on this organism, there is no licensed vaccine to protect against pneumonic forms of Y. pestis disease. In the present study, plasmids were constructed to express cholera toxin A(2)/B chimeric molecules containing the LcrV protective antigen from Yersinia enterocolitica and Y. pestis. These chimeras were expressed and purified to high yields from the supernatant of transformed Escherichia coli. Western and GM(1) ELISA assays were used to characterize the composition, receptor-binding and relative stability of the LcrV-CTA(2)/B chimera in comparison to cholera toxin. In addition, we investigated the ability of the Y. pestis LcrV-CTA(2)/B chimera to bind to and internalize into cultured epithelial cells and macrophages by confocal microscopy. These studies indicate that the uptake and trafficking of the LcrV antigen from the chimera is comparable to the trafficking of native toxin. Together these findings report that stable, receptor-binding, non-toxic LcrV-cholera toxin A(2)/B chimeras can be expressed at high levels in E. coli and purified from the supernatant. In addition, the internalization of antigen in vitro reported here supports the development of these molecules as novel mucosal vaccine candidates. Copyright 2010 Elsevier Inc. All rights reserved.

  18. Solution and crystallographic studies of branched multivalent ligands that inhibit the receptor-binding of cholera toxin.

    Science.gov (United States)

    Zhang, Zhongsheng; Merritt, Ethan A; Ahn, Misol; Roach, Claudia; Hou, Zheng; Verlinde, Christophe L M J; Hol, Wim G J; Fan, Erkang

    2002-11-06

    The structure-based design of multivalent ligands offers an attractive strategy toward high affinity protein inhibitors. The spatial arrangement of the receptor-binding sites of cholera toxin, the causative agent of the severe diarrheal disease cholera and a member of the AB(5) bacterial toxin family, provides the opportunity of designing branched multivalent ligands with 5-fold symmetry. Our modular synthesis enabled the construction of a family of complex ligands with five flexible arms each ending with a bivalent ligand. The largest of these ligands has a molecular weight of 10.6 kDa. These ligands are capable of simultaneously binding to two toxin B pentamer molecules with high affinity, thus blocking the receptor-binding process of cholera toxin. A more than million-fold improvement over the monovalent ligand in inhibitory power was achieved with the best branched decavalent ligand. This is better than the improvement observed earlier for the corresponding nonbranched pentavalent ligand. Dynamic light scattering studies demonstrate the formation of concentration-dependent unique 1:1 and 1:2 ligand/toxin complexes in solution with no sign of nonspecific aggregation. This is in complete agreement with a crystal structure of the branched multivalent ligand/toxin B pentamer complex solved at 1.45 A resolution that shows the specific 1:2 ligand/toxin complex formation in the solid state. These results reiterate the power of the structure-based design of multivalent protein ligands as a general strategy for achieving high affinity and potent inhibition.

  19. Proinflammatory responses in the murine brain after intranasal delivery of cholera toxin: implications for the use of AB toxins as adjuvants in intranasal vaccines.

    Science.gov (United States)

    Armstrong, Michelle E; Lavelle, Ed C; Loscher, Christine E; Lynch, Marina A; Mills, Kingston H G

    2005-11-01

    Intranasal delivery of vaccines provides an attractive alternative to parenteral delivery, but it requires appropriate mucosal adjuvants. Cholera toxin (CT) is a powerful mucosal adjuvant, but it can undergo retrograde transport to the brain via the olfactory system after intranasal delivery. We demonstrate that intranasal delivery of CT increases the expression of interleukin-1 beta , cyclooxygenase-2, and chemokine messenger RNA in the murine hypothalamus, whereas parenterally delivered CT has little effect. Our findings suggest that CT can induce proinflammatory mediators in the brain when it is administered intranasally but not parenterally, and they raise concerns about the use of AB toxins as adjuvants in intranasal vaccines.

  20. A nanocoaxial-based electrochemical sensor for the detection of cholera toxin.

    Science.gov (United States)

    Archibald, Michelle M; Rizal, Binod; Connolly, Timothy; Burns, Michael J; Naughton, Michael J; Chiles, Thomas C

    2015-12-15

    Sensitive, real-time detection of biomarkers is of critical importance for rapid and accurate diagnosis of disease for point of care (POC) technologies. Current methods do not allow for POC applications due to several limitations, including sophisticated instrumentation, high reagent consumption, limited multiplexing capability, and cost. Here, we report a nanocoaxial-based electrochemical sensor for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV) or square wave voltametry (SWV). The device architecture is composed of vertically-oriented, nanoscale coaxial electrodes in array format (~10(6) coaxes per square millimeter). The coax cores and outer shields serve as integrated working and counter electrodes, respectively, exhibiting a nanoscale separation gap corresponding to ~100 nm. Proof-of-concept was demonstrated for the detection of cholera toxin (CT). The linear dynamic range of detection was 10 ng/ml-1 µg/ml, and the limit of detection (LOD) was found to be 2 ng/ml. This level of sensitivity is comparable to the standard optical ELISA used widely in clinical applications, which exhibited a linear dynamic range of 10 ng/ml-1 µg/ml and a LOD of 1 ng/ml. In addition to matching the detection profile of the standard ELISA, the nanocoaxial array provides a simple electrochemical readout and a miniaturized platform with multiplexing capabilities for the simultaneous detection of multiple biomarkers, giving the nanocoax a desirable advantage over the standard method towards POC applications.

  1. Identification of a Campylobacter jejuni protein that cross-reacts with cholera toxin.

    Science.gov (United States)

    Albert, M John; Haridas, Shilpa; Steer, David; Dhaunsi, Gursev S; Smith, A Ian; Adler, Ben

    2007-06-01

    The question of whether Campylobacter jejuni produces a cholera toxin-like toxin (CTLT) has been controversial. The objective of this study was to identify the factor that cross-reacts with CT from C. jejuni. Filtrates of C. jejuni grown in four different liquid media reported to promote CTLT production were tested by Chinese hamster ovary (CHO) cell elongation assay and for reactivity with CT antibody using GM1 ganglioside enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Protein sequence was determined by matrix-assisted laser desorption ionization-time of flight (MALDI TOF-TOF). Filtrates from seven reference strains reported to produce CTLT and from 80 clinical strains were negative in the CHO cell assay, but those from three reference strains and 16 clinical strains were positive by GM1 ELISA. All strains tested, including C. jejuni NCTC 11168, which does not contain a CT gene homologue, possessed a 53-kDa protein which reacted with CT antibody by immunoblotting. This band was identified as the major outer membrane protein, PorA, of C. jejuni. CT antibody reacted by immunoblotting with a recombinant PorA, but antibody to the recombinant PorA did not react with CT. Our results indicate that C. jejuni does not produce a functional CTLT, but the reactivity of PorA with CT antibody would lead to the erroneous conclusion that C. jejuni produces a functional CTLT.

  2. A Nanocoaxial-Based Electrochemical Sensor for the Detection of Cholera Toxin

    Science.gov (United States)

    Archibald, Michelle; Rizal, Binod; Connolly, Timothy; Burns, Michael J.; Naughton, Michael J.; Chiles, Thomas C.; Biology; Physics Collaboration

    We report a nanocoax-based electrochemical sensor for the detection of bacterial toxins using an electrochemical enzyme-linked immunosorbent assay (ELISA) and differential pulse voltammetry (DPV). The device architecture is composed of vertically-oriented, nanoscale coaxial electrodes, with coax cores and shields serving as integrated working and counter electrodes, respectively. Proof-of-concept was demonstrated for the detection of cholera toxin (CT), with a linear dynamic range of detection was 10 ng/ml - 1 µg/ml, and a limit of detection (LOD) of 2 ng/ml. This level of sensitivity is comparable to the standard optical ELISA used widely in clinical applications. The nanocoax array thus matches the detection profile of the standard ELISA while providing a simple electrochemical readout and a miniaturized platform with multiplexing capabilities, toward point-of-care (POC) implementation. In addition, next generation nanocoax devices with extended cores are currently under development, which would provide a POC platform amenable for biofunctionalization of ELISA receptor proteins directly onto the device. This work was supported by the National Institutes of Health (National Cancer Institute Award No. CA137681 and National Institute of Allergy and Infectious Diseases Award No. AI100216).

  3. Cholera toxin-B (ctxB) antigen expressing Salmonella Typhimurium polyvalent vaccine exerts protective immune response against Vibrio cholerae infection.

    Science.gov (United States)

    Vishwakarma, Vikalp; Sahoo, Sushree Sangita; Das, Susmita; Ray, Shilpa; Hardt, Wolf-Dietrich; Suar, Mrutyunjay

    2015-04-08

    Live attenuated vaccines are cost effective approach for preventing a broad range of infectious diseases, and thus are of great interest. However, immune-defects can predispose the patient to infections by the vaccine candidate itself. So far, few live vaccine candidates have been designed specifically for immune compromised individuals. Recently, we reported a new Salmonella Typhimurium Z234-vaccine strain (Periaswamy et al., PLoS ONE 2012;7:e45433), which was specifically attenuated in the NADPH-oxidase deficient host. In the present study, the Z234-vaccine strain was further engineered to express heterologous antigen (Vibrio cholerae toxin antigen subunit-B, i.e. CtxB) with the intention of creating a vector for simultaneous protection against Cholera and Salmonellosis. The primary aim of this study was to ensure the expression of CtxB antigen by the recombinant vaccine strain Z234-pMS101. The antigen CtxB was expressed through Z234 as a fusion protein with N-terminal signal sequence of Salmonella outer protein (SopE), an effector protein from Salmonella under the control of SopE promoter. The CtxB-expressing plasmid construct pMS101 (pM968-pSopE-ctxB) was found to be stable both in vitro and in vivo. In an oral mouse infection model, the vaccine strain Z234-pMS101 efficiently colonized the host gut. The extent of protection was confirmed after challenging the immunized hosts with live V. cholerae. Vaccinated mice showed reduced gut colonization by V. cholerae. Further assessment of immunological parameters supported the possibility of conferring effective immune response by Z234-pMS101 vaccine strain. Overall, the Z234-pMS101 vaccine strain showed potential as a promising polyvalent vaccine candidate to protect against S. Typhimurium and V. cholerae infection simultaneously. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, B; Pallesen, L; Jensen, LB;

    1997-01-01

    The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions...... in the fimA gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fimA in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted...... with respect to host background in three different Escherichia coli strains, i.e. an isogenic set of K-12 strains, differing in the presence of an indigenous fim gene cluster, as well as a wild-type isolate. Immunization of rabbits with purified chimeric fimbriae resulted in serum which specifically recognized...

  5. Intranasal immunization with influenza antigens conjugated with cholera toxin subunit B stimulates broad spectrum immunity against influenza viruses.

    Science.gov (United States)

    Li, Junwei; Arévalo, Maria T; Chen, Yanping; Posadas, Olivia; Smith, Jacob A; Zeng, Mingtao

    2014-01-01

    Frequent mutation of influenza viruses keep vaccinated and non-vaccinated populations vulnerable to new infections, causing serious burdens to public health and the economy. Vaccination with universal influenza vaccines would be the best way to effectively protect people from infection caused by mismatched or unforeseen influenza viruses. Presently, there is no FDA approved universal influenza vaccine. In this study, we expressed and purified a fusion protein comprising of influenza matrix 2 protein ectodomain peptides, a centralized influenza hemagglutinin stem region, and cholera toxin subunit B. Vaccination of BALB/c mice with this novel artificial antigen resulted in potent humoral immune responses, including induction of specific IgA and IgG, and broad protection against infection by multiple influenza viruses. Furthermore, our results demonstrated that when used as a mucosal antigen, cholera toxin subunit B improved antigen-stimulated T cell and memory B cell responses.

  6. Immunochromatographic detection of the heat-labile enterotoxin of enterotoxigenic Escherichia coli with cross-detection of cholera toxin.

    Science.gov (United States)

    Arimitsu, Hideyuki; Sasaki, Keiko; Tsuji, Takao

    2017-01-01

    Here, we report the development of an immunochromatographic test strip that can detect heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli. Five types of monoclonal antibody (mAb)-producing hybridomas were isolated: three mAbs were A subunit specific and two were B subunit specific. Four mAbs also cross-reacted with both LT proteins derived from swine and human E. coli strains, but only one mAb 57B9 additionally cross-reacted with cholera toxin. Thus, mAb 57B9 was used to form a gold colloid-conjugated antibody for the immunochromatographic test by combination with polyclonal anti-LT rabbit IgG. This test strip detected not only LT in the culture supernatant of LT gene-positive strains, but also cholera toxin in the culture supernatant of Vibrio cholerae. These results indicate that this test strip is suitable for the diagnosis of both enterotoxigenic E. coli and V. cholerae infection.

  7. Cholera outbreaks caused by an altered Vibrio cholerae O1 El Tor biotype strain producing classical cholera toxin B in Vietnam in 2007 to 2008.

    Science.gov (United States)

    Nguyen, Binh Minh; Lee, Je Hee; Cuong, Ngo Tuan; Choi, Seon Young; Hien, Nguyen Tran; Anh, Dang Duc; Lee, Hye Ri; Ansaruzzaman, M; Endtz, Hubert P; Chun, Jongsik; Lopez, Anna Lena; Czerkinsky, Cecil; Clemens, John D; Kim, Dong Wook

    2009-05-01

    Vibrio cholerae O1 isolates collected during cholera outbreaks occurring from late 2007 to early 2008 in northern Vietnam were revealed to represent an altered strain containing the RS1 element followed by a CTX prophage harboring El Tor type rstR and classical ctxB on the large chromosome.

  8. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin.

    OpenAIRE

    Eisinger, M.; Marko, O

    1982-01-01

    Cultures consisting almost entirely of human melanocytes were obtained from epidermal single-cell suspensions by using phorbol 12-myristate 13-acetate (10 ng/ml) in the culture medium. At this concentration, phorbol ester is toxic to human keratinocytes but not to melanocytes. When the seeding density was optimal (0.8-2 x 10(4)/cm2) and the medium contained both phorbol ester and cholera toxin, melanocytes proliferated extensively. Under these conditions, human melanocytes could be passaged s...

  9. Crystallization of two operator complexes from the Vibrio cholerae HigBA2 toxin-antitoxin module

    DEFF Research Database (Denmark)

    Hadzi, San; Garcia-Pino, Abel; Gerdes, Kenn

    2015-01-01

    The HigA2 antitoxin and the HigBA2 toxin-antitoxin complex from Vibrio cholerae were crystallized in complex with their operator box. Screening of 22 different DNA duplexes led to two crystal forms of HigA2 complexes and one crystal form of a HigBA2 complex. Crystals of HigA2 in complex with a 17...

  10. The nucleotide exchange factors Grp170 and Sil1 induce cholera toxin release from BiP to enable retrotranslocation.

    Science.gov (United States)

    Williams, Jeffrey M; Inoue, Takamasa; Chen, Grace; Tsai, Billy

    2015-06-15

    Cholera toxin (CT) intoxicates cells by trafficking from the cell surface to the endoplasmic reticulum (ER), where the catalytic CTA1 subunit hijacks components of the ER-associated degradation (ERAD) machinery to retrotranslocate to the cytosol and induce toxicity. In the ER, CT targets to the ERAD machinery composed of the E3 ubiquitin ligase Hrd1-Sel1L complex, in part via the activity of the Sel1L-binding partner ERdj5. This J protein stimulates BiP's ATPase activity, allowing BiP to capture the toxin. Presumably, toxin release from BiP must occur before retrotranslocation. Here, using loss-and gain-of-function approaches coupled with binding studies, we demonstrate that the ER-resident nucleotide exchange factors (NEFs) Grp170 and Sil1 induce CT release from BiP in order to promote toxin retrotranslocation. In addition, we find that after NEF-dependent release from BiP, the toxin is transferred to protein disulfide isomerase; this ER redox chaperone is known to unfold CTA1, which allows the toxin to cross the Hrd1-Sel1L complex. Our data thus identify two NEFs that trigger toxin release from BiP to enable successful retrotranslocation and clarify the fate of the toxin after it disengages from BiP.

  11. Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination.

    Science.gov (United States)

    Tamaki, Yukihiro; Harakuni, Tetsuya; Yamaguchi, Rui; Miyata, Takeshi; Arakawa, Takeshi

    2016-03-04

    The cholera toxin B subunit (CTB) is secreted in its pentameric form from Escherichia coli if its leader peptide is replaced with one of E. coli origin. However, the secretion of the pentamer is generally severely impaired when the molecule is mutated or fused to a foreign peptide. Therefore, we attempted to regenerate pentameric CTB from the inclusion bodies (IBs) of E. coli. Stepwise dialysis of the IBs solubilized in guanidine hydrochloride predominantly generated soluble high-molecular-mass (HMM) aggregates and only a small fraction of pentamer. Three methods to reassemble homogeneous pentameric molecules were evaluated: (i) using a pentameric coiled-coil fusion partner, expecting it to function as an assembly core; (ii) optimizing the protein concentration during refolding; and (iii) eliminating contaminants before refolding. Coiled-coil fusion had some effect, but substantial amounts of HMM aggregates were still generated. Varying the protein concentration from 0.05 mg/mL to 5mg/mL had almost no effect. In contrast, eliminating the contaminants before refolding had a robust effect, and only the pentamer was regenerated, with no detectable HMM aggregates. Surprisingly, the protein concentration at refolding was up to 5mg/mL when the contaminants were removed, with no adverse effects on refolding. The regenerated pentamer was indistinguishable in its biochemical and immunological characteristics from CTB secreted from E. coli or choleragenoid from Vibrio cholerae. This study provides a simple but very efficient strategy for pentamerizing CTB with a highly homogeneous molecular conformation, with which it may be feasible to engineer CTB derivatives and CTB fusion antigens.

  12. A novel fluorescent retrograde neural tracer: cholera toxin B conjugated carbon dots

    Science.gov (United States)

    Zhou, Nan; Hao, Zeyu; Zhao, Xiaohuan; Maharjan, Suraj; Zhu, Shoujun; Song, Yubin; Yang, Bai; Lu, Laijin

    2015-09-01

    The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde tracers.The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde

  13. Proteolysis of virulence regulator ToxR is associated with entry of Vibrio cholerae into a dormant state.

    Directory of Open Access Journals (Sweden)

    Salvador Almagro-Moreno

    2015-04-01

    Full Text Available Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the diarrheal disease, cholera. Two of its primary virulence regulators, TcpP and ToxR, are localized in the inner membrane. TcpP is encoded on the Vibrio Pathogenicity Island (VPI, a horizontally acquired mobile genetic element, and functions primarily in virulence gene regulation. TcpP has been shown to undergo regulated intramembrane proteolysis (RIP in response to environmental conditions that are unfavorable for virulence gene expression. ToxR is encoded in the ancestral genome and is present in non-pathogenic strains of V. cholerae, indicating it has roles outside of the human host. In this study, we show that ToxR undergoes RIP in V. cholerae in response to nutrient limitation at alkaline pH, a condition that occurs during the stationary phase of growth. This process involves the site-2 protease RseP (YaeL, and is dependent upon the RpoE-mediated periplasmic stress response, as deletion mutants for the genes encoding these two proteins cannot proteolyze ToxR under nutrient limitation at alkaline pH. We determined that the loss of ToxR, genetically or by proteolysis, is associated with entry of V. cholerae into a dormant state in which the bacterium is normally found in the aquatic environment called viable but nonculturable (VBNC. Strains that can proteolyze ToxR, or do not encode it, lose culturability, experience a change in morphology associated with cells in VBNC, yet remain viable under nutrient limitation at alkaline pH. On the other hand, mutant strains that cannot proteolyze ToxR remain culturable and maintain the morphology of cells in an active state of growth. Overall, our findings provide a link between the proteolysis of a virulence regulator and the entry of a pathogen into an environmentally persistent state.

  14. Proteolysis of Virulence Regulator ToxR Is Associated with Entry of Vibrio cholerae into a Dormant State

    Science.gov (United States)

    Almagro-Moreno, Salvador; Kim, Tae K.; Skorupski, Karen; Taylor, Ronald K.

    2015-01-01

    Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the diarrheal disease, cholera. Two of its primary virulence regulators, TcpP and ToxR, are localized in the inner membrane. TcpP is encoded on the Vibrio Pathogenicity Island (VPI), a horizontally acquired mobile genetic element, and functions primarily in virulence gene regulation. TcpP has been shown to undergo regulated intramembrane proteolysis (RIP) in response to environmental conditions that are unfavorable for virulence gene expression. ToxR is encoded in the ancestral genome and is present in non-pathogenic strains of V. cholerae, indicating it has roles outside of the human host. In this study, we show that ToxR undergoes RIP in V. cholerae in response to nutrient limitation at alkaline pH, a condition that occurs during the stationary phase of growth. This process involves the site-2 protease RseP (YaeL), and is dependent upon the RpoE-mediated periplasmic stress response, as deletion mutants for the genes encoding these two proteins cannot proteolyze ToxR under nutrient limitation at alkaline pH. We determined that the loss of ToxR, genetically or by proteolysis, is associated with entry of V. cholerae into a dormant state in which the bacterium is normally found in the aquatic environment called viable but nonculturable (VBNC). Strains that can proteolyze ToxR, or do not encode it, lose culturability, experience a change in morphology associated with cells in VBNC, yet remain viable under nutrient limitation at alkaline pH. On the other hand, mutant strains that cannot proteolyze ToxR remain culturable and maintain the morphology of cells in an active state of growth. Overall, our findings provide a link between the proteolysis of a virulence regulator and the entry of a pathogen into an environmentally persistent state. PMID:25849031

  15. Cholera toxin adjuvant promotes a balanced Th1/Th2/Th17 response independently of IL-12 and IL-17 by acting on Gsα in CD11b⁺ DCs

    National Research Council Canada - National Science Library

    Mattsson, J; Schön, K; Ekman, L; Fahlén-Yrlid, L; Yrlid, U; Lycke, N Y

    Despite an extensive literature on the mechanism of action of cholera toxin (CT), we still lack critical information about how the toxin acts as an adjuvant and, especially, which dendritic cells (DCs...

  16. Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler's diarrhea.

    Science.gov (United States)

    Karaman, S; Cunnick, J; Wang, K

    2012-03-01

    The non-toxic B subunit (CT-B) of cholera toxin from Vibrio cholerae is a strong immunogen and amplifies the immune reaction to conjugated antigens. In this work, a synthetic gene encoding for CT-B was expressed under control of a γ-zein promoter in maize seeds. Levels of CT-B in maize plants were determined via ganglioside dependent ELISA. The highest expression level recorded in T(1) generation seeds was 0.0014% of total aqueous soluble protein (TASP). Expression level of the same event in the T(2) generation was significantly increased to 0.0197% of TASP. Immunogenicity of maize derived CT-B was evaluated in mice with an oral immunization trial. Anti-CTB IgG and anti-CTB IgA were detected in the sera and fecal samples of the orally immunized mice, respectively. The mice were protected against holotoxin challenge with CT. An additional group of mice was administrated with an equal amount (5 μg per dose each) of mixed maize-derived CT-B and LT-B (B subunit of E. coli heat labile toxin). In the sera and fecal samples obtained from this group, the specific antibody levels were enhanced compared to either the same or a higher amount of CT-B alone. These results suggest that a synergistic action may be achieved using a CT-B and LT-B mixture that can lead to a more efficacious combined vaccine to target diarrhea induced by both cholera and enterotoxigenic strains of Escherichia coli.

  17. O139霍乱弧菌肠毒素核苷酸序列分析%Nucleotide sequence analysis of cholera toxin in Vibrio cholerae O139

    Institute of Scientific and Technical Information of China (English)

    王军; 王玺华; 白文林; 施红; 金磊

    2000-01-01

    目的 探讨O139霍乱弧菌肠毒素(CTX)核苷酸与O1群霍乱CTX毒素核苷酸序列异同.方法 用聚合酶链反应、DNA序列分析测定2株O139群、2株O1群古典型、2株O1群E1 Tor型霍乱弧菌CTX A2-B亚单位核苷酸.结果 2株O139群霍乱弧菌均含有CTX A2-B亚单位基因,O139群与O1群CTX A2-B核苷酸同源性为97.1%~98.9%.结论 O139群与O1群霍乱弧菌CTX A2-B核苷酸基本同源.进一步证实两者CTX核苷酸序列一致.%Objective To investigate the difference of nucleotide sequence between cholera toxin (CTX)of O139 and O1 Vibrio cholerae.Methods Polymerase chain reaction and DNA sequence analysis were used to study 2 strains of O139.2 strains of classical biotype and 2 strains of EI Tor hiotype.Results Both the 2 strains of cholerae O139 contained the fragment of CTX A2-B gene,and the homology befween O1 and O139 serogroups was 98.9%~97.1%.Conclusion The nucleotide sequence of CTX A2-B in Vibrio cholerae O139 was almost consistent with that in O1,which reconfirmed the consistence of the nucleotide sequence of CTX in the 2 serogroups of Vibrio cholorae.

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

    Science.gov (United States)

    Sinclair, Haydn R; de Slegte, Jaap; Gibson, Glenn R; Rastall, Robert A

    2009-04-22

    It is widely reported that cholera toxin (Ctx) remains a significant cause of gastrointestinal disease globally, particularly in developing countries where access to clean drinking water is at a premium. Vaccines are prohibitively expensive and have shown only short-term protection. Consequently, there is scope for continued development of novel treatment strategies. One example is the use of galactooligosaccharides (GOS) as functional mimics for the cell-surface toxin receptor (GM1). In this study, GOS fractions were fractionated using cation exchange chromatography followed by structural characterization using a combination of hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization mass spectrometry (ESI-MS) such that their molecular weight profiles were known. Each profile was correlated against biological activity measured using a competitive inhibitory GM1-linked ELISA. GOS fractions containing >5% hexasaccharides (DP(6)) exhibited >90% binding, with EC(50) values between 29.27 and 56.04 mg/mL. Inhibition by GOS DP(6) was dose dependent, with an EC(50) value of 5.10 mg/mL (5.15 microM MW of 990 Da). In removing low molecular weight carbohydrates that do possess prebiotic, nutraceutical, and/or biological properties and concentrating GOS DP(5) and/or DP(6), Ctx antiadhesive activity per unit of (dry) weight was improved. This could be advantageous in the manufacture of pharmaceutical or nutraceutical formulations for the treatment or prevention of an acute or chronic disease associated with or caused by the adhesion and/or uptake of a Ctx or HLT.

  19. Mutants of Escherichia coli heat-labile enterotoxin and cholera toxin as mucosal adjuvants

    Institute of Scientific and Technical Information of China (English)

    FENG Qiang; CAI Shaoxi; ZOU Quanming

    2003-01-01

    Mucosal vaccination has been getting more and more recognition because of its compliance and low risk of spreading infectious disease by contaminated syringes used in subcutaneous immunization. However, most vaccines are unable to induce immune responses when given mucosally, and require the use of strong adjuvant for effective delivery systems. Heat-labile enterotoxin (LT) and Cholera toxin(CT) are powerful mucosal adjuvants when co-administered with soluble antigens. But high toxicity hampers their use in humans. Thanks to the fine knowledge of the structure-function relationship of LT and CT, many nontoxic or low toxic mutants have been generated, part of them retain high adjuvanticity of mucosal immunization. Among these mutants, LTS63K, LTA72R, LTR192G and CTE29H, CTE112K have been widely investigated. LTS63K and CTE112K are fully non toxic, whereas LTA72R and CTE29H are low toxic, and LTR192G is nontoxic in vitro(it remains the same toxicity as wild type LT in vivo). These mutants are extremely active as mucosal adjuvants when co-administrated with a variety of antigens in different animal models. They will be investigated more widely and deeply in the future. Some of them will be tested soon in human bodies.

  20. Activation of AMPK inhibits cholera toxin stimulated chloride secretion in human and murine intestine.

    Directory of Open Access Journals (Sweden)

    Ailín C Rogers

    Full Text Available Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR, is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK, can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX mediated diarrhea. Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK. In order to substantiate our findings on the whole tissue level, short-circuit current (SCC was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops. CTX and forskolin (FSK significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments. The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.

  1. Effect of pressure on refolding of recombinant pentameric cholera toxin B.

    Science.gov (United States)

    Rodrigues, D; Farinha-Arcieri, L E; Ventura, A M; Chura-Chambi, R M; Malavasi, N V; Lemke, L S; Guimarães, J S; Ho, P L; Morganti, L

    2014-03-10

    The production of recombinant proteins is an essential tool for the expansion of modern biological research and biotechnology. The expression of heterologous proteins in Escherichia coli often results in an incomplete folding process that leads to the accumulation of inclusion bodies (IB), aggregates that hold a certain degree of native-like secondary structure. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, leading to dissociation of aggregates under non-denaturing conditions and is therefore a useful tool to solubilize proteins for posterior refolding. Cholera toxin (CT) is composed of a non-toxic pentamer of B subunits (CTB), a useful adjuvant in vaccines, and a toxic subunit A (CTA). We studied the process of refolding of CTB using HHP. HHP was shown to be effective for dissociation of CTB monomers from IB. Posterior incubation at atmospheric pressure of concentrated CTB (1mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4kbar for 16h in the presence of Tween 20 and incubated at 1bar for 120h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structure.

  2. Repeatability of ellipsometric data in cholera toxin G M1-ELISA structures

    Science.gov (United States)

    Castro, Leon G.; Thompson, Daniel W.; Tiwald, Thomas; Berberov, Emil M.; Woollam, John A.

    2007-04-01

    The need for repeated measurements in a wide range of biological testing due to statistical variations is well known. In this paper, we discuss a specific example in which the measurement probe is a spectroscopic ellipsometer. Repeatable results are needed in a wide range of applications such as drug testing, immunoassays and other tests for disease, and fundamental biomaterial research. The present paper seeks to help reduce the non-meaningful causes of lack of repeatability by identifying a large number of externally controllable factors. Another goal of this work was to quantify the effects of many of these factors on ellipsometric measurements. By exploiting the sensitivity of spectroscopic ellipsometry to ultrathin layers, improved ways to detect and quantitatively differentiate biological events can be explored. This initial work was motivated from an interest to distinguish one disease from another or discern effects of one drug from another using the high surface sensitivity of spectroscopic ellipsometry. In this paper, we investigate the example biological system of cholera toxin (CT) in an ELISA structure with monosialoganglioside (G M1).

  3. Glycolipid Crosslinking Is Required for Cholera Toxin to Partition Into and Stabilize Ordered Domains.

    Science.gov (United States)

    Raghunathan, Krishnan; Wong, Tiffany H; Chinnapen, Daniel J; Lencer, Wayne I; Jobling, Michael G; Kenworthy, Anne K

    2016-12-20

    Current models of lipid rafts propose that lipid domains exist as nanoscale compositional fluctuations and these fluctuations can potentially be stabilized into larger domains, consequently better compartmentalizing cellular functions. However, the mechanisms governing stabilized raft assembly and function remain unclear. Here, we test the role of glycolipid crosslinking as a raft targeting and ordering mechanism using the well-studied raft marker cholera toxin B pentamer (CTxB) that binds up to five GM1 glycosphingolipids to enter host cells. We show that when applied to cell-derived giant plasma membrane vesicles, a variant of CTxB containing only a single functional GM1 binding site exhibits significantly reduced partitioning to the ordered phase compared to wild-type CTxB with five binding sites. Moreover, monovalent CTxB does not stabilize membrane domains, unlike wild-type CTxB. These results support the long-held hypothesis that CTxB stabilizes raft domains via a lipid crosslinking mechanism and establish a role for crosslinking in the partitioning of CTxB to ordered domains.

  4. Intranasal delivery of cholera toxin induces th17-dominated T-cell response to bystander antigens.

    Directory of Open Access Journals (Sweden)

    Jee-Boong Lee

    Full Text Available Cholera toxin (CT is a potent vaccine adjuvant, which promotes mucosal immunity to protein antigen given by nasal route. It has been suggested that CT promotes T helper type 2 (Th2 response and suppresses Th1 response. We here report the induction of Th17-dominated responses in mice by intranasal delivery of CT. This dramatic Th17-driving effect of CT, which was dependent on the B subunit, was observed even in Th1 or Th2-favored conditions of respiratory virus infection. These dominating Th17 responses resulted in the significant neutrophil accumulation in the lungs of mice given CT. Both in vitro and in vivo treatment of CT induced strongly augmented IL-6 production, and Th17-driving ability of CT was completely abolished in IL-6 knockout mice, indicating a role of this cytokine in the Th17-dominated T-cell responses by CT. These data demonstrate a novel Th17-driving activity of CT, and help understand the mechanisms of CT adjuvanticity to demarcate T helper responses.

  5. Nod2-mediated recognition of the microbiota is critical for mucosal adjuvant activity of cholera toxin

    Science.gov (United States)

    Kim, Donghyun; Kim, Yun-Gi; Seo, Sang-Uk; Kim, Dong-Jae; Kamada, Nobuhiko; Prescott, Dave; Philpott, Dana J.; Rosenstiel, Philip; Inohara, Naohiro; Núñez, Gabriel

    2016-01-01

    Cholera toxin (CT) is a potent adjuvant for inducing mucosal immune responses. However, the mechanism by which CT induces adjuvant activity remains unclear. Here we show that the microbiota is critical for inducing antigen-specific IgG production after intranasal immunization. After mucosal vaccination with CT, both antibiotic-treated mice and germ-free (GF) had reduced antigen-specific IgG, recall-stimulated cytokine responses, an impaired follicular helper T (TFH) response and reduced plasma cells. Recognition of symbiotic bacteria via Nod2 in CD11c+ cells was required for the adjuvanticity of CT. Reconstitution of GF mice with a Nod2 agonist or Staphylococcus sciuri having high Nod2-stimulatory activity was sufficient to promote robust CT adjuvant activity whereas bacteria with low Nod2-stimulatory activity did not. Mechanistically, CT enhanced Nod2-mediated cytokine production in DCs via intracellular cAMP. These results show an important role for the microbiota and the intracellular receptor Nod2 in promoting the mucosal adjuvant activity of CT. PMID:27064448

  6. Purification and characterization of a novel cell-penetrating carrier similar to cholera toxin chimeric protein.

    Science.gov (United States)

    Lin, Weiping; Zheng, Xi; Wang, Huaqian; Yu, Lin; Zhou, Xiaofen; Sun, Yunxiao; Zhao, Suqing; Du, Zhiyun; Zhang, Kun

    2017-01-01

    Developing a recombinant vector for noninvasively delivering biological macromolecules into the brain is important. This study constructed and purified a protein complex based on the cholera toxin (CT) molecular structure. Enhanced green fluorescent protein (EGFP)-modified A2 subunits of CT (CTA2) were used as tracer molecules for introduction of transactivator of transcription (TAT) through the A subunit into cells. The protein complex EGFP-CTA2-TAT/(CTB)5 (CTB: B subunit of CT) was obtained using an in vitro recombination method and verified by monosialoganglioside-enzyme-linked immunosorbent assay and high performance liquid chromatography assay. The protein complexes bound more strongly to monosialoganglioside (GM1) than (CTB)5 at low concentrations (0.625-1.25 μg/mL). In vitro assays revealed that the transmembrane function of TAT was also maintained. The GM1-binding activity and cell membrane-penetrating ability suggested that a CT structure-based protein complexes could be used to design a delivery carrier for intranasal administration through GM1 binding. The expression vector introduced in this study provides a feasible expression frame for constructing several new macromolecular protein drugs for effective cell penetration.

  7. Fighting Cholera One-on-One: The Development and Efficacy of Multivalent Cholera-Toxin-Binding Molecules

    NARCIS (Netherlands)

    Zuilhof, H.

    2016-01-01

    A series of diseases, ranging from cholera via travelers’ diarrhea to hamburger disease, are caused by bacterially produced toxic proteins. In particular, a toxic protein unit is brought into the host cell upon binding to specific membrane-bound oligosaccharides on the host cell membrane. For exampl

  8. Fighting Cholera One-on-One: The Development and Efficacy of Multivalent Cholera-Toxin-Binding Molecules

    NARCIS (Netherlands)

    Zuilhof, H.

    2016-01-01

    A series of diseases, ranging from cholera via travelers’ diarrhea to hamburger disease, are caused by bacterially produced toxic proteins. In particular, a toxic protein unit is brought into the host cell upon binding to specific membrane-bound oligosaccharides on the host cell membrane. For

  9. N-Glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential

    OpenAIRE

    Hamorsky, Krystal Teasley; Kouokam, J. Calvin; Jurkiewicz, Jessica M.; Nelson, Bailey; Moore, Lauren J.; Husk, Adam S.; Kajiura, Hiroyuki; Fujiyama, Kazuhito; Matoba, Nobuyuki

    2015-01-01

    Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduc...

  10. Transcutaneous immunization with toxin-coregulated pilin A induces protective immunity against Vibrio cholerae O1 El Tor challenge in mice.

    Science.gov (United States)

    Rollenhagen, Julianne E; Kalsy, Anuj; Cerda, Francisca; John, Manohar; Harris, Jason B; Larocque, Regina C; Qadri, Firdausi; Calderwood, Stephen B; Taylor, Ronald K; Ryan, Edward T

    2006-10-01

    Toxin-coregulated pilin A (TcpA) is the main structural subunit of a type IV bundle-forming pilus of Vibrio cholerae, the cause of cholera. Toxin-coregulated pilus is involved in formation of microcolonies of V. cholerae at the intestinal surface, and strains of V. cholerae deficient in TcpA are attenuated and unable to colonize intestinal surfaces. Anti-TcpA immunity is common in humans recovering from cholera in Bangladesh, and immunization against TcpA is protective in murine V. cholerae models. To evaluate whether transcutaneously applied TcpA is immunogenic, we transcutaneously immunized mice with 100 mug of TcpA or TcpA with an immunoadjuvant (cholera toxin [CT], 50 mug) on days 0, 19, and 40. Mice immunized with TcpA alone did not develop anti-TcpA responses. Mice that received transcutaneously applied TcpA and CT developed prominent anti-TcpA immunoglobulin G (IgG) serum responses but minimal anti-TcpA IgA. Transcutaneous immunization with CT induced prominent IgG and IgA anti-CT serum responses. In an infant mouse model, offspring born to dams transcutaneously immunized either with TcpA and CT or with CT alone were challenged with 10(6) CFU (one 50% lethal dose) wild-type V. cholerae O1 El Tor strain N16961. At 48 h, mice born to females transcutaneously immunized with CT alone had 36% +/- 10% (mean +/- standard error of the mean) survival, while mice born to females transcutaneously immunized with TcpA and CT had 69% +/- 6% survival (P < 0.001). Our results suggest that transcutaneous immunization with TcpA and an immunoadjuvant induces protective anti-TcpA immune responses. Anti-TcpA responses may contribute to an optimal cholera vaccine.

  11. Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin

    Directory of Open Access Journals (Sweden)

    Masaya Takehara

    2017-08-01

    Full Text Available Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins.

  12. Autophagy and endosomal trafficking inhibition by Vibrio cholerae MARTX toxin phosphatidylinositol-3-phosphate-specific phospholipase A1 activity.

    Science.gov (United States)

    Agarwal, Shivani; Kim, Hyunjin; Chan, Robin B; Agarwal, Shivangi; Williamson, Rebecca; Cho, Wonhwa; Paolo, Gilbert Di; Paolo, Gilbert D; Satchell, Karla J F

    2015-10-26

    Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser-His-Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae.

  13. Protein-disulfide Isomerase Displaces the Cholera Toxin A1 Subunit from the Holotoxin without Unfolding the A1 Subunit*

    OpenAIRE

    Taylor, Michael; Banerjee, Tuhina; Ray, Supriyo; Tatulian, Suren A.; Teter, Ken

    2011-01-01

    Protein-disulfide isomerase (PDI) has been proposed to exhibit an “unfoldase” activity against the catalytic A1 subunit of cholera toxin (CT). Unfolding of the CTA1 subunit is thought to displace it from the CT holotoxin and to prepare it for translocation to the cytosol. To date, the unfoldase activity of PDI has not been demonstrated for any substrate other than CTA1. An alternative explanation for the putative unfoldase activity of PDI has been suggested by recent structural studies demons...

  14. Immunomodulatory activity of recombinant α-gliadin conjugated to cholera toxin in DQ8 transgenic mice.

    Science.gov (United States)

    Rossi, Stefano; Luongo, Diomira; Maurano, Francesco; Bergamo, Paolo; Rossi, Mauro

    2017-07-01

    Coeliac disease (CD) is characterized by an intestinal lesion sustained by an abnormal mucosal T-cell response to wheat gliadin. An immunological approach that is able to suppress this immune response is a perspective worth pursuing. Several strategies of antigen administration have been aimed at the downregulation of pathogenic T-cells. In particular, we previously reported a significant suppression of the systemic cell-mediated response toward wheat gliadin in DQ8 transgenic mice receiving nasally a recombinant α-gliadin. To gain further insight about the cellular mechanisms underlying the tolerogenic properties of this molecule, we analysed different preparations of the recombinant α-gliadin, alone or conjugated to the adjuvant cholera toxin (CT), by in vitro challenge with spleen CD4(+) T cells from gliadin-sensitized DQ8 tg mice. We found that a partially purified preparation of recombinant α-gliadin (r-gliadin) induced a significantly higher production of IFN-γ than native gliadin as well as HPLC purified r-gliadin. Interestingly, r-gliadin, but not HPLC purified r-gliadin, stimulated the gliadin-specific expression of IL-10 in CD4(+) T cells. No significant cytotoxic effect was induced by r-gliadin in MODE-K cells, a murine model of enterocytes. Notably, a conjugate CT-r-gliadin failed in stimulating IFN-γ, whereas IL-10 secretion was still induced in gliadin-specific CD4(+) T cells. In conclusion, our results showed that DCs, pulsed with CT-r-gliadin in vitro, could modulate the ongoing Th1-like T cell response toward wheat gliadin. This finding provides new insight into the design of immunomodulatory protocols potentially useful for CD. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  15. Cholera Toxin B Subunit Shows Transneuronal Tracing after Injection in an Injured Sciatic Nerve.

    Directory of Open Access Journals (Sweden)

    Bi-Qin Lai

    Full Text Available Cholera toxin B subunit (CTB has been extensively used in the past for monosynaptic mapping. For decades, it was thought to lack the ability of transneuronal tracing. In order to investigate whether biotin conjugates of CTB (b-CTB would pass through transneurons in the rat spinal cord, it was injected into the crushed left sciatic nerve. For experimental control, the first order afferent neuronal projections were defined by retrograde transport of fluorogold (FG, a non-transneuronal labeling marker as an experimental control injected into the crushed right sciatic nerve in the same rat. Neurons containing b-CTB or FG were observed in the dorsal root ganglia (DRG at the L4-L6 levels ipsilateral to the tracer injection. In the spinal cord, b-CTB labeled neurons were distributed in all laminae ipsilaterally between C7 and S1 segments, but labeling of neurons at the cervical segment was abolished when the T10 segment was transected completely. The interneurons, distributed in the intermediate gray matter and identified as gamma-aminobutyric acid-ergic (GABAergic, were labeled by b-CTB. In contrast, FG labeling was confined to the ventral horn neurons at L4-L6 spinal segments ipsilateral to the injection. b-CTB immunoreactivity remained to be restricted to the soma of neurons and often appeared as irregular patches detected by light and electron microscopy. Detection of monosialoganglioside (GM1 in b-CTB labeled neurons suggests that GM1 ganglioside may specifically enhance the uptake and transneuronal passage of b-CTB, thus supporting the notion that it may be used as a novel transneuronal tracer.

  16. Chloramphenicol improved expression of recombinant cholera toxin B subunit in Escherichia coli and its adjuvanticity

    Institute of Scientific and Technical Information of China (English)

    XIE Xiao-yan; WAN Yan-min; ZHU Zhao-qin; ZHANG Huan-xiang; XU Jian-qing

    2011-01-01

    Background Cholera toxin B subunit (CTB) was shown to be a potent adjuvant for protein immunogen,especially when inoculated through mucosal route.We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine.Methods Wild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a,and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl β-D-thiogalactoside.Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection.Interferon y (IFN-γ) enzyme-linked immunospot (Elispot) assay was used to read out the specific T-cell immunity.Results Chloramphenicol was essential for the efficient expression of recombinant CTB (rCTB) in pET-30a/BL21 (DE3)system and could be optimized at the concentration of 0.625 μg/ml in the presence of chloramphenicol.The purified rCTB could bind with GM1 efficiently.INF-y Elispot data showed the T-cell response induced in CTB adjuvanted group ((734±240) spot forming cells/106 splenocytes) was higher than that induced by non-adjuvanted ((520±150) spot forming cells/106 splenocytes),ail responses against different antigens were enhanced in parallel.Conclusion CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancing the specific T cell responses elicited by DNA vaccine,the mechanism needs to be explored in the future.

  17. Mucosally induced immunological tolerance, regulatory T cells and the adjuvant effect by cholera toxin B subunit.

    Science.gov (United States)

    Sun, J-B; Czerkinsky, C; Holmgren, J

    2010-01-01

    Induction of peripheral immunological tolerance by mucosal administration of selected antigens (Ags) ('oral tolerance') is an attractive, yet medically little developed, approach to prevent or treat selected autoimmune or allergic disorders. A highly effective way to maximize oral tolerance induction for immunotherapeutic purposes is to administer the relevant Ag together with, and preferably linked to the non-toxic B subunit protein of cholera toxin (CTB). Oral, nasal or sublingual administration of such Ag/CTB conjugates or gene fusion proteins have been found to induce tolerance with superior efficiency compared with administration of Ag alone, including the suppression of various autoimmune disorders and allergies in animal models. In a proof-of-concept clinical trial in patients with Behcet's disease, this was extended with highly promising results to prevent relapse of autoimmune uveitis. Tolerization by mucosal Ag/CTB administration results in a strong increase in Ag-specific regulatory CD4(+) T cells, apparently via two separate pathways: one using B cells as APCs and leading to a strong expansion of Foxp3(+) Treg cells which can both suppress and mediate apoptotic depletion of effector T cells, and one being B cell-independent and associated with development of Foxp3(-) regulatory T cells that express membrane latency-associated peptide and transforming growth factor (TGF-beta) and/or IL-10. The ability of CTB to dramatically increase mucosal Ag uptake and presentation by different APCs through binding to GM1 ganglioside (which makes most B cells effective APCs irrespective of their Ag specificity), together with CTB-mediated stimulation of TGF-beta and IL-10 production and inhibition of IL-6 formation may explain the dramatic potentiation of oral tolerance by mucosal Ags presented with CTB.

  18. Cholera Toxin B Subunit Shows Transneuronal Tracing after Injection in an Injured Sciatic Nerve

    Science.gov (United States)

    Lai, Bi-Qin; Qiu, Xue-Chen; Zhang, Ke; Zhang, Rong-Yi; Jin, Hui; Li, Ge; Shen, Hui-Yong; Wu, Jin-Lang; Ling, Eng-Ang; Zeng, Yuan-Shan

    2015-01-01

    Cholera toxin B subunit (CTB) has been extensively used in the past for monosynaptic mapping. For decades, it was thought to lack the ability of transneuronal tracing. In order to investigate whether biotin conjugates of CTB (b-CTB) would pass through transneurons in the rat spinal cord, it was injected into the crushed left sciatic nerve. For experimental control, the first order afferent neuronal projections were defined by retrograde transport of fluorogold (FG, a non-transneuronal labeling marker as an experimental control) injected into the crushed right sciatic nerve in the same rat. Neurons containing b-CTB or FG were observed in the dorsal root ganglia (DRG) at the L4-L6 levels ipsilateral to the tracer injection. In the spinal cord, b-CTB labeled neurons were distributed in all laminae ipsilaterally between C7 and S1 segments, but labeling of neurons at the cervical segment was abolished when the T10 segment was transected completely. The interneurons, distributed in the intermediate gray matter and identified as gamma-aminobutyric acid-ergic (GABAergic), were labeled by b-CTB. In contrast, FG labeling was confined to the ventral horn neurons at L4-L6 spinal segments ipsilateral to the injection. b-CTB immunoreactivity remained to be restricted to the soma of neurons and often appeared as irregular patches detected by light and electron microscopy. Detection of monosialoganglioside (GM1) in b-CTB labeled neurons suggests that GM1 ganglioside may specifically enhance the uptake and transneuronal passage of b-CTB, thus supporting the notion that it may be used as a novel transneuronal tracer. PMID:26640949

  19. Molecular simulation of N-acetylneuraminic acid analogs and molecular dynamics studies of cholera toxin-Neu5Gc complex.

    Science.gov (United States)

    Blessy, J Jino; Sharmila, D Jeya Sundara

    2015-01-01

    Cholera toxin (CT) is an AB5 protein complex secreted by the pathogen Vibrio cholera, which is responsible for cholera infection. N-acetylneuraminic acid (NeuNAc) is a derivative of neuraminic acid with nine-carbon backbone. NeuNAc is distributed on the cell surface mainly located in the terminal components of glycoconjugates, and also plays an important role in cell-cell interaction. In our current study, molecular docking and molecular dynamic (MD) simulations were implemented to identify the potent NeuNAc analogs with high-inhibitory activity against CT protein. Thirty-four NeuNAc analogs, modified in different positions C-1/C-2/C-4/C-5/C-7/C-8/C-9, were modeled and docked against the active site of CT protein. Among the 34 NeuNAc analogs, the analog Neu5Gc shows the least extra precision glide score of -9.52 and glide energy of -44.71 kcal/mol. NeuNAc analogs block the CT active site residues HIS:13, ASN:90, LYS:91, GLN:56, GLN:61, and TRP:88 through intermolecular hydrogen bonding. The MD simulation for CT-Neu5Gc docking complex was performed using Desmond. MD simulation of CT-Neu5Gc complex reveals the stable nature of docking interaction.

  20. Novel cholix toxin variants, ADP-ribosylating toxins in Vibrio cholerae non-O1/non-O139 strains, and their pathogenicity.

    Science.gov (United States)

    Awasthi, Sharda Prasad; Asakura, Masahiro; Chowdhury, Nityananda; Neogi, Sucharit Basu; Hinenoya, Atsushi; Golbar, Hossain M; Yamate, Jyoji; Arakawa, Eiji; Tada, Toshiji; Ramamurthy, T; Yamasaki, Shinji

    2013-02-01

    Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.

  1. Ganglioside GM1-mediated transcytosis of cholera toxin bypasses the retrograde pathway and depends on the structure of the ceramide domain.

    Science.gov (United States)

    Saslowsky, David E; te Welscher, Yvonne M; Chinnapen, Daniel J-F; Wagner, Jessica S; Wan, Joy; Kern, Eli; Lencer, Wayne I

    2013-09-06

    Cholera toxin causes diarrheal disease by binding ganglioside GM1 on the apical membrane of polarized intestinal epithelial cells and trafficking retrograde through sorting endosomes, the trans-Golgi network (TGN), and into the endoplasmic reticulum. A fraction of toxin also moves from endosomes across the cell to the basolateral plasma membrane by transcytosis, thus breeching the intestinal barrier. Here we find that sorting of cholera toxin into this transcytotic pathway bypasses retrograde transport to the TGN. We also find that GM1 sphingolipids can traffic from apical to basolateral membranes by transcytosis in the absence of toxin binding but only if the GM1 species contain cis-unsaturated or short acyl chains in the ceramide domain. We found previously that the same GM1 species are needed to efficiently traffic retrograde into the TGN and endoplasmic reticulum and into the recycling endosome, implicating a shared mechanism of action for sorting by lipid shape among these pathways.

  2. Hemocyte–hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit

    Science.gov (United States)

    Lapointe, Jason F.; Dunphy, Gary B.; Mandato, Craig A.

    2012-01-01

    Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell–bacteria adhesion and hemocyte–hemocyte adhesion (microaggregation). Hemocyte–microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte–hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2 nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6–120 nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2–120 nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2–6 nM) cause reduced hemocyte adhesion, while high levels (12–120 nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6–120 nM) and in vivo (12–120 nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the

  3. Hemocyte-hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit.

    Science.gov (United States)

    Lapointe, Jason F; Dunphy, Gary B; Mandato, Craig A

    2012-01-01

    Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell-bacteria adhesion and hemocyte-hemocyte adhesion (microaggregation). Hemocyte-microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte-hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2 nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6-120 nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2-120 nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2-6 nM) cause reduced hemocyte adhesion, while high levels (12-120 nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6-120 nM) and in vivo (12-120 nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the hemocytes, suggesting

  4. Novel GM1 ganglioside-like peptide mimics prevent the association of cholera toxin to human intestinal epithelial cells in vitro.

    Science.gov (United States)

    Yu, Robert K; Usuki, Seigo; Itokazu, Yutaka; Wu, Han-Chung

    2016-01-01

    Cholera is an acute diarrheal disease caused by infection in the gastrointestinal tract by the gram-negative bacterium, Vibrio cholerae, and is a serious public health threat worldwide. There has not been any effective treatment for this infectious disease. Cholera toxin (CT), which is secreted by V. cholerae, can enter host cells by binding to GM1, a monosialoganglioside widely distributed on the plasma membrane surface of various animal epithelial cells. The present study was undertaken to generate peptides that are conformationally similar to the carbohydrate epitope of GM1 for use in the treatment of cholera and related bacterial infection. For this purpose, we used cholera toxin B (CTB) subunit to select CTB-binding peptides that structurally mimic GM1 from a dodecamer phage-display library. Six GM1-replica peptides were selected by biopanning based on CTB recognition. Five of the six peptides showed inhibitory activity for GM1 binding to CTB. To test the potential of employing the peptide mimics for intervening with the bacterial infection, those peptides were examined for their binding capacity, functional inhibitory activity and in vitro effects using a human intestinal epithelial cell line, Caco-2 cells. One of the peptides, P3 (IPQVWRDWFKLP), was most effective in inhibiting cellular uptake of CTB and suppressing CT-stimulated cyclic adenosine monophosphate production in the cells. Our results thus provide convincing evidence that GM1-replica peptides could serve as novel agents to block CTB binding on epithelial cells and prevent the ensuing physiological effects of CT.

  5. Manipulation of electrostatic and saccharide linker interactions in the design of efficient glycopolypeptide-based cholera toxin inhibitors.

    Science.gov (United States)

    Maheshwari, Ronak; Levenson, Eric A; Kiick, Kristi L

    2010-01-11

    Multivalent, glycopolymer inhibitors designed for the treatment of disease and pathogen infection have shown improvements in binding correlated with general changes in glycopolymer architecture and composition. We have previously demonstrated that control of glycopolypeptide backbone extension and ligand spacing significantly impacts the inhibition of the cholera toxin B subunit pentamer (CT B(5)) by these polymers. In the studies reported here, we elucidate the role of backbone charge and linker length in modulating the inhibition event. Peptides of the sequence AXPXG (where X is a positive, neutral or negative amino acid), equipped with the alkyne functionality of propargyl glycine, were designed and synthesized via solid-phase peptide synthetic methods and glycosylated via Cu(I)-catalyzed alkyne-azide cycloaddition reactions. The capacity of the glycopeptides to inhibit the binding of the B(5) subunit of cholera toxin was evaluated. These studies indicated that glycopeptides with a negatively charged backbone show improved inhibition of the binding event relative to the other glycopeptides. In addition, variations in the length of the linker between the peptide and the saccharide ligand also affected the inhibition of CT by the glycopeptides. Our findings suggest that, apart from appropriate saccharide spacing and polypeptide chain extension, saccharide linker conformation and the systematic placement of charges on the polypeptide backbone are also significant variables that can be tuned to improve the inhibitory potencies of glycopolypeptide-based multivalent inhibitors.

  6. Cholera toxin B subunit acts as a potent systemic adjuvant for HIV-1 DNA vaccination intramuscularly in mice.

    Science.gov (United States)

    Hou, Jue; Liu, Ying; Hsi, Jenny; Wang, Hongzhi; Tao, Ran; Shao, Yiming

    2014-01-01

    Cholera toxin B subunit (CTB) was investigated as a classical mucosal adjuvant that can increase vaccine immunogenicity. In this study, we found out the in vitro efficacy of cholera toxin B subunit (CTB) in activating mice bone marrow-derived dendritic cells (BMDCs) through Toll-like receptor signaling pathways. In vitro RNA and transcriptional level profiling arrays revealed that CTB guides high levels of Th1 and Th2 type cytokines, inflammatory cytokines, and chemokines. Based on the robustness of these profiling results, we examined the induction of HIV Env-specific immunity by CTB co-inoculated with HIV Env DNA vaccine intramuscularly in vivo. CTB enhanced HIV-Env specific cellular immune responses in Env-specific IFN-γ ELISPOT, compared with DNA vaccine alone. Moreover, CTB induced high levels of Env specific humoral response and promoted antibody maturation after the third round of vaccination. This combination immunization strategy induced a Th2-type bias response which is indicative of a high ratio of IgG1/IgG2a. This study reports that CTB as a classical mucosal adjuvant could enhance HIV-1 DNA-based vaccine immunogenicity intramuscularly; therefore, these findings suggest that CTB could serve as an effective candidate adjuvant for DNA vaccination.

  7. Cholera Toxin Discriminates Between T Helper 1 and 2 Cells in T Cell Receptor-Mediated Activation : Role of cAMP in T Cell Proliferation

    NARCIS (Netherlands)

    Muñoz, Eduardo; Zubiaga, Ana M.; Merrow, Martha; Sauter, Nicholas P.; Huber, Brigitte T.

    1990-01-01

    CD4+ T helper (Th) clones can be divided into interleukin 2 (IL2)-secreting Th1 and IL-4-secreting Th2 cells. We show in the present report that these two Th subsets have different activation requirements for lymphokine production and proliferation: namely, cholera toxin (CT) as well as forskolin in

  8. Cholera Toxin Discriminates Between T Helper 1 and 2 Cells in T Cell Receptor-Mediated Activation : Role of cAMP in T Cell Proliferation

    NARCIS (Netherlands)

    Muñoz, Eduardo; Zubiaga, Ana M.; Merrow, Martha; Sauter, Nicholas P.; Huber, Brigitte T.

    1990-01-01

    CD4+ T helper (Th) clones can be divided into interleukin 2 (IL2)-secreting Th1 and IL-4-secreting Th2 cells. We show in the present report that these two Th subsets have different activation requirements for lymphokine production and proliferation: namely, cholera toxin (CT) as well as forskolin

  9. The Live Attenuated Cholera Vaccine CVD 103-HgR Primes Responses to the Toxin-Coregulated Pilus Antigen TcpA in Subjects Challenged with Wild-Type Vibrio cholerae.

    Science.gov (United States)

    Mayo-Smith, Leslie M; Simon, Jakub K; Chen, Wilbur H; Haney, Douglas; Lock, Michael; Lyon, Caroline E; Calderwood, Stephen B; Kirkpatrick, Beth D; Cohen, Mitchell; Levine, Myron M; Gurwith, Marc; Harris, Jason B

    2017-01-01

    One potential advantage of live attenuated bacterial vaccines is the ability to stimulate responses to antigens which are only expressed during the course of infection. To determine whether the live attenuated cholera vaccine CVD 103-HgR (Vaxchora) results in antibody responses to the in vivo-induced toxin-coregulated pilus antigen TcpA, we measured IgA and IgG responses to Vibrio cholerae O1 El Tor TcpA in a subset of participants in a recently reported experimental challenge study. Participants were challenged with V. cholerae O1 El Tor Inaba N16961 either 10 days or 90 days after receiving the vaccine or a placebo. Neither vaccination nor experimental infection with V. cholerae alone resulted in a robust TcpA IgG or IgA response, but each did elicit a strong response to cholera toxin. However, compared to placebo recipients, vaccinees had a marked increase in IgG TcpA antibodies following the 90-day challenge, suggesting that vaccination with CVD 103-HgR resulted in priming for a subsequent response to TcpA. No such difference between vaccine and placebo recipients was observed for volunteers challenged 10 days after vaccination, indicating that this was insufficient time for vaccine-induced priming of the TcpA response. The priming of the response to TcpA and potentially other antigens expressed in vivo by attenuated V. cholerae may have relevance to the maintenance of immunity in areas where cholera is endemic.

  10. Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

    Science.gov (United States)

    Critchley, D R; Streuli, C H; Kellie, S; Ansell, S; Patel, B

    1982-04-15

    Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].

  11. Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks.

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    Krystal Teasley Hamorsky

    Full Text Available INTRODUCTION: Cholera toxin B subunit (CTB is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants. METHODOLOGY/PRINCIPAL FINDINGS: In a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro. CONCLUSIONS/SIGNIFICANCE: Taken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major

  12. Synthesis, biological evaluation, WAC and NMR studies of S-galactosides and non-carbohydrate ligands of cholera toxin based on polyhydroxyalkylfuroate moieties.

    Science.gov (United States)

    Ramos-Soriano, Javier; Niss, Ulf; Angulo, Jesús; Angulo, Manuel; Moreno-Vargas, Antonio J; Carmona, Ana T; Ohlson, Sten; Robina, Inmaculada

    2013-12-23

    The synthesis of several non-carbohydrate ligands of cholera toxin based on polyhydroxyalkylfuroate moieties is reported. Some of them have been linked to D-galactose through a stable and well-tolerated S-glycosidic bond. They represent a novel type of non-hydrolyzable bidentate ligand featuring galactose and polyhydroxyalkylfuroic esters as pharmacophoric residues, thus mimicking the GM1 ganglioside. The affinity of the new compounds towards cholera toxin was measured by weak affinity chromatography (WAC). The interaction of the best candidates with this toxin was also studied by saturation transfer difference NMR experiments, which allowed identification of the binding epitopes of the ligands interacting with the protein. Interestingly, the highest affinity was shown by non-carbohydrate mimics based on a polyhydroxyalkylfuroic ester structure.

  13. Oral immunization with cholera toxin provides protection against Campylobacter jejuni in an adult mouse intestinal colonization model.

    Science.gov (United States)

    Albert, M John; Mustafa, Abu Salim; Islam, Anjum; Haridas, Shilpa

    2013-05-07

    Immunity to Campylobacter jejuni, a major diarrheal pathogen, is largely Penner serotype specific. For broad protection, a vaccine should be based on a common antigen(s) present in all strains. In our previous study (M. J. Albert, S. Haridas, D. Steer, G. S. Dhaunsi, A. I. Smith, and B. Adler, Infect. Immun. 75:3070-3073, 2007), we demonstrated that antibody to cholera toxin (CT) cross-reacted with the major outer membrane proteins (MOMPs) of all Campylobacter jejuni strains tested. In the current study, we investigated whether immunization with CT protects against intestinal colonization by C. jejuni in an adult mouse model and whether the nontoxic subunit of CT (CT-B) is the portion mediating cross-reaction. Mice were orally immunized with CT and later challenged with C. jejuni strains (48, 75, and 111) of different serotypes. Control animals were immunized with phosphate-buffered saline. Fecal shedding of challenge organisms was studied daily for 9 days. Serum and fecal antibody responses were studied by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The cross-reactivity of rabbit CT-B antibody to MOMP was studied by immunoblotting. The reactivity of 21 overlapping 30-mer oligopeptides (based on MOMP's sequence) against rabbit CT antibody was tested by ELISA. Test animals produced antibodies to CT and MMP in serum and feces and showed resistance to colonization, the vaccine efficacies being 49% (for strain 48), 37% (for strain 75), and 34% (for strain 111) (P, ≤0.05 to ≤0.001). One peptide corresponding to a variable region of MOMP showed significant reactivity. CT-B antibody cross-reacted with MOMP. Since CT-B is a component of oral cholera vaccines, it might be possible to control C. jejuni diarrhea with these vaccines. Campylobacter jejuni is a major cause of diarrhea worldwide. Patients who recover from C. jejuni diarrhea develop immunity to the infecting serotype and remain susceptible to infection with other serotypes. A vaccine based on

  14. Development of a PCR-restriction fragment length polymorphism assay for detection and subtyping of cholix toxin variant genes of Vibrio cholerae.

    Science.gov (United States)

    Awasthi, Sharda Prasad; Asakura, Masahiro; Neogi, Sucharit Basu; Hinenoya, Atsushi; Ramamurthy, T; Yamasaki, Shinji

    2014-05-01

    Cholix toxin (ChxA) is an exotoxin reported in Vibrio cholerae non-O1/non-O139. Apart from its prototype (ChxA I) we have recently identified two novel variants of this toxin, ChxA II and ChxA III. Our previous investigations indicated that the first two variants may instigate extra-intestinal infections and ChxA II can be more lethal than ChxA I in mice. However, all three cholix toxins (ChxA I to III) failed to show any enterotoxicity in rabbit ileal loops. In this study we developed a PCR-restriction fragment length polymorphism (RFLP) assay to differentiate all three chxA variants to further understand the importance of each subtype. By using 53 V. cholerae non-O1/non-O139 strains harbouring chxA genes, which were previously categorized by sequencing, and various other strains as negative controls, the PCR-RFLP assay showed 100 % typability and specificity. Furthermore, when applied to differentiate additional V. cholerae strains, which were also screened for the chxA gene by colony hybridization, this assay identified chxA I and chxA II genes among 18.5 % and 4.5 % of non-O1/non-O139 strains (n = 178), respectively. One non-O1/non-O139 strain was untypable due to the insertion of an IS911-like element. Interestingly, the chxA I gene was detected in 10 out of 137 cholera toxin gene-negative V. cholerae O1 strains. These results suggest that the PCR-RFLP assay developed in this study can be a rapid and simple method to differentiate the chxA subtypes.

  15. Multiple neuroanatomical tract-tracing using fluorescent Alexa Fluor conjugates of cholera toxin subunit B in rats.

    Science.gov (United States)

    Conte, William L; Kamishina, Hiroaki; Reep, Roger L

    2009-01-01

    Cholera toxin subunit B (CTB) is a highly sensitive retrograde neuroanatomical tracer. With the new availability of fluorescent Alexa Fluor (AF) conjugates of CTB, multiple neuroanatomical connections can be reliably studied and compared in the same animal. Here we provide a protocol that describes the use of AF-CTB for studying connections in the central nervous system of rats. The viscous properties of CTB allow small and discreet injection sites yet still show robust retrograde labeling. Furthermore, the AF conjugates are extremely bright and photostable, compared with other conventional fluorescent tracers. This protocol can also be adapted for use with other neuroanatomical tracers. Including a 7-d survival period, this protocol takes approximately 11 to 12 d to complete in its entirety.

  16. The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus

    Science.gov (United States)

    Harn, Tony; Spielman, Ingrid; Gao, Yang; Kovacikova, Gabriela; Biais, Nicolas

    2016-01-01

    Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system. PMID:27992883

  17. 霍乱毒素佐剂应用研究进展%Cholera toxin as potent immuno-adjuvant

    Institute of Scientific and Technical Information of China (English)

    解晓燕; 朱召芹

    2010-01-01

    霍乱毒素(CT)是霍乱弧菌分泌的一种不耐热肠毒素,具有很强的免疫原性和佐剂活性.CT本身有很强的毒副作用,如何既能具备优良的佐剂活性而又没有明显的毒副作用是当前研究的主要目标.CT及其亚单位因其独特的生理功能已被广泛地应用到很多新的免疫与疫苗策略中,已成为当前重要的佐剂之一.%Cholera toxin (CT) is a cholera-secreted, heat-labile enterotoxin with potent adjuvant and immunomodulatory activities. It has been well studied as a mucosal immunity adjuvant. It seems that the adjuvant efffect of CT is tightly associated with its toxicity. How to balance its adjuvant activity and toxicity becomes the major concern in the recent studies. The unique advantage of both CT and its subunits has been extensively employed in new vaccine development strategy.

  18. Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.

    Science.gov (United States)

    Ma, Tonghui; Thiagarajah, Jay R; Yang, Hong; Sonawane, Nitin D; Folli, Chiara; Galietta, Luis J V; Verkman, A S

    2002-12-01

    Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

  19. The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus.

    Directory of Open Access Journals (Sweden)

    Dixon Ng

    2016-12-01

    Full Text Available Type IV pilus (T4P systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system.

  20. Immunization with the Recombinant Cholera Toxin B Fused to Fimbria 2 Protein Protects against Bordetella pertussis Infection

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    Noelia Olivera

    2014-01-01

    Full Text Available This study examined the immunogenic properties of the fusion protein fimbria 2 of Bordetella pertussis (Fim2—cholera toxin B subunit (CTB in the intranasal murine model of infection. To this end B. pertussis Fim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2 were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside enzyme-linked immunosorbent assay (ELISA. To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 μg of CTB-Fim2. Recombinant (rFim2 or purified (BpFim2 Fim2, CTB, and phosphate-buffered saline (PBS were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (P<0.01 or P<0.001, resp.. Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen against Bordetella pertussis infection.

  1. Cholera toxin, a typical protein kinase A activator, induces G1 phase growth arrest in human bladder transitional cell carcinoma cells via inhibiting the c-Raf/MEK/ERK signaling pathway.

    Science.gov (United States)

    Zheng, Xiaoke; Ou, Yanqiu; Shu, Minfeng; Wang, Youqiong; Zhou, Yuxi; Su, Xingwen; Zhu, Wenbo; Yin, Wei; Li, Shifeng; Qiu, Pengxin; Yan, Guangmei; Zhang, Jingxia; Hu, Jun; Xu, Dong

    2014-05-01

    The biotoxin cholera toxin has been demonstrated to have anti-tumor activity in numerous types of cancer, including glioma. However, the role of cholera toxin in the tumorigenesis of transitional cell carcinoma (TCC), the most common malignant tumor of the bladder, remains to be elucidated. To address this, in the present study, two TCC cell lines, T24 and UM-UC-3, were treated with cholera toxin [protein kinase A (PKA) activator] and KT5720 (PKA inhibitor). Cell survival and proliferation, cell cycle alterations and apoptosis were analyzed using Hoechst staining, the MTT assay, fluorescence microscopy and flow cytometry. Western blot analysis was used to detect the expression of proteins involved in cell cycle regulation. The results revealed that cholera toxin significantly induced G1 arrest and downregulated the expression of cyclin D1 and cyclin-dependent kinase 4/6 in the TCC cell lines, and this was rescued by KT5720. Furthermore, it was demonstrated that cholera toxin downregulated the activation of the c-Raf/Mek/Erk cascade, an important mediator of tumor cell proliferation, via the PKA-dependent c-Raf phosphorylation at Ser-43. Furthermore, inhibition of Mek activity with UO126 mimicked the effects of cholera toxin. In conclusion, these results confirmed that cholera toxin specifically inhibited proliferation and induced G1 phase arrest in human bladder TCC cells. This effect was due to PKA-dependent inactivation of the c-Raf/Mek/Erk pathway. This suggested that cholera toxin may be a viable therapeutic treatment against tumorigenesis and proliferation in bladder cancer.

  2. Revisiting the membrane interaction mechanism of a membrane-damaging β-barrel pore-forming toxin Vibrio cholerae cytolysin.

    Science.gov (United States)

    Rai, Anand Kumar; Chattopadhyay, Kausik

    2015-09-01

    Vibrio cholerae cytolysin (VCC) permeabilizes target cell membranes by forming transmembrane oligomeric β-barrel pores. VCC has been shown to associate with the target membranes via amphipathicity-driven spontaneous partitioning into the membrane environment. More specific interaction(s) of VCC with the membrane components have also been documented. In particular, specific binding of VCC with the membrane lipid components is believed to play a crucial role in determining the efficacy of the pore-formation process. However, the structural basis and the functional implications of the VCC interaction with the membrane lipids remain unclear. Here we show that the distinct loop sequences within the membrane-proximal region of VCC play critical roles to determine the functional interactions of the toxin with the membrane lipids. Alterations of the loop sequences via structure-guided mutagenesis allow amphipathicity-driven partitioning of VCC to the membrane lipid bilayer. Alterations of the loop sequences, however, block specific interactions of VCC with the membrane lipids and abort the oligomerization, membrane insertion, pore-formation and cytotoxic activity of the toxin. Present study identifies the structural signatures in VCC implicated for its functional interactions with the membrane lipid components, a process that presumably acts to drive the subsequent steps of the oligomeric β-barrel pore-formation and cytotoxic responses.

  3. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. (Burroughs Wellcome Co., Research Triangle Park, NC (USA))

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

  4. Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains.

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    Andreas Androutsellis-Theotokis

    Full Text Available BACKGROUND: New mechanisms that regulate neural stem cell (NSC expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen. CONCLUSIONS/SIGNIFICANCE: Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer.

  5. Vibrio cholerae Infection of Drosophilamelanogaster Mimics the Human Disease Cholera.

    Directory of Open Access Journals (Sweden)

    2005-09-01

    Full Text Available Cholera, the pandemic diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, continues to be a major public health challenge in the developing world. Cholera toxin, which is responsible for the voluminous stools of cholera, causes constitutive activation of adenylyl cyclase, resulting in the export of ions into the intestinal lumen. Environmental studies have demonstrated a close association between V. cholerae and many species of arthropods including insects. Here we report the susceptibility of the fruit fly, Drosophila melanogaster, to oral V. cholerae infection through a process that exhibits many of the hallmarks of human disease: (i death of the fly is dependent on the presence of cholera toxin and is preceded by rapid weight loss; (ii flies harboring mutant alleles of either adenylyl cyclase, Gsalpha, or the Gardos K channel homolog SK are resistant to V. cholerae infection; and (iii ingestion of a K channel blocker along with V. cholerae protects wild-type flies against death. In mammals, ingestion of as little as 25 mug of cholera toxin results in massive diarrhea. In contrast, we found that ingestion of cholera toxin was not lethal to the fly. However, when cholera toxin was co-administered with a pathogenic strain of V. cholerae carrying a chromosomal deletion of the genes encoding cholera toxin, death of the fly ensued. These findings suggest that additional virulence factors are required for intoxication of the fly that may not be essential for intoxication of mammals. Furthermore, we demonstrate for the first time the mechanism of action of cholera toxin in a whole organism and the utility of D. melanogaster as an accurate, inexpensive model for elucidation of host susceptibility to cholera.

  6. Influence of Exogenous Reproductive Hormones on Specific Antibody Production in Genital Secretions after Vaginal Vaccination with Recombinant Cholera Toxin B Subunit in Humans

    OpenAIRE

    Wassen, Lotta; Jertborn, Marianne

    2006-01-01

    The objective of this study was to investigate the influence of exogenous reproductive hormones on the local and systemic production of specific immunoglobulin A (IgA) and IgG antibodies after vaginal vaccination with recombinant cholera toxin subunit B (CTB). Three groups of women using either progesterone-containing intrauterine devices (n = 9), oral contraceptives (n = 8), or no hormonal contraceptive methods (n = 9) were vaginally immunized twice, 2 weeks apart. Cervical secretions, vagin...

  7. Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose.

    Science.gov (United States)

    Kirkeby, S

    2016-01-01

    FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini remained unaffected even after increased oxidation. Staining with the subunit was also reduced significantly by adding galactose to the incubation medium. Binding of CTB to cell surfaces apparently requires intact sialic groups on most, but not all, cell surfaces. Oxidation of the sialic acid residues may influence the structure of the sialylated GM1 molecules on the cell surface in different ways. It is possible that both the sialic acid residue and the terminal galactose are oxidized. Alternatively, the sialic acid may be resistant to acid hydrolysis in gangliosides in which the sialic acid is attached to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human milk contains high levels of sialic acid glycoconjugates that may provide defense mechanisms.

  8. A single native ganglioside GM1-binding site is sufficient for cholera toxin to bind to cells and complete the intoxication pathway.

    Science.gov (United States)

    Jobling, Michael G; Yang, Zhijie; Kam, Wendy R; Lencer, Wayne I; Holmes, Randall K

    2012-10-30

    Cholera toxin (CT) from Vibrio cholerae is responsible for the majority of the symptoms of the diarrheal disease cholera. CT is a heterohexameric protein complex with a 240-residue A subunit and a pentameric B subunit of identical 103-residue B polypeptides. The A subunit is proteolytically cleaved within a disulfide-linked loop to generate the A1 and A2 fragments. The B subunit of wild-type (wt) CT binds 5 cell surface ganglioside GM(1) (GM(1)) molecules, and the toxin-GM(1) complex traffics from the plasma membrane (PM) retrograde through endosomes and the Golgi apparatus to the endoplasmic reticulum (ER). From the ER, the enzymatic A1 fragment retrotranslocates to the cytosol to cause disease. Clustering of GM(1) by multivalent toxin binding can structurally remodel cell membranes in ways that may assist toxin uptake and retrograde trafficking. We have recently found, however, that CT may traffic from the PM to the ER by exploiting an endogenous glycosphingolipid pathway (A. A. Wolf et al., Infect. Immun. 76:1476-1484, 2008, and D. J. F. Chinnapen et al., Dev. Cell 23:573-586, 2012), suggesting that multivalent binding to GM(1) is dispensable. Here we formally tested this idea by creating homogenous chimeric holotoxins with defined numbers of native GM(1) binding sites from zero (nonbinding) to five (wild type). We found that a single GM(1) binding site is sufficient for activity of the holotoxin. Therefore, remodeling of cell membranes by mechanisms that involve multivalent binding of toxin to GM(1) receptors is not essential for toxicity of CT. Through multivalent binding to its lipid receptor, cholera toxin (CT) can remodel cell membranes in ways that may assist host cell invasion. We recently found that CT variants which bind no more than 2 receptor molecules do exhibit toxicity, suggesting that CT may be able to enter cells by coopting an endogenous lipid sorting pathway without clustering receptors. We tested this idea directly by using purified variants

  9. Chloroquine derivatives block the translocation pores and inhibit cellular entry of Clostridium botulinum C2 toxin and Bacillus anthracis lethal toxin.

    Science.gov (United States)

    Kreidler, Anna-Maria; Benz, Roland; Barth, Holger

    2017-03-01

    The pathogenic bacteria Clostridium botulinum and Bacillus anthracis produce the binary protein toxins C2 and lethal toxin (LT), respectively. These toxins consist of a binding/transport (B7) component that delivers the separate enzyme (A) component into the cytosol of target cells where it modifies its specific substrate and causes cell death. The B7 components of C2 toxin and LT, C2IIa and PA63, respectively, are ring-shaped heptamers that bind to their cellular receptors and form complexes with their A components C2I and lethal factor (LF), respectively. After receptor-mediated endocytosis of the toxin complexes, C2IIa and PA63 insert into the membranes of acidified endosomes and form trans-membrane pores through which C2I and LF translocate across endosomal membranes into the cytosol. C2IIa and PA63 also form channels in planar bilayer membranes, and we used this approach earlier to identify chloroquine as a potent blocker of C2IIa and PA63 pores. Here, a series of chloroquine derivatives was investigated to identify more efficient toxin inhibitors with less toxic side effects. Chloroquine, primaquine, quinacrine, and fluphenazine blocked C2IIa and PA63 pores in planar lipid bilayers and in membranes of living epithelial cells and macrophages, thereby preventing the pH-dependent membrane transport of the A components into the cytosol and protecting cells from intoxication with C2 toxin and LT. These potent inhibitors of toxin entry underline the central role of the translocation pores for cellular uptake of binary bacterial toxins and as relevant drug targets, and might be lead compounds for novel pharmacological strategies against severe enteric diseases and anthrax.

  10. Mediodorsal thalamic nucleus receives a direct retinal input in marmoset monkey (Callithrix jacchus): a subunit B cholera toxin study.

    Science.gov (United States)

    de Sousa, Twyla Barros; de Santana, Melquisedec Abiaré Dantas; Silva, Alane de Medeiros; Guzen, Fausto Pierdoná; Oliveira, Francisco Gilberto; Cavalcante, Judney Cley; Cavalcante, Jeferson de Souza; Costa, Miriam Stela Maris Oliveira; Nascimento, Expedito Silva do

    2013-01-01

    The mediodorsal thalamic nucleus is a prominent nucleus in the thalamus, positioned lateral to the midline nuclei and medial to the intralaminar thalamic complex in the dorsal thalamus. Several studies identify the mediodorsal thalamic nucleus as a key structure in learning and memory, as well as in emotional mechanisms and alertness due to reciprocal connections with the limbic system and prefrontal cortex. Fibers from the retina to the mediodorsal thalamic nucleus have recently been described for the first time in a crepuscular rodent, suggesting a possible regulation of the mediodorsal thalamic nucleus by visual activity. The present study shows retinal afferents in the mediodorsal thalamic nucleus of a new world primate, the marmoset (Callithrix jacchus), using B subunit of cholera toxin (CTb) as an anterograde tracer. A small population of labeled retinofugal axonal arborizations is consistently labeled in small domains of the medial and lateral periphery of the caudal half of the mediodorsal nucleus. Retinal projections in the mediodorsal thalamic nucleus are exclusively contralateral and the morphology of the afferent endings was examined. Although the functional significance of this projection remains unknown, this retina-mediodorsal thalamic nucleus pathway may be involved in a wide possibility of functional implications.

  11. Transport of the cholera toxin B-subunit from recycling endosomes to the Golgi requires clathrin and AP-1.

    Science.gov (United States)

    Matsudaira, Tatsuyuki; Niki, Takahiro; Taguchi, Tomohiko; Arai, Hiroyuki

    2015-08-15

    The retrograde pathway is defined by the transport of proteins and lipids from the plasma membrane through endosomes to the Golgi complex, and is essential for a variety of cellular activities. Recycling endosomes are important sorting stations for some retrograde cargo. SMAP2, a GTPase-activating protein (GAP) for Arf1 with a putative clathrin-binding domain, has previously been shown to participate in the retrograde transport of the cholera toxin B-subunit (CTxB) from recycling endosomes. Here, we found that clathrin, a vesicle coat protein, and clathrin adaptor protein complex 1 (AP-1) were present at recycling endosomes and were needed for the retrograde transport of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes. SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling endosomes. Moreover, knockdown of Arf1 suppressed the retrograde transport of CTxB from recycling endosomes to the Golgi. These findings suggest that retrograde transport is mediated by clathrin-coated vesicles from recycling endosomes and that the role of the coat proteins is in the recruitment of Arf GAP to transport vesicles.

  12. Oral administration of a cholera toxin B subunit-insulin fusion protein produced in silkworm protects against autoimmune diabetes.

    Science.gov (United States)

    Gong, Zhaohui; Jin, Yongfeng; Zhang, Yaozhou

    2005-09-22

    The oral administration of disease-specific autoantigens can induce oral immune tolerance and prevent or delay the onset of autoimmune disease symptoms. Here, we describe the construction of an edible vaccine consisting of a fusion protein composed of cholera toxin B subunit (CTB) and insulin that is produced in silkworm larvae at levels of up to 0.3 mg/ml of hemolymph. The silkworm bioreactor produced this fusion protein vaccine as the pentameric CTB-insulin form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and insulin. Non-obese diabetic mice fed hemolymph containing microgram quantities of the CTB-insulin fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of symptoms of clinical diabetes. These results demonstrate that the silkworm bioreactor is a feasible production and delivery system for an oral protein vaccine designed to develop immunological tolerance against T-cell-mediated autoimmune diabetes by regulatory T-cell induction.

  13. Dorsal lateral geniculate substructure in the Long-Evans rat: A cholera toxin B-subunit study

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    Claire B. Discenza

    2012-09-01

    Full Text Available The pigmented rat is an increasingly important model in visual neuroscience research, yet the lamination of retinal projections in the dLGN has not been examined in sufficient detail. From previous studies it was known that most of the rat dLGN receives monocular input from the contralateral eye, with a small island receiving predominantly ipsilateral projections. Here we revisit the question using cholera toxin B subunit (CTB, a tracer that efficiently fills retinal terminals after intra-ocular injection. We imaged retinal termini throughout the dLGN at 0.5 um resolution and traced areas of ipsilateral and contralateral terminals to obtain a high resolution 3D reconstruction of the projection pattern. Retinal termini in the dLGN are well segregated by eye of origin, as expected. We find, however, that the ipsilateral projections form multiple discrete projection zones in three dimensions, not the single island previously described. It remains to be determined whether these subdomains represent distinct functional sublaminae, as is the case in other mammals.

  14. Immunogenicity and protective efficacy of rotavirus VP8* fused to cholera toxin B subunit in a mouse model.

    Science.gov (United States)

    Xue, Miaoge; Yu, Linqi; Jia, Lianzhi; Li, Yijian; Zeng, Yuanjun; Li, Tingdong; Ge, Shengxiang; Xia, Ningshao

    2016-11-01

    In attempts to develop recombinant subunit vaccines against rotavirus disease, it was previously shown that the N-terminal truncated VP8* protein, VP8-1 (aa26-231), is a good vaccine candidate when used for immunization in combination with Freund's adjuvant. However, this protein stimulated only weak immune response when aluminum hydroxide was used as an adjuvant. In this study, the nontoxic B subunit of cholera toxin (CTB) was employed as intra-molecular adjuvant to improve the immunogenicity of VP8-1. Both, the N-terminal and C-terminal fusion proteins, were purified to homogeneity, at which stage they formed pentamers, and showed significantly higher immunogenicity and protective efficacy than a VP8-1/aluminum hydroxide mixture in a mouse model. Compared to VP8-1-CTB, CTB-VP8-1 showed higher binding activity to both, GM1 and the conformation sensitive neutralizing monoclonal antibodies specific to VP8. More importantly, CTB-VP8-1 elicited higher titers of neutralizing antibodies and conferred higher protective efficacy than VP8-1-CTB. Therefore, the protein CTB-VP8-1, with enhanced immunogenicity and immunoprotectivity, could be considered as a viable candidate for further development of an alternative, replication-incompetent, parenterally administered vaccine against rotavirus disease.

  15. Induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin B subunit vaccine without plant-associated sugar modification.

    Science.gov (United States)

    Yuki, Yoshikazu; Mejima, Mio; Kurokawa, Shiho; Hiroiwa, Tomoko; Takahashi, Yuko; Tokuhara, Daisuke; Nochi, Tomonori; Katakai, Yuko; Kuroda, Masaharu; Takeyama, Natsumi; Kashima, Koji; Abe, Michiyo; Chen, Yingju; Nakanishi, Ushio; Masumura, Takehiro; Takeuchi, Yoji; Kozuka-Hata, Hiroko; Shibata, Hiroaki; Oyama, Masaaki; Tanaka, Kunisuke; Kiyono, Hiroshi

    2013-09-01

    Plants have been used as expression systems for a number of vaccines. However, the expression of vaccines in plants sometimes results in unexpected modification of the vaccines by N-terminal blocking and sugar-chain attachment. Although MucoRice-CTB was thought to be the first cold-chain-free and unpurified oral vaccine, the molecular heterogeneity of MucoRice-CTB, together with plant-based sugar modifications of the CTB protein, has made it difficult to assess immunological activity of vaccine and yield from rice seed. Using a T-DNA vector driven by a prolamin promoter and a signal peptide added to an overexpression vaccine cassette, we established MucoRice-CTB/Q as a new generation oral cholera vaccine for humans use. We confirmed that MucoRice-CTB/Q produces a single CTB monomer with an Asn to Gln substitution at the 4th glycosylation position. The complete amino acid sequence of MucoRice-CTB/Q was determined by MS/MS analysis and the exact amount of expressed CTB was determined by SDS-PAGE densitometric analysis to be an average of 2.35 mg of CTB/g of seed. To compare the immunogenicity of MucoRice-CTB/Q, which has no plant-based glycosylation modifications, with that of the original MucoRice-CTB/N, which is modified with a plant N-glycan, we orally immunized mice and macaques with the two preparations. Similar levels of CTB-specific systemic IgG and mucosal IgA antibodies with toxin-neutralizing activity were induced in mice and macaques orally immunized with MucoRice-CTB/Q or MucoRice-CTB/N. These results show that the molecular uniformed MucoRice-CTB/Q vaccine without plant N-glycan has potential as a safe and efficacious oral vaccine candidate for human use.

  16. Effects of tcpB Mutations on Biogenesis and Function of the Toxin-Coregulated Pilus, the Type IVb Pilus of Vibrio cholerae.

    Science.gov (United States)

    Gao, Yang; Hauke, Caitlyn A; Marles, Jarrad M; Taylor, Ronald K

    2016-10-15

    Vibrio cholerae is the etiological agent of the acute intestinal disorder cholera. The toxin-coregulated pilus (TCP), a type IVb pilus, is an essential virulence factor of V. cholerae Recent work has shown that TcpB is a large minor pilin encoded within the tcp operon. TcpB contributes to efficient pilus formation and is essential for all TCP functions. Here, we have initiated a detailed targeted mutagenesis approach to further characterize this salient TCP component. We have identified (thus far) 20 residues of TcpB which affect either the steady-state level of TcpB or alter one or more TCP functions. This study provides a solid framework for further understanding of the complex role of TcpB and will be of use upon determination of the crystal structure of TcpB or related minor pilin orthologs of type IVb pilus systems. Type IV pili, such as the toxin-coregulated pilus (TCP) in V. cholerae, are bacterial appendages that often act as essential virulence factors. Minor pilins, like TcpB, of these pili systems often play integral roles in pilus assembly and function. In this study, we have generated mutations in tcpB to determine residues of importance for TCP stability and function. Combined with a predicted tertiary structure, characterization of these mutants allows us to better understand critical residues in TcpB and the role they may play in the mechanisms underlying minor pilin functions. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Toxins

    Science.gov (United States)

    Toxins are substances created by plants and animals that are poisonous to humans. Toxins also include some medicines that are helpful in small doses, but poisonous in large amounts. Most toxins that cause problems ...

  18. Safety of the recombinant cholera toxin B subunit, killed whole-cell (rBS-WC oral cholera vaccine in pregnancy.

    Directory of Open Access Journals (Sweden)

    Ramadhan Hashim

    Full Text Available Mass vaccinations are a main strategy in the deployment of oral cholera vaccines. Campaigns avoid giving vaccine to pregnant women because of the absence of safety data of the killed whole-cell oral cholera (rBS-WC vaccine. Balancing this concern is the known higher risk of cholera and of complications of pregnancy should cholera occur in these women, as well as the lack of expected adverse events from a killed oral bacterial vaccine.From January to February 2009, a mass rBS-WC vaccination campaign of persons over two years of age was conducted in an urban and a rural area (population 51,151 in Zanzibar. Pregnant women were advised not to participate in the campaign. More than nine months after the last dose of the vaccine was administered, we visited all women between 15 and 50 years of age living in the study area. The outcome of pregnancies that were inadvertently exposed to at least one oral cholera vaccine dose and those that were not exposed was evaluated. 13,736 (94% of the target women in the study site were interviewed. 1,151 (79% of the 1,453 deliveries in 2009 occurred during the period when foetal exposure to the vaccine could have occurred. 955 (83% out of these 1,151 mothers had not been vaccinated; the remaining 196 (17% mothers had received at least one dose of the oral cholera vaccine. There were no statistically significant differences in the odds ratios for birth outcomes among the exposed and unexposed pregnancies.We found no statistically significant evidence of a harmful effect of gestational exposure to the rBS-WC vaccine. These findings, along with the absence of a rational basis for expecting a risk from this killed oral bacterial vaccine, are reassuring but the study had insufficient power to detect infrequent events.ClinicalTrials.gov NCT00709410.

  19. Identification of a TcpC-TcpQ Outer Membrane Complex Involved in the Biogenesis of the Toxin-Coregulated Pilus of Vibrio cholerae

    OpenAIRE

    Bose, Niranjan; Taylor, Ronald K.

    2005-01-01

    The toxin-coregulated pilus (TCP) of Vibrio cholerae and the soluble TcpF protein that is secreted via the TCP biogenesis apparatus are essential for intestinal colonization. The TCP biogenesis apparatus is composed of at least nine proteins but is largely uncharacterized. TcpC is an outer membrane lipoprotein required for TCP biogenesis that is a member of the secretin protein superfamily. In the present study, analysis of TcpC in a series of strains deficient in each of the TCP biogenesis p...

  20. The β-subunit of cholera toxin has a high affinity for ganglioside GM1 embedded into solid supported lipid membranes with a lipid raft-like composition.

    Science.gov (United States)

    Margheri, G; D'Agostino, R; Trigari, S; Sottini, S; Del Rosso, M

    2014-02-01

    In this communication, we report on the fabrication of GM1-rich solid-supported bilayer lipid membranes (ssBLM) made of sphingomyelin and cholesterol, the main components of lipid rafts,which are the physiological hosting microenvironment of GM1 on the cell membrane. The functionality of the ganglioside has been checked by measuring the apparent dissociation constant K(D) of the complex formed by the β-subunit of the cholera toxin and GM1. The value found deviates less than one order of magnitude from that measured for in vivo cells, indicating the potential of these ssBLM as optimized in vitro biomimetic platforms.

  1. Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

    Science.gov (United States)

    Liu, Jinny L; Anderson, George P; Delehanty, James B; Baumann, Richard; Hayhurst, Andrew; Goldman, Ellen R

    2007-03-01

    Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

  2. Immunogenicity of a West Nile Virus DIII-Cholera Toxin A2/B Chimera after Intranasal Delivery

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    Juliette K. Tinker

    2014-04-01

    Full Text Available West Nile virus (WNV causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT CTA2/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA2/B, DIII, DIII mixed with CTA2/B, or CTA2/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA2/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA2/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA2/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA2/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate.

  3. Immunogenicity of a West Nile virus DIII-cholera toxin A2/B chimera after intranasal delivery.

    Science.gov (United States)

    Tinker, Juliette K; Yan, Jie; Knippel, Reece J; Panayiotou, Panos; Cornell, Kenneth A

    2014-04-22

    West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT) CTA2/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA2/B, DIII, DIII mixed with CTA2/B, or CTA2/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA2/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA2/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA2/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA2/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate.

  4. Cholera toxin subunit B inhibits IL-12 and IFN-{gamma} production and signaling in experimental colitis and Crohn's disease.

    Science.gov (United States)

    Coccia, E M; Remoli, M E; Di Giacinto, C; Del Zotto, B; Giacomini, E; Monteleone, G; Boirivant, M

    2005-11-01

    Cholera toxin B subunit (CT-B) is a powerful modulator of immune responses. The authors have previously demonstrated that oral administration of recombinant CT-B (rCT-B) is able to prevent and cure the Crohn's disease (CD)-like trinitrobenzene sulfonic acid (TNBS) mediated colitis. In this study they extended their observations and examined if rCT-B interferes with the molecular signaling underlying the Th1 type response both in TNBS colitis and in ex vivo human CD explants. TNBS treated mice were fed with rCT-B, and IFN-gamma and IL-12 production by colonic lamina propria mononuclear cells (LPMC) was examined by ELISA. In vitro culture of mucosal explants from CD patients and non-inflammatory bowel disease controls, pre-incubated with rCT-B, were examined for IFN-gamma and IL-12 production by ELISA and semiquantitative reverse transcription polymerase chain reactions. STAT-1, -4, -6 activation and T-bet expression were examined following rCT-B treatment by western blotting both in TNBS treated mice and in human mucosal explants. rCT-B significantly reduced IL-12 and IFN-gamma secretion by LPMC from TNBS treated mice. Consistent with this, rCT-B inhibited both STAT-4 and STAT-1 activation and downregulated T-bet expression. Inhibition of Th1 signaling by CT-B associated with no change in IL-4 synthesis and expression of active STAT-6 indicating that rCT-B does not enhance Th2 cell responses. Moreover, in vitro treatment of CD mucosal explants with rCT-B resulted in reduced secretion of IL-12/IFN-gamma and inhibition of STAT-4/STAT-1 activation and T-bet expression. These studies indicate that CT-B inhibits mucosal Th1 cell signaling and suggest that rCT-B may be a promising candidate for CD therapy.

  5. M-cell targeted delivery of recombinant hepatitis B surface antigen using cholera toxin B subunit conjugated bilosomes.

    Science.gov (United States)

    Shukla, Anshuman; Katare, O P; Singh, Bhupinder; Vyas, Suresh P

    2010-01-29

    The present study aims to improve upon our earlier findings with bilosomes as potential delivery vehicle through oral route for recombinant hepatitis B surface antigen (HBsAg). The work entails the conjugation of bilosomal system with cholera toxin B subunit (CTB) to increase transmucosal uptake via M-cell specific delivery approach. The study encompasses the development and characterization of HBsAg-loaded CTB-conjugated system for percent antigen entrapment, size, shape, and stability in SGF (USP, pH 1.2), SIF (USP, pH 7.5) and in bile salt solutions. Biological activity of CTB, subsequent to conjugation, was verified by hemagglutination test. Anti-HBsAg IgG response in serum and anti-HBsAg sIgA in various body secretions were estimated using ELISA, following oral immunization with 10 microg dose-loaded CTB-conjugated bilosomes (CTB2) and 20 microg dose-loaded CTB-conjugated bilosomes (CTB1) in BALB/c mice. The results showed that CTB1 produced anti-HBsAg IgG antibody titre response comparable to that of the intramuscular (i.m.) injection of 10 microg of alum-adsorbed HBsAg. Moreover, all the bilosomal preparations elicited measurable sIgA vis-à-vis negligible response with i.m. administered HBsAg. Thus, HBsAg-loaded CTB-conjugated bilosomes provide a promising potential for targeted oral immunization against hepatitis B. 2009 Elsevier B.V. All rights reserved.

  6. Measuring Positive Cooperativity Using the Direct ESI-MS Assay. Cholera Toxin B Subunit Homopentamer Binding to GM1 Pentasaccharide

    Science.gov (United States)

    Lin, Hong; Kitova, Elena N.; Klassen, John S.

    2014-01-01

    Direct electrospray ionization mass spectrometry (ESI-MS) assay was used to investigate the stepwise binding of the GM1 pentasaccharide β- D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β- D-Gal p-(1→4)-β-D-Glc p (GM1os) to the cholera toxin B subunit homopentamer (CTB5) and to establish conclusively whether GM1os binding is cooperative. Apparent association constants were measured for the stepwise addition of one to five GM1os to CTB5 at pH 6.9 and 22 °C. The intrinsic association constant, which was established from the apparent association constant for the addition of a single GM1os to CTB5, was found to be (3.2 ± 0.2) × 106 M-1. This is in reasonable agreement with the reported value of (6.4 ± 0.3) × 106 M-1, which was measured at pH 7.4 and 25 °C using isothermal titration calorimetry (ITC). Analysis of the apparent association constants provides direct and unambiguous evidence that GM1os binding exhibits small positive cooperativity. Binding was found to be sensitive to the number of ligand-bound nearest neighbor subunits, with the affinities enhanced by a factor of 1.7 and 2.9 when binding occurs next to one or two ligand-bound subunits, respectively. These findings, which provide quantitative support for the binding model proposed by Homans and coworkers [14], highlight the unique strengths of the direct ESI-MS assay for measuring cooperative ligand binding.

  7. Luminal cholera toxin alters motility in isolated guinea-pig jejunum via a pathway independent of 5-HT3 receptors

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    Candice eFung

    2010-09-01

    Full Text Available Cholera toxin (CT is well established to produce diarrhoea by producing hyperactivity of the enteric neural circuits that regulate water and electrolyte secretion. Its effects on intestinal motor patterns are less well understood. We examined the effects of luminal CT on motor activity of guinea-pig jejunum in vitro. Segments of jejunum were cannulated at either end and mounted horizontally.Their contractile activity was video-imaged and the recordings were used to construct spatiotemporal maps of contractile activity with CT (1.25 μg/ml or 12.5 μg/ml in the lumen. Both concentrations of CT induced propulsive motor activity in jejunal segments. The effect of 1.25 μg/ml CT was markedly enhanced by coincubation with granisetron (5-HT3 antagonist, 1 μM, which prevents the hypersecretion induced by CT. The increased propulsive activity was not accompanied by increased segmentation and occurred very early after exposure to CT in the presence of granisetron. Luminal CT also reduced the pressure threshold for saline distension evoked propulsive reflexes, an effect resistant to granisetron. In contrast, CT prevented the induction of segmenting contractions by luminal decanoic acid, so its effects on propulsive and segmenting contractile activity are distinctly different. Thus, in addition to producing hypersecretion, CT excites propulsive motor activity with an entirely different time course and pharmacology, but inhibits nutrient induced segmentation. This suggests that CT excites more than one enteric neural circuit and that propulsive and segmenting motor patterns are differentially regulated.

  8. Vibrio cholerae in an Historically Cholera-Free Country.

    Science.gov (United States)

    Haley, Bradd J; Chen, Arlene; Grim, Christopher J; Clark, Philip; Diaz, Celia Municio; Taviani, Elisa; Hasan, Nur A; Sancomb, Elizabeth; Elnemr, Wessam Mahmoud; Islam, Muhammad A; Huq, Anwar; Colwell, Rita R; Benediktsdóttir, Eva

    2012-08-01

    We report the autochthonous existence of Vibrio cholerae in coastal waters of Iceland, a geothermally active country where cholera is absent and has never been reported. Seawater, mussel, and macroalgae samples were collected close to and distant from sites where geothermal activity causes a significant increase in water temperature during low tides. V. cholerae was detected only at geothermal-influenced sites during low-tides. None of the V. cholerae isolates encoded cholera toxin (ctxAB) and all were non-O1/non-O139 serogroups. However, all isolates encoded other virulence factors that are associated with cholera as well as extra-intestinal V. cholerae infections. The virulence factors were functional at temperatures of coastal waters of Iceland, suggesting an ecological role. It is noteworthy that V. cholerae was isolated from samples collected at sites distant from anthropogenic influence, supporting the conclusion that V. cholerae is autochthonous to the aquatic environment of Iceland.

  9. In vitro evaluation of capsaicin inhibitory effects on zonula occludens toxin in vibrio cholerae ATCC14035 strain

    Directory of Open Access Journals (Sweden)

    Soroor Erfanimanesh

    2014-10-01

    Conclusion: Capsaicin is one of the active compounds of red chili that can drastically suppress zot gene expression and shows promising inhibitory effect against V. cholerae zot production. Thus, routine intake of red chilli, which is easily available and inexpensive, may be an alternative approach to prevent and control symptoms of cholera.

  10. Trapping of Vibrio cholerae cytolysin in the membrane-bound monomeric state blocks membrane insertion and functional pore formation by the toxin.

    Science.gov (United States)

    Rai, Anand Kumar; Chattopadhyay, Kausik

    2014-06-13

    Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging cytolytic toxin that belongs to the family of β barrel pore-forming protein toxins. VCC induces lysis of its target eukaryotic cells by forming transmembrane oligomeric β barrel pores. The mechanism of membrane pore formation by VCC follows the overall scheme of the archetypical β barrel pore-forming protein toxin mode of action, in which the water-soluble monomeric form of the toxin first binds to the target cell membrane, then assembles into a prepore oligomeric intermediate, and finally converts into the functional transmembrane oligomeric β barrel pore. However, there exists a vast knowledge gap in our understanding regarding the intricate details of the membrane pore formation process employed by VCC. In particular, the membrane oligomerization and membrane insertion steps of the process have only been described to a limited extent. In this study, we determined the key residues in VCC that are critical to trigger membrane oligomerization of the toxin. Alteration of such key residues traps the toxin in its membrane-bound monomeric state and abrogates subsequent oligomerization, membrane insertion, and functional transmembrane pore-formation events. The results obtained from our study also suggest that the membrane insertion of VCC depends critically on the oligomerization process and that it cannot be initiated in the membrane-bound monomeric form of the toxin. In sum, our study, for the first time, dissects membrane binding from the subsequent oligomerization and membrane insertion steps and, thus, defines the exact sequence of events in the membrane pore formation process by VCC.

  11. N-glycosylation of cholera toxin B subunit in Nicotiana benthamiana: impacts on host stress response, production yield and vaccine potential.

    Science.gov (United States)

    Hamorsky, Krystal Teasley; Kouokam, J Calvin; Jurkiewicz, Jessica M; Nelson, Bailey; Moore, Lauren J; Husk, Adam S; Kajiura, Hiroyuki; Fujiyama, Kazuhito; Matoba, Nobuyuki

    2015-01-23

    Plant-based transient overexpression systems enable rapid and scalable production of subunit vaccines. Previously, we have shown that cholera toxin B subunit (CTB), an oral cholera vaccine antigen, is N-glycosylated upon expression in transgenic Nicotiana benthamiana. Here, we found that overexpression of aglycosylated CTB by agroinfiltration of a tobamoviral vector causes massive tissue necrosis and poor accumulation unless retained in the endoplasmic reticulum (ER). However, the re-introduction of N-glycosylation to its original or an alternative site significantly relieved the necrosis and provided a high CTB yield without ER retention. Quantitative gene expression analysis of PDI, BiP, bZIP60, SKP1, 26Sα proteasome and PR1a, and the detection of ubiquitinated CTB polypeptides revealed that N-glycosylation significantly relieved ER stress and hypersensitive response, and facilitated the folding/assembly of CTB. The glycosylated CTB (gCTB) was characterized for potential vaccine use. Glycan profiling revealed that gCTB contained approximately 38% plant-specific glycans. gCTB retained nanomolar affinity to GM1-ganglioside with only marginal reduction of physicochemical stability and induced an anti-cholera holotoxin antibody response comparable to native CTB in a mouse oral immunization study. These findings demonstrated gCTB's potential as an oral immunogen and point to a potential role of N-glycosylation in increasing recombinant protein yields in plants.

  12. Studies on nonidet P40 lysis of murine lymphoid cells. I. Use of cholera toxin and cell surface Ig to determine degree of dissociation of the plasma membrane.

    Science.gov (United States)

    Hart, D A

    1975-09-01

    Lymphoid cells from A/J mice were iodinated (125I) by the lactoperoxidase lysed with the non-ionic detergent NP-40. The plasma membrane glycolipid receptor for cholera toxin and cell surface immunoglobulin were utilized in immune precipitation systems to characterize the degree of dissociation of the plasma membrane under various conditions. It was found that at 0.1% NP-40 and at cell concentration from 5 to 10 times 10(7) cells/ml, lipid-protein and protein-lipid-protein complexes formed in NP-40 which were soluble after centrifugation at 10(5) times G. Column chromatography of 125I-cell lysates on agarose A-0.5 M in 0.1% or 0.5% NP-40/PBS indicated that the majority of iodinated cell surface material existed as aggregates in detergent micelles. The availability of the oligosaccharide moiety of the glycolipid to interact with the cholera toxin was dependent on both the detergent concentration and the cell concentration used for cell lysis. However, the cell surface immunoglobulin was immunoprecipitable under all conditions of lysis tested.

  13. Evidence for TLR4 and FcRγ-CARD9 activation by cholera toxin B subunit and its direct bindings to TREM2 and LMIR5 receptors.

    Science.gov (United States)

    Phongsisay, Vongsavanh; Iizasa, Ei'ichi; Hara, Hiromitsu; Yoshida, Hiroki

    2015-08-01

    Cholera toxin (CTX) is a virulent factor of Vibrio cholerae that causes life-threatening diarrheal disease. Its non-toxic subunit CTB has been extensively studied for vaccine delivery. In immune cells, CTB induces a number of signaling molecules related to cellular activation and cytokine production. The mechanisms by which CTB exerts its immunological effects are not understood. We report here the immunological targets of CTB. The unexpected finding that GM1 ganglioside inhibited NF-κB activation in human monocytes stimulated with CTX and agonists of Toll-like receptors (TLR) suggests the possibility of CTX-TLR interaction. Indeed, CTX-induced IL-6 production was substantially reduced in MyD88(-/-) or TLR4(-/-) macrophages. Ectopic expression of TLR4 was required for CTX-induced NF-κB activation in HEK 293 cells. Furthermore, the inflammatory capacity of CTB was lost in the absence of TLR4, adaptor protein FcRγ, or its downstream signaling molecule CARD9. Attempts have been made to identify CTB-binding targets from various C-type lectin and immunoglobulin-like receptors. CTB targeted not only GM1 and TLR4 but also TREM2 and LMIR5/CD300b. CTB-TREM2 interaction initiated signal transduction through adaptor protein DAP12. The binding of CTB inhibited LMIR5 activation induced by its endogenous ligand 3-O-sulfo-β-d-galactosylceramide C24:1. In summary, CTB targets TLR4, FcRγ-CARD9, TREM2, and LMIR5. These findings provide new insights into the immunobiology of cholera toxin.

  14. Advances in the study of cholera toxin as a mucosal adjuvant%霍乱毒素作为黏膜佐剂的研究进展

    Institute of Scientific and Technical Information of China (English)

    郝海霞; 李润花; 殷国荣

    2012-01-01

    霍乱毒素(cholera toxin,CT)是霍乱弧菌分泌的一种不耐热肠毒素,既是强黏膜免疫原,又具有很强的黏膜佐剂活性,是当今研究最多且最深入的黏膜免疫佐剂之一.然而,由于CT其毒性,限制了在人体的使用.很多研究致力于使CT的佐剂性与毒性分离,CT亚基的佐剂性的研究就是方向之一.困扰黏膜疫苗的一个重要问题是很多抗原的黏膜免疫原性较弱,不能刺激有效的免疫反应,这也与黏膜免疫耐受有关,以CT为佐剂能消除机体对这些共免疫原的耐受.%Cholera toxin (CT) secreted by Vibrio cholerae is a heat-labile enterotoxin and is also an exceptionally potent mucosal immunogen. CT has also been found to be a strong adjuvant for many coadministered antigens, making it one of the most intensively studied mucosal adjuvants. Although CT is a powerful mucosal adjuvant, its toxicity is likely to preclude usage in human vaccines. Many studies have sought to separate CTs adjuvanticity from its toxicity; one way to do so is through the adjuvanticity of its subunits. A problem hampering mucosal vaccines is the weak immunogenicity of many antigens, causing the vaccine to fail to stimulate an effective immune response. This phenomenon is related to immune tolerance, and use of CT as a mucosal adjuvant will eliminate tolerance to the co-immunogen.

  15. Activation of a pro-survival pathway IL-6/JAK2/STAT3 contributes to glial fibrillary acidic protein induction during the cholera toxin-induced differentiation of C6 malignant glioma cells.

    Science.gov (United States)

    Shu, Minfeng; Zhou, Yuxi; Zhu, Wenbo; Wu, Sihan; Zheng, Xiaoke; Yan, Guangmei

    2011-06-01

    Differentiation-inducing therapy has been proposed to be a novel potential approach to treat malignant gliomas. Glial fibrillary acidic protein (GFAP) is a well-known specific astrocyte biomarker and acts as a tumor suppressor gene (TSG) in glioma pathogenesis. Previously we reported that a traditional biotoxin cholera toxin could induce malignant glioma cell differentiation characterized by morphologic changes and dramatic GFAP expression. However, the molecular mechanisms underlying GFAP induction are still largely unknown. Here we demonstrate that an oncogenic pathway interleukin-6/janus kinase-2/signal transducer and activator of transcription 3 (IL-6/JAK2/STAT3) cascade mediates the cholera toxin-induced GFAP expression. Cholera toxin dramatically stimulated GFAP expression at the transcriptional level in C6 glioma cells. Meanwhile, phosphorylation of STAT3 and JAK2 was highly induced in a time-dependent manner after cholera toxin incubation, whereas no changes of STAT3 and JAK2 were observed. Furthermore, the IL-6 gene was quickly induced by cholera toxin and subsequent IL-6 protein secretion was stimulated. Importantly, exogenous recombinant rat IL-6 can also induce phosphorylation of STAT3 concomitant with GFAP expression while JAK2 specific inhibitor AG490 could effectively block both cholera toxin- and IL-6-induced GFAP expression. Given that the methylation of the STAT3 binding element can suppress GFAP expression, we detected the methylation status of the critical recognition sequence of STAT3 in the promoter of GFAP gene (-1518 ∼ -1510) and found that it was unmethylated in C6 glioma cells. In addition, neither DNA methyltransferase1 (DNMT1) inhibitor 5-Aza-2'-deoxycytidine (5-AZa-CdR) nor silencing DNMT1 can stimulate GFAP expression, indicating that the loss of GFAP expression in C6 cells is not caused by its promoter hypermethylation. Taken together, our findings suggest that activation of a pro-survival IL-6/JAK2/STAT3 cascade contributes to

  16. Molecular dynamics of sialic acid analogues complex with cholera toxin and DFT optimization of ethylene glycol-mediated zinc nanocluster conjugation.

    Science.gov (United States)

    Sharmila, D Jeya Sundara; Jino Blessy, J

    2017-01-01

    Cholera is an infectious disease caused by cholera toxin (CT) protein of bacterium Vibrio cholerae. A sequence of sialic acid (N-acetylneuraminic acid, NeuNAc or Neu5Ac) analogues modified in its C-5 position is modelled using molecular modelling techniques and docked against the CT followed by molecular dynamics simulations. Docking results suggest better binding affinity of NeuNAc analogue towards the binding site of CT. The NeuNAc analogues interact with the active site residues GLU:11, TYR:12, HIS:13, GLY:33, LYS:34, GLU:51, GLN:56, HIE:57, ILE:58, GLN:61, TRP:88, ASN:90 and LYS:91 through intermolecular hydrogen bonding. Analogues N-glycolyl-NeuNAc, N-Pentanoyl-NeuNAc and N-Propanoyl-NeuNAc show the least XPGscore (docking score) of -9.90, -9.16, and -8.91, respectively, and glide energy of -45.99, -42.14 and -41.66 kcal/mol, respectively. Stable nature of CT-N-glycolyl-NeuNAc, CT-N-Pentanoyl-NeuNAc and CT-N-Propanoyl-NeuNAc complexes was verified through molecular dynamics simulations, each for 40 ns using the software Desmond. All the nine NeuNAc analogues show better score for drug-like properties, so could be considered as suitable candidates for drug development for cholera infection. To improve the enhanced binding mode of NeuNAc analogues towards CT, the nine NeuNAc analogues are conjugated with Zn nanoclusters through ethylene glycol (EG) as carriers. The NeuNAc analogues conjugated with EG-Zn nanoclusters show better binding energy towards CT than the unconjugated nine NeuNAc analogues. The electronic structural optimization of EG-Zn nanoclusters was carried out for optimizing their performance as better delivery vehicles for NeuNAc analogues through density functional theory calculations. These sialic acid analogues may be considered as novel leads for the design of drug against cholera and the EG-Zn nanocluster may be a suitable carrier for sialic acid analogues.

  17. IFN-gamma-induced chemokines synergize with pertussis toxin to promote T cell entry to the central nervous system

    DEFF Research Database (Denmark)

    Millward, Jason M; Caruso, Maria; Campbell, Iain L

    2007-01-01

    was predominantly localized to meningeal and ependymal cells, and was also seen in astrocytes and microglia. IFN-gamma-induced chemokine expression did not lead to inflammation. However, when pertussis toxin was given i.p. to mice infected with the IFN-gamma vector, there was a dramatic increase in the number of T...... that by itself is insufficient to promote inflammation, and that IFN-gamma-induced CNS chemoattractant signals can synergize with a peripheral infectious stimulus to drive T cell entry into the CNS Udgivelsesdato: 2007-Jun-15...

  18. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii

    OpenAIRE

    Shanan, Salah; Bayoumi, Magdi; Saeed, Amir; Sandström, Gunnar; Abd, Hadi

    2016-01-01

    Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11,...

  19. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii

    OpenAIRE

    Shanan, Salah; Bayoumi, Magdi; Saeed, Amir; Sandström, Gunnar; Abd, Hadi

    2016-01-01

    Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11,...

  20. Analysis of a Cholera Toxin B Subunit (CTB) and Human Mucin 1 (MUC1) Conjugate Protein in a MUC1 Tolerant Mouse Model

    Science.gov (United States)

    Pinkhasov, Julia; Alvarez, M. Lucrecia; Pathangey, Latha B.; Tinder, Teresa L.; Mason, Hugh S.; Walmsley, Amanda M.; Gendler, Sandra J.; Mukherjee, Pinku

    2011-01-01

    Since epithelial mucin 1 (MUC1) is associated with several adenocarcinomas at mucosal sites, it is pertinent to test the efficacy of a mucosally targeted vaccine formulation. The B subunit of the Vibrio cholerae cholera toxin (CTB) has great potential to act as a mucosal carrier for subunit vaccines. In the present study we evaluated whether a MUC1 tandem repeat (TR) peptide chemically linked to CTB would break self-antigen tolerance in the transgenic MUC1 tolerant mouse model (MUC1.Tg) through oral or parenteral immunizations. We report that oral immunization with the CTB-MUC1 conjugate along with mucosal adjuvant, unmethylated CpG oligodeoxynucleotide (ODN) and interleukin-12 (IL-12), did not break self-antigen tolerance in MUC1.Tg mice, but induced a strong humoral response in wild-type C57BL/6 mice. However, self-antigen tolerance in the MUC1.Tg mouse model was broken after parenteral immunizations with different doses of the CTB-MUC1 conjugate protein and with the adjuvant CpG ODN co-delivered with CTB-MUC1. Importantly, mice immunized systemically with CpG ODN alone and with CTB-MUC1 exhibited decreased tumor burden when challenged with a mammary gland tumor cell line that expresses human MUC1. PMID:20824430

  1. Analysis of a cholera toxin B subunit (CTB) and human mucin 1 (MUC1) conjugate protein in a MUC1-tolerant mouse model.

    Science.gov (United States)

    Pinkhasov, Julia; Alvarez, M Lucrecia; Pathangey, Latha B; Tinder, Teresa L; Mason, Hugh S; Walmsley, Amanda M; Gendler, Sandra J; Mukherjee, Pinku

    2010-12-01

    Since epithelial mucin 1 (MUC1) is associated with several adenocarcinomas at the mucosal sites, it is pertinent to test the efficacy of a mucosally targeted vaccine formulation. The B subunit of the Vibrio cholerae cholera toxin (CTB) has great potential to act as a mucosal carrier for subunit vaccines. In the present study we evaluated whether a MUC1 tandem repeat (TR) peptide chemically linked to CTB would break self-antigen tolerance in the transgenic MUC1-tolerant mouse model (MUC1.Tg) through oral or parenteral immunizations. We report that oral immunization with the CTB-MUC1 conjugate along with mucosal adjuvant, unmethylated CpG oligodeoxynucleotide (ODN) and interleukin-12 (IL-12) did not break self-antigen tolerance in MUC1.Tg mice, but induced a strong humoral response in wild-type C57BL/6 mice. However, self-antigen tolerance in the MUC1.Tg mouse model was broken after parenteral immunizations with different doses of the CTB-MUC1 conjugate protein and with the adjuvant CpG ODN co-delivered with CTB-MUC1. Importantly, mice immunized systemically with CpG ODN alone and with CTB-MUC1 exhibited decreased tumor burden when challenged with a mammary gland tumor cell line that expresses human MUC1.

  2. Cholera Toxin Promotes Th17 Cell Differentiation by Modulating Expression of Polarizing Cytokines and the Antigen-Presenting Potential of Dendritic Cells.

    Directory of Open Access Journals (Sweden)

    Jung-Ok Kang

    Full Text Available Cholera toxin (CT, an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN. Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1β, and activin A had a synergic role with TGF-β1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines.

  3. Cholera Toxin Promotes Th17 Cell Differentiation by Modulating Expression of Polarizing Cytokines and the Antigen-Presenting Potential of Dendritic Cells

    Science.gov (United States)

    Kang, Jung-Ok; Lee, Jee-Boong

    2016-01-01

    Cholera toxin (CT), an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC) populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN). Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1β, and activin A had a synergic role with TGF-β1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines. PMID:27271559

  4. A Mutational Analysis of Residues in Cholera Toxin A1 Necessary for Interaction with Its Substrate, the Stimulatory G Protein Gsα

    Directory of Open Access Journals (Sweden)

    Michael G. Jobling

    2015-03-01

    Full Text Available Pathogenesis of cholera diarrhea requires cholera toxin (CT-mediated adenosine diphosphate (ADP-ribosylation of stimulatory G protein (Gsα in enterocytes. CT is an AB5 toxin with an inactive CTA1 domain linked via CTA2 to a pentameric receptor-binding B subunit. Allosterically activated CTA1 fragment in complex with NAD+ and GTP-bound ADP-ribosylation factor 6 (ARF6-GTP differs conformationally from the CTA1 domain in holotoxin. A surface-exposed knob and a short α-helix (formed, respectively, by rearranging “active-site” and “activation” loops in inactive CTA1 and an ADP ribosylating turn-turn (ARTT motif, all located near the CTA1 catalytic site, were evaluated for possible roles in recognizing Gsα. CT variants with one, two or three alanine substitutions at surface-exposed residues within these CTA1 motifs were tested for assembly into holotoxin and ADP-ribosylating activity against Gsα and diethylamino-(benzylidineamino-guanidine (DEABAG, a small substrate predicted to fit into the CTA1 active site. Variants with single alanine substitutions at H55, R67, L71, S78, or D109 had nearly wild-type activity with DEABAG but significantly decreased activity with Gsα, suggesting that the corresponding residues in native CTA1 participate in recognizing Gsα. As several variants with multiple substitutions at these positions retained partial activity against Gsα, other residues in CTA1 likely also participate in recognizing Gsα.

  5. Temperature and cholera toxin B are factors that influence formation of membrane nanotubes in RT4 and T24 urothelial cancer cell lines

    Directory of Open Access Journals (Sweden)

    Doron Kabaso

    2011-03-01

    Full Text Available Doron Kabaso1*, Maruša Lokar1*, Veronika Kralj-Iglic2, Peter Veranic3, Aleš Iglic11Laboratory of Biophysics, Faculty of Electrical Engineering, 2Laboratory of Clinical Biophysics, Faculty of Medicine, 3Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; *These two authors equally share the first authorshipAbstract: The growth of membrane nanotubes is crucial for intercellular communication in both normal development and pathological conditions. Therefore, identifying factors that influence their stability and formation are important for both basic research and in development of potential treatments of pathological states. Here we investigate the effect of cholera toxin B (CTB and temperature on two pathological model systems: urothelial cell line RT4, as a model system of a benign tumor, and urothelial cell line T24, as a model system of a metastatic tumor. In particular, the number of intercellular membrane nanotubes (ICNs; ie, membrane nanotubes that bridge neighboring cells was counted. In comparison with RT4 cells, we reveal a significantly higher number in the density of ICNs in T24 cells not derived from RT4 without treatments (P = 0.005, after 20 minutes at room temperature (P = 0.0007, and following CTB treatment (P = 0.000025. The binding of CTB to GM1–lipid complexes in membrane exvaginations or tips of membrane nanotubes may reduce the positive spontaneous (intrinsic curvature of GM1–lipid complexes, which may lead to lipid mediated attractive interactions between CTB–GM1–lipid complexes, their aggregation and consequent formation of enlarged spherical tips of nanotubes. The binding of CTB to GM1 molecules in the outer membrane leaflet of membrane exvaginations and tips of membrane nanotubes may also increase the area difference between the two leaflets and in this way facilitate the growth of membrane nanotubes.Keywords: cancer cells, membrane nanotubes, cholera toxin

  6. A mutational analysis of residues in cholera toxin A1 necessary for interaction with its substrate, the stimulatory G protein Gsα.

    Science.gov (United States)

    Jobling, Michael G; Gotow, Lisa F; Yang, Zhijie; Holmes, Randall K

    2015-03-18

    Pathogenesis of cholera diarrhea requires cholera toxin (CT)-mediated adenosine diphosphate (ADP)-ribosylation of stimulatory G protein (Gsα) in enterocytes. CT is an AB5 toxin with an inactive CTA1 domain linked via CTA2 to a pentameric receptor-binding B subunit. Allosterically activated CTA1 fragment in complex with NAD+ and GTP-bound ADP-ribosylation factor 6 (ARF6-GTP) differs conformationally from the CTA1 domain in holotoxin. A surface-exposed knob and a short α-helix (formed, respectively, by rearranging "active-site" and "activation" loops in inactive CTA1) and an ADP ribosylating turn-turn (ARTT) motif, all located near the CTA1 catalytic site, were evaluated for possible roles in recognizing Gsα. CT variants with one, two or three alanine substitutions at surface-exposed residues within these CTA1 motifs were tested for assembly into holotoxin and ADP-ribosylating activity against Gsα and diethylamino-(benzylidineamino)-guanidine (DEABAG), a small substrate predicted to fit into the CTA1 active site). Variants with single alanine substitutions at H55, R67, L71, S78, or D109 had nearly wild-type activity with DEABAG but significantly decreased activity with Gsα, suggesting that the corresponding residues in native CTA1 participate in recognizing Gsα. As several variants with multiple substitutions at these positions retained partial activity against Gsα, other residues in CTA1 likely also participate in recognizing Gsα.

  7. Bile and unsaturated fatty acids inhibit the binding of cholera toxin and Escherichia coli heat-labile enterotoxin to GM1 receptor.

    Science.gov (United States)

    Chatterjee, Arpita; Chowdhury, Rukhsana

    2008-01-01

    Cholera toxin (CT) is an archetypal bacterial toxin that binds with a high affinity to the receptor ganglioside GM1 on the intestinal epithelial surface and that causes the severe watery diarrhea characteristic of the disease cholera. Blockage of the interaction of CT with the GM1 receptor is an attractive approach for therapeutic intervention. We report here that crude bile prevents the interaction of CT with GM1 and reduces CT-mediated fluid accumulation in the rabbit intestine. The unsaturated fatty acids detected in crude bile, arachidonic, linoleic, and oleic acids, were found to be the most effective. Crude bile and the unsaturated fatty acids interacted with CT but not GM1 to prevent CT-GM1 binding. Neither crude bile nor the unsaturated fatty acids had any effect on the subunit structure of CT. The binding of CT to unsaturated fatty acids resulted in a shift of the apparent pI of CT from 6.8 to 8.2 and a marked decrease in intrinsic fluorescence. The Kd was calculated from fluorescence quenching assays. It was demonstrated by the rabbit ileal loop model that practically no fluid accumulated in the intestinal loops when CT was administered together with inhibitory concentrations of linoleic acid. The bile present in the intestine was sufficient to inhibit the activity of up to 300 ng CT. Bile and unsaturated fatty acids also inhibited the binding of Escherichia coli heat-labile enterotoxin (LT) to GM1, and no fluid accumulation was observed in rabbit ileal loops when LT was administered together with linoleic acid.

  8. Comparison of Vibrio cholerae O139 with V. cholerae O1 classical and El Tor biotypes.

    OpenAIRE

    Calia, K E; Murtagh, M.; Ferraro, M J; Calderwood, S B

    1994-01-01

    Vibrio cholerae O139 is a recently identified non-O1 V. cholerae strain responsible for outbreaks of epidemic cholera in India, Bangladesh, and Thailand in the past 2 years. Other workers have demonstrated the presence of the cholera toxin genetic element in V. cholerae O139, unlike the situation for other non-O1 V. cholerae strains. We sought to compare further this strain with strains of V. cholerae O1, classical and El Tor biotypes, by classic microbiologic methods, Southern blot analysis ...

  9. Draft Genome Sequence of Environmental Vibrio cholerae 2012EL-1759 with Similarities to the V. cholerae O1 Classical Biotype

    OpenAIRE

    Katz, Lee S.; Turnsek, Maryann; Kahler, Amy; Hill, Vincent R.; Boyd, E. Fidelma; Tarr, Cheryl L.

    2014-01-01

    Vibrio cholerae 2012EL-1759 is an environmental isolate from Haiti that was recovered in 2012 during a cholera outbreak. The genomic backbone is similar to that of the prototypical V. cholerae O1 classical biotype strain O395, and it carries the Vibrio pathogenicity islands (VPI-1 and VPI-2) and a cholera toxin (CTX) prephage.

  10. The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Stephen Vadia

    2011-11-01

    Full Text Available Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2. Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

  11. The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes.

    Science.gov (United States)

    Vadia, Stephen; Arnett, Eusondia; Haghighat, Anne-Cécile; Wilson-Kubalek, Elisabeth M; Tweten, Rodney K; Seveau, Stephanie

    2011-11-01

    Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

  12. Fowl cholera.

    Science.gov (United States)

    Christensen, J P; Bisgaard, M

    2000-08-01

    Pasteurella multocida subspecies multocida is the most common cause of fowl cholera, although P. multocida subspecies septica and gallicida may also cause fowl cholera-like disease to some extent. However, the virulence properties of the different subspecies for various hosts have not been elucidated. The severity and incidence of P. multocida infections may vary considerably depending on several factors associated with the host (including species and age of infected birds), the environment and the bacterial strain. No single virulence factor has been associated with the observed variation in virulence among strains. Possible virulence factors include the following: the capsule, endotoxin, outer membrane proteins, iron binding systems, heat shock proteins, neuraminidase production and antibody cleaving enzymes. No RTX toxins (repeats in toxin) appear to be produced by P. multocida, but P. multocida exotoxin (PMT) could contribute to virulence in some avian infections. The epidemiology of fowl cholera appears complex. Traditional serotyping systems are only of limited use in epidemiological studies. In recent years, molecular typing methods have been applied to avian strains of P. multocida of different origin. The results obtained using these newer methods indicate that wild birds may be a source of infection to commercial poultry. Documentation suggesting that mammals play a similar role is not as comprehensive, but the possibility cannot be excluded. Carrier birds seem to play a major role in the transmission of cholera. Surviving birds from diseased flocks appear to represent a risk, but more recent investigations indicate that carriers of P. multocida may exist within poultry flocks with no history of previous outbreaks of fowl cholera. The significance of this awaits further investigation. The site of infection for P. multocida is generally believed to be the respiratory tract. The outcome of infections may range from peracute/acute infections to chronic

  13. A Cholera Conjugate Vaccine Containing O-specific Polysaccharide (OSP of V. cholerae O1 Inaba and Recombinant Fragment of Tetanus Toxin Heavy Chain (OSP:rTTHc Induces Serum, Memory and Lamina Proprial Responses against OSP and Is Protective in Mice.

    Directory of Open Access Journals (Sweden)

    Md Abu Sayeed

    Full Text Available Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP component of lipopolysaccharide (LPS.Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc. We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg, vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1, effect of an adjuvant, and route of immunization.Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 μg. We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model.We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens.

  14. A Cholera Conjugate Vaccine Containing O-specific Polysaccharide (OSP) of V. cholerae O1 Inaba and Recombinant Fragment of Tetanus Toxin Heavy Chain (OSP:rTTHc) Induces Serum, Memory and Lamina Proprial Responses against OSP and Is Protective in Mice

    Science.gov (United States)

    Eckhoff, Grace; Charles, Richelle C.; Alam, Mohammad Murshid; Sultana, Tania; Rashu, Md. Rasheduzzaman; Berger, Amanda; Gonzalez-Escobedo, Geoffrey; Mandlik, Anjali; Bhuiyan, Taufiqur Rahman; Leung, Daniel T.; LaRocque, Regina C.; Harris, Jason B.; Calderwood, Stephen B.; Qadri, Firdausi; Vann, W. F.; Kováč, Pavol; Ryan, Edward T.

    2015-01-01

    Background Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). Methodology Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization. Principle Findings Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 μg). We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model. Conclusion We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens. PMID:26154421

  15. Vibrio cholerae MARTX toxin heterologous translocation of beta-lactamase and roles of individual effector domains on cytoskeleton dynamics.

    Science.gov (United States)

    Dolores, Jazel S; Agarwal, Shivani; Egerer, Martina; Satchell, Karla J F

    2015-02-01

    The Vibrio cholerae MARTXVc toxin delivers three effector domains to eukaryotic cells. To study toxin delivery and function of individual domains, the rtxA gene was modified to encode toxin with an in-frame beta-lactamase (Bla) fusion. The hybrid RtxA::Bla toxin was Type I secreted from bacteria; and then Bla was translocated into eukaryotic cells and delivered by autoprocessing, demonstrating that the MARTXVc toxin is capable of heterologous protein transfer. Strains that produce hybrid RtxA::Bla toxins that carry one effector domain in addition to Bla were found to more efficiently translocate Bla. In cell biological assays, the actin cross-linking domain (ACD) and Rho-inactivation domain (RID) are found to cross-link actin and inactivate RhoA, respectively, when other effector domains are absent, with toxin autoprocessing required for high efficiency. The previously unstudied alpha-beta hydrolase domain (ABH) is shown here to activate CDC42, although the effect is ameliorated when RID is also present. Despite all effector domains acting on cytoskeleton assembly, the ACD was sufficient to rapidly inhibit macrophage phagocytosis. Both the ACD and RID independently disrupted polarized epithelial tight junction integrity. The sufficiency of ACD but strong selection for retention of RID and ABH suggests these two domains may primarily function by modulating cell signaling.

  16. 霍乱毒素无毒B亚单位(CTB)黏膜免疫佐剂的研究进展%Advance on the immuno-adjuvant of cholera toxin B subunit

    Institute of Scientific and Technical Information of China (English)

    严小芳; 张佩; 董硕

    2009-01-01

    cholera toxin(CT) is an effective adjuvant in mucosal immunity,therefore it becomes the focus in the immunity study.Just now the mechanisms of CT have been not solved.This article reviews the function of cholera toxin B subunit in mucosal immuno-adjutant.%霍乱毒素(CT)具有很强的黏膜免疫佐剂活性,是当今研究热点之一,但CT的黏膜免疫佐剂效应机理尚未完全弄清.本文主要就霍乱毒素无毒B亚单位(CTB)的黏膜免疫佐剂作用进行综述.

  17. Intranasal Immunization with the Cholera Toxin B Subunit-Pneumococcal Surface Antigen A Fusion Protein Induces Protection against Colonization with Streptococcus pneumoniae and Has Negligible Impact on the Nasopharyngeal and Oral Microbiota of Mice

    OpenAIRE

    F.C. Pimenta; Miyaji, E. N.; Arêas, A. P. M.; Oliveira, M. L. S.; de Andrade, A. L. S. S.; Ho, P.L.; Hollingshead, S. K.; Leite, L. C. C.

    2006-01-01

    One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identity in other viridans group streptococci. In this work, we demonstrate that intranasal immunization with a cholera toxin B subunit-PsaA fusion protein is able to...

  18. Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus.

    Science.gov (United States)

    Ji, Xianliang; Ren, Zhiguang; Xu, Na; Meng, Lingnan; Yu, Zhijun; Feng, Na; Sang, Xiaoyu; Li, Shengnan; Li, Yuanguo; Wang, Tiecheng; Zhao, Yongkun; Wang, Hualei; Zheng, Xuexing; Jin, Hongli; Li, Nan; Yang, Songtao; Cao, Jinshan; Liu, Wensen; Gao, Yuwei; Xia, Xianzhu

    2016-04-21

    Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs) composed of the hemagglutinin (HA), neuraminidase (NA) and matrix protein (M1) of A/Changchun/01/2009 (H1N1) with or without either membrane-anchored cholera toxin B (CTB) or ricin toxin B (RTB) as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival). Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses.

  19. Targeting bacterial toxins.

    Science.gov (United States)

    Ivarsson, Mattias E; Leroux, Jean-Christophe; Castagner, Bastien

    2012-04-23

    Protein toxins constitute the main virulence factors of several species of bacteria and have proven to be attractive targets for drug development. Lead candidates that target bacterial toxins range from small molecules to polymeric binders, and act at each of the multiple steps in the process of toxin-mediated pathogenicity. Despite recent and significant advances in the field, a rationally designed drug that targets toxins has yet to reach the market. This Review presents the state of the art in bacterial toxin targeted drug development with a critical consideration of achieved breakthroughs and withstanding challenges. The discussion focuses on A-B-type protein toxins secreted by four species of bacteria, namely Clostridium difficile (toxins A and B), Vibrio cholerae (cholera toxin), enterohemorrhagic Escherichia coli (Shiga toxin), and Bacillus anthracis (anthrax toxin), which are the causative agents of diseases for which treatments need to be improved. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Structure of the cytoplasmic domain of TcpE, the inner membrane core protein required for assembly of the Vibrio cholerae toxin-coregulated pilus.

    Science.gov (United States)

    Kolappan, Subramaniapillai; Craig, Lisa

    2013-04-01

    Type IV pili are long thin surface-displayed polymers of the pilin subunit that are present in a diverse group of bacteria. These multifunctional filaments are critical to virulence for pathogens such as Vibrio cholerae, which use them to form microcolonies and to secrete the colonization factor TcpF. The type IV pili are assembled from pilin subunits by a complex inner membrane machinery. The core component of the type IV pilus-assembly platform is an integral inner membrane protein belonging to the GspF superfamily of secretion proteins. These proteins somehow convert chemical energy from ATP hydrolysis by an assembly ATPase on the cytoplasmic side of the inner membrane to mechanical energy for extrusion of the growing pilus filament out of the inner membrane. Most GspF-family inner membrane core proteins are predicted to have N-terminal and central cytoplasmic domains, cyto1 and cyto2, and three transmembrane segments, TM1, TM2 and TM3. Cyto2 and TM3 represent an internal repeat of cyto1 and TM1. Here, the 1.88 Å resolution crystal structure of the cyto1 domain of V. cholerae TcpE, which is required for assembly of the toxin-coregulated pilus, is reported. This domain folds as a monomeric six-helix bundle with a positively charged membrane-interaction face at one end and a hydrophobic groove at the other end that may serve as a binding site for partner proteins in the pilus-assembly complex.

  1. Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

    Directory of Open Access Journals (Sweden)

    Aria Eshraghi

    2014-07-01

    Full Text Available Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs, are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD machinery, Derlin-2 (Derl2, the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT. In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

  2. Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

    Science.gov (United States)

    Eshraghi, Aria; Dixon, Shandee D; Tamilselvam, Batcha; Kim, Emily Jin-Kyung; Gargi, Amandeep; Kulik, Julia C; Damoiseaux, Robert; Blanke, Steven R; Bradley, Kenneth A

    2014-07-01

    Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

  3. The detection of cholera toxin based on agglutination reaction with amplification of silica nanoparticles%纳米二氧化硅颗粒免疫凝聚用于霍乱毒素的检测研究

    Institute of Scientific and Technical Information of China (English)

    余红伟; 王源升; 李瑜; 蒋健晖; 吴海龙; 魏徵; 沈国励; 俞汝勤

    2011-01-01

    提出了一种基于SiO2纳米颗粒免疫凝集反应的压电传感器对霍乱毒素的直接检测,研究发现待测物霍乱毒素会同时被晶振表面固定的霍乱毒素抗体和检测体系中SiO:标记的霍乱毒素抗体识别,引起检测体系中的SiO:在传感界面上特异性凝集,从而引起晶振表面的巨大质量变化和检测介质密度和粘度变化。实验结果表明,该方法修饰的探针能够有效地对霍乱毒素进行检测。%A simple piezoelectric immunoagglutination assay technique has been developed for direct detection of cholera toxin. It was based on the specific agglutination of cholera toxin antibody-coated silica nanoparticles in the presence of cholera toxin. The antibody modified on the probe surface would combine with antibody-coated nanoparticles in the presence of antigen (cholera toxin) when the surface agglutinationreaction took place, which couples both the mass effect and the viscoelastic effect acting on the probe. The results indicated that the probe signal can be observably multiplied.

  4. The β-prism lectin domain of Vibrio cholerae hemolysin promotes self-assembly of the β-pore-forming toxin by a carbohydrate-independent mechanism.

    Science.gov (United States)

    Ganguly, Sreerupa; Mukherjee, Amarshi; Mazumdar, Budhaditya; Ghosh, Amar N; Banerjee, Kalyan K

    2014-02-14

    Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa β-pore-forming toxin with a C-terminal β-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC(50)) without the lectin domain, and mutant VCC(D617A) with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10(7) M(-1). However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the β-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC(50) was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the β1-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the β-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.

  5. DETECTION OF BACTERIAL TOXINS WITH MONOSACCHARIDE ARRAYS

    Science.gov (United States)

    Ngundi, Miriam M.; Taitt, Chris R.; McMurry, Scott A.; Kahne, Daniel; Ligler, Frances S.

    2006-01-01

    A large number of bacterial toxins, viruses and bacteria target carbohydrate derivatives on the cell surface to attach to and gain entry into the cell. We report here the use of a monosaccharide-based array to detect protein toxins. The array-based technique provides the capability to perform simultaneous multianalyte analyses. Arrays of N-acetyl galactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) derivatives were immobilized on the surface of a planar waveguide and were used as receptors for protein toxins. These arrays were probed with fluorescently labeled bacterial cells and protein toxins. While Salmonella typhimurium, Listeria monocytogenes, Escherichia coli and staphylococcal enterotoxin B (SEB) did not bind to either of the monosaccharides, both cholera toxin and tetanus toxin bound to GalNAc and Neu5Ac. The results show that the binding of the toxins to the carbohydrates is density dependent and semi-selective. Both toxins were detectable at 100 ng/ml. PMID:15946840

  6. Construction of supported lipid membrane modified piezoelectric biosensor for sensitive assay of cholera toxin based on surface-agglutination of ganglioside-bearing liposomes.

    Science.gov (United States)

    Chen, Huan; Hu, Qing-Yuan; Yue-Zheng; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2010-01-11

    A novel piezoelelctric biosensor has been developed for cholera toxin (CT) detection based on the analyte-mediated surface-agglutination of ganglioside (GM1)-functionalized liposomes. To achieve a CT-specific agglutination at the surface, the gold electrode is modified by a GM1-functionalized supported lipid membrane via spontaneous spread of the liposomes on a self-assembled monolayer of a long-chain alkanethiol. In the presence of CT, the GM1-incorporated liposomes in assay medium will rapidly specifically agglutinate at the electrode surface through the binding of CT to GM1 on the electrode surface and the liposome interface. This results in an enormous mass loading on the piezoelelctric crystal as well as a significant increase of density and viscosity at the interface, thereby generating a decrease in frequency of the piezoelelctric crystal. The combination of mass loading with interfacial change in the surface-agglutination reaction allows the developed piezoelelctric biosensor to show substantial signal amplification in response to the analyte CT. The detection limit can be achieved as low as 25 ng mL(-1) CT. This is the first demonstration on CT detection based on specific surface-agglutination of GM1-modified liposomes. The supported lipid layer based sensing interface can be prepared readily and renewably, making the developed technique especially useful for simple, reusable and sensitive determination of proteins.

  7. Intradermal immunization in the ear with cholera toxin and its non-toxic β subunit promotes efficient Th1 and Th17 differentiation dependent on migrating DCs.

    Science.gov (United States)

    Meza-Sánchez, David; Pérez-Montesinos, Gibrán; Sánchez-García, Javier; Moreno, José; Bonifaz, Laura C

    2011-10-01

    The nature of CD4(+) T-cell responses after skin immunization and the role of migrating DCs in the presence of adjuvants in the elicited response are interesting issues to be investigated. Here, we evaluated the priming of CD4(+) T cells following ear immunization with low doses of model antigens in combination with either cholera toxin (CT) or the non-toxic β CT subunit (CTB) as an adjuvant. Following immunization with CT, we found efficient antigen presentation that is reflected in the production of IFN-γ and IL-17 by CD4(+) T cells over IL-4 or IL-5 production. The CTB-induced activation of DCs in the ear occurred without visible inflammation, which reflects a similar type of CD4(+) T-cell differentiation. In both cases, the elicited response was dependent on the presence of migrating skin cells. Remarkably, immunization with CT or with CTB led to the induction of a delayed-type hypersensitivity (DTH) response in the ear. The DTH response that was induced by CT immunization was dependent on IL-17 and partially dependent on IFN-γ activity. These results indicate that both CT and CTB induce an efficient CD4(+) T-cell response to a co-administered antigen following ear immunization that is dependent on migrating DCs.

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

    Science.gov (United States)

    Sinclair, Haydn R; Kemp, Fred; Slegte, Jaap de; Gibson, Glenn R; Rastall, Robert A

    2009-10-12

    We have studied 'food grade' sialyloligosaccharides (SOS) as anti-adhesive drugs or receptor analogues, since the terminal sialic acid residue has already been shown to contribute significantly to the adhesion and pathogenesis of the Vibrio cholerae toxin (Ctx). GM1-oligosaccharide (GM1-OS) was immobilized into a supporting POPC lipid bilayer onto a surface plasmon resonance (SPR) chip, and the interaction between uninhibited Ctx and GM1-OS-POPC was measured. SOS inhibited 94.7% of the Ctx binding to GM1-OS-POPC at 10mg/mL. The SOS EC(50) value of 5.521mg/mL is high compared with 0.2811microg/mL (182.5rhoM or 1.825x10(-10)M) for GM1-OS. The commercially available sialyloligosaccharide (SOS) mixture Sunsial E((R)) is impure, containing one monosialylated and two disialylated oligosaccharides in the ratio 9.6%, 6.5% and 17.5%, respectively, and 66.4% protein. However, these inexpensive food-grade molecules are derived from egg yolk and could be used to fortify conventional food additives, by way of emulsifiers, sweeteners and/or preservatives. The work further supports our hypothesis that SOS could be a promising natural anti-adhesive glycomimetic against Ctx and prevent subsequent onset of disease.

  9. Cholera toxin B subunit-five-stranded α-helical coiled-coil fusion protein: "five-to-five" molecular chimera displays robust physicochemical stability.

    Science.gov (United States)

    Arakawa, Takeshi; Harakuni, Tetsuya

    2014-09-03

    To create a physicochemically stable cholera toxin (CT) B subunit (CTB), it was fused to the five-stranded α-helical coiled-coil domain of cartilage oligomeric matrix protein (COMP). The chimeric fusion protein (CTB-COMP) was expressed in Pichia pastoris, predominantly as a pentamer, and retained its affinity for the monosialoganglioside GM1, a natural receptor of CT. The fusion protein displayed thermostability, tolerating the boiling temperature of water for 10min, whereas unfused CTB readily dissociated to its monomers and lost its affinity for GM1. The fusion protein also displayed resistance to strong acid at pHs as low as 0.1, and to the protein denaturant sodium dodecyl sulfate at concentrations up to 10%. Intranasal administration of the fusion protein to mice induced anti-B subunit serum IgG, even after the protein was boiled, whereas unfused CTB showed no thermostable mucosal immunogenicity. This study demonstrates that CTB fused to a pentameric α-helical coiled coil has a novel physicochemical phenotype, which may provide important insight into the molecular design of enterotoxin-B-subunit-based vaccines and vaccine delivery molecules.

  10. NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine.

    Science.gov (United States)

    Luci, Carmelo; Bekri, Selma; Bihl, Franck; Pini, Jonathan; Bourdely, Pierre; Nouhen, Kelly; Malgogne, Angélique; Walzer, Thierry; Braud, Véronique M; Anjuère, Fabienne

    2015-01-01

    Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46+ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46+ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46+ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46+ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46+ ILC in the regulation of mucosal CD8 T cell responses.

  11. Oral immunisation of mice with transgenic rice calli expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes.

    Science.gov (United States)

    Kim, Mi-Young; Kim, Byeong-Young; Oh, Sun-Mi; Reljic, Rajko; Jang, Yong-Suk; Yang, Moon-Sik

    2016-10-01

    Dengue virus (DENV) infection is an emerging global health threat. DENV consists of four distinct serotypes, necessitating a tetravalent vaccine. In this study, expression of consensus envelope protein domain III (cEDIII) fused to cholera toxin B subunit (CTB) in transgenic rice calli was improved using the luminal binding protein BiP at the N-terminus and the SEKDEL signal sequences at the C-terminus, targeting the recombinant protein to endoplasmic reticulum (ER). We found that the fusion protein showed higher levels of expression when compared to the fusion proteins using rice amylase 3D (RAmy3D) or CTB native signal sequence only. The CTB-cEDIII fusion protein was evaluated as an oral dengue vaccine candidate in mice. Serotype specific systemic IgG antibodies and specific IgA response in feces were detected and furthermore, T cell proliferation and high frequency antibody-secreting B cells were detected in the spleen. These results suggest the possible use of plant-based dengue tetravalent vaccine targeted to the mucosal immune system for induction of systemic and mucosal immune responses to DENV infection.

  12. Use of flagellin and cholera toxin as adjuvants in intranasal vaccination of mice to enhance protective immune responses against uropathogenic Escherichia coli antigens.

    Science.gov (United States)

    Asadi Karam, Mohammad Reza; Habibi, Mehri; Bouzari, Saeid

    2016-09-01

    Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most common infections in human. Antibiotics are common therapy for UTIs, but increase in antibiotic resistance will complicate future treatment of the infections, making the development of an efficacious UTI vaccine more urgent. In this study, we have evaluated intranasally the efficacy of FliC and FimH antigens of UPEC in different vaccine formulations with and without cholera toxin (CT) adjuvant. Immunization of mice with FliC in fusion form or admixed with FimH elicited higher levels of serum, mucosal and cell-mediated responses than FimH alone. Furthermore, the use of CT in synergism with FliC resulted in the stimulation of a mixed Th1 and Th2 responses against FimH and FliC as antigen and maintained the antibody responses for at least 24 weeks following the last vaccine dose. Of the vaccine preparations, Fusion, Fusion + CT, and FimH admixed with FliC and CT showed the best protection against UPEC. These data indicated that intranasal administration of a FliC and CT adjuvant-based vaccine has the potential to provide protective responses against UPEC strains.

  13. NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

    Science.gov (United States)

    Luci, Carmelo; Bekri, Selma; Bihl, Franck; Pini, Jonathan; Bourdely, Pierre; Nouhen, Kelly; Malgogne, Angélique; Walzer, Thierry; Braud, Véronique M.; Anjuère, Fabienne

    2015-01-01

    Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46+ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46+ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46+ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46+ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46+ ILC in the regulation of mucosal CD8 T cell responses. PMID:26630176

  14. 霍乱毒素B亚单位作为免疫佐剂的研究进展%Recent advances in the development of cholera toxin B subunit as immune adjuvant

    Institute of Scientific and Technical Information of China (English)

    贾岚; 洪艳

    2015-01-01

    霍乱肠毒素(CT)是霍乱弧菌重要的毒力因子,内含A和B两个亚单位.A亚单位(CTA)为毒性亚基,B亚单位(CTB)不具毒性.CTB因其独特的生理功能已成为重要的佐剂之一,近年来备受各国学者重视.深入探究CTB的免疫调节机制有助于更好地设计疫苗并优化其效果,此文将对CTB作为免疫佐剂在疫苗研究中的进展进行综述.%Cholera toxin (CT) is the important virulence factor of Vibrio cholerae which is composed of A and B subunits.Cholera toxin A subunit (CTA) is toxic,while B subunit (CTB) is nontoxic and has become one of the important adjuvants and been paid much attention by scholars in recent years because of its unique physiological function.Better understanding of immunomodulatory mechanism of CTB would be helpful to develop and improve novel vaccines.This paper discusses the latest progress of CTB as immune adjuvant in vaccine research.

  15. Piperine treatment suppresses Helicobacter pylori toxin entry in to gastric epithelium and minimizes β-catenin mediated oncogenesis and IL-8 secretion in vitro

    OpenAIRE

    2016-01-01

    Helicobacter pylori related gastric cancer initiation has been studied widely. The objective of our present study was to evaluate the effect of a single compound piperine on H. pylori infection and its anti-inflammatory and anti-cancer effects in vitro. Cytotoxicity was tested by Ez-cytox cell viability assay kit. Effects of piperine on H. pylori toxin gene expression and IL-8 expression in mammalian cells during infection were assessed by RT-PCR. Effects of piperine on toxin entry into host ...

  16. 1.65 Å resolution structure of the AraC-family transcriptional activator ToxT from Vibrio cholerae.

    Science.gov (United States)

    Li, Jiaqin; Wehmeyer, Graham; Lovell, Scott; Battaile, Kevin P; Egan, Susan M

    2016-09-01

    ToxT is an AraC-family transcriptional activator protein that controls the expression of key virulence factors in Vibrio cholerae, the causative agent of cholera. ToxT directly activates the expression of the genes that encode the toxin-coregulated pilus and cholera toxin, and also positively auto-regulates its own expression from the tcp promoter. The crystal structure of ToxT has previously been solved at 1.9 Å resolution (PDB entry 3gbg). In this study, a crystal structure of ToxT at 1.65 Å resolution with a similar overall structure to the previously determined structure is reported. However, there are distinct differences between the two structures, particularly in the region that extends from Asp101 to Glu110. This region, which can influence ToxT activity but was disordered in the previous structure, can be traced entirely in the current structure.

  17. Holliday junction affinity of the base excision repair factor Endo III contributes to cholera toxin phage integration.

    Science.gov (United States)

    Bischerour, Julien; Spangenberg, Claudia; Barre, François-Xavier

    2012-09-12

    Toxigenic conversion of Vibrio cholerae bacteria results from the integration of a filamentous phage, CTX phage. Integration is driven by the bacterial Xer recombinases, which catalyse the exchange of a single pair of strands between the phage single-stranded DNA and the host double-stranded DNA genomes; replication is thought to convert the resulting pseudo-Holliday junction (HJ) intermediate into the final recombination product. The natural tendency of the Xer recombinases to recycle HJ intermediates back into substrate should thwart this integration strategy, which prompted a search for additional co-factors aiding directionality of the process. Here, we show that Endo III, a ubiquitous base excision repair enzyme, facilitates CTX phage-integration in vivo. In vitro, we show that it prevents futile Xer recombination cycles by impeding new rounds of strand exchanges once the pseudo-HJ is formed. We further demonstrate that this activity relies on the unexpected ability of Endo III to bind to HJs even in the absence of the recombinases. These results explain how tandem copies of the phage genome can be created, which is crucial for subsequent virion production.

  18. Vibrio cholerae-induced inflammation in the neonatal mouse cholera model.

    Science.gov (United States)

    Bishop, Anne L; Patimalla, Bharathi; Camilli, Andrew

    2014-06-01

    Vibrio cholerae is the causative agent of the acute diarrheal disease of cholera. Innate immune responses to V. cholerae are not a major cause of cholera pathology, which is characterized by severe, watery diarrhea induced by the action of cholera toxin. Innate responses may, however, contribute to resolution of infection and must be required to initiate adaptive responses after natural infection and oral vaccination. Here we investigated whether a well-established infant mouse model of cholera can be used to observe an innate immune response. We also used a vaccination model in which immunized dams protect their pups from infection through breast milk antibodies to investigate innate immune responses after V. cholerae infection for pups suckled by an immune dam. At the peak of infection, we observed neutrophil recruitment accompanied by induction of KC, macrophage inflammatory protein 2 (MIP-2), NOS-2, interleukin-6 (IL-6), and IL-17a. Pups suckled by an immunized dam did not mount this response. Accessory toxins RtxA and HlyA played no discernible role in neutrophil recruitment in a wild-type background. The innate response to V. cholerae deleted for cholera toxin-encoding phage (CTX) and part of rtxA was significantly reduced, suggesting a role for CTX-carried genes or for RtxA in the absence of cholera toxin (CTX). Two extracellular V. cholerae DNases were not required for neutrophil recruitment, but DNase-deficient V. cholerae caused more clouds of DNA in the intestinal lumen, which appeared to be neutrophil extracellular traps (NETs), suggesting that V. cholerae DNases combat NETs. Thus, the infant mouse model has hitherto unrecognized utility for interrogating innate responses to V. cholerae infection.

  19. Vibrio cholerae-Induced Inflammation in the Neonatal Mouse Cholera Model

    Science.gov (United States)

    Bishop, Anne L.; Patimalla, Bharathi

    2014-01-01

    Vibrio cholerae is the causative agent of the acute diarrheal disease of cholera. Innate immune responses to V. cholerae are not a major cause of cholera pathology, which is characterized by severe, watery diarrhea induced by the action of cholera toxin. Innate responses may, however, contribute to resolution of infection and must be required to initiate adaptive responses after natural infection and oral vaccination. Here we investigated whether a well-established infant mouse model of cholera can be used to observe an innate immune response. We also used a vaccination model in which immunized dams protect their pups from infection through breast milk antibodies to investigate innate immune responses after V. cholerae infection for pups suckled by an immune dam. At the peak of infection, we observed neutrophil recruitment accompanied by induction of KC, macrophage inflammatory protein 2 (MIP-2), NOS-2, interleukin-6 (IL-6), and IL-17a. Pups suckled by an immunized dam did not mount this response. Accessory toxins RtxA and HlyA played no discernible role in neutrophil recruitment in a wild-type background. The innate response to V. cholerae deleted for cholera toxin-encoding phage (CTXϕ) and part of rtxA was significantly reduced, suggesting a role for CTXϕ-carried genes or for RtxA in the absence of cholera toxin (CTX). Two extracellular V. cholerae DNases were not required for neutrophil recruitment, but DNase-deficient V. cholerae caused more clouds of DNA in the intestinal lumen, which appeared to be neutrophil extracellular traps (NETs), suggesting that V. cholerae DNases combat NETs. Thus, the infant mouse model has hitherto unrecognized utility for interrogating innate responses to V. cholerae infection. PMID:24686062

  20. Effect of Dietary Minerals on Virulence Attributes of Vibrio cholerae.

    Science.gov (United States)

    Bhattaram, Varunkumar; Upadhyay, Abhinav; Yin, Hsin-Bai; Mooyottu, Shankumar; Venkitanarayanan, Kumar

    2017-01-01

    Vibrio cholerae is a water-borne pathogen responsible for causing a toxin-mediated profuse diarrhea in humans, leading to severe dehydration and death in unattended patients. With increasing reports of antibiotic resistance in V. cholerae, there is a need for alternate interventional strategies for controlling cholera. A potential new strategy for treating infectious diseases involves targeting bacterial virulence rather than growth, where a pathogen's specific mechanisms critical for causing infection in hosts are inhibited. Since bacterial motility, intestinal colonization and cholera toxin are critical components in V. cholerae pathogenesis, attenuating these virulence factors could potentially control cholera in humans. In this study, the efficacy of sub-inhibitory concentration (SIC, highest concentration not inhibiting bacterial growth) of essential minerals, zinc (Zn), selenium (Se), and manganese (Mn) in reducing V. cholerae motility and adhesion to intestinal epithelial cells (Caco-2), cholera toxin production, and toxin binding to the ganglioside receptor (GM1) was investigated. Additionally, V. cholerae attachment and toxin production in an ex vivo mouse intestine model was determined. Further, the effect of Zn, Se and Mn on V. cholerae virulence genes, ctxAB (toxin production), fliA (motility), tcpA (intestinal colonization), and toxR (master regulon) was determined using real-time quantitative PCR. All three minerals significantly reduced V. cholerae motility, adhesion to Caco-2 cells, and cholera toxin production in vitro, and decreased adhesion and toxin production in mouse intestine ex vivo (P cholerae virulence. However, in vivo studies in an animal model are necessary to validate these results.

  1. Induction of melanin biosynthesis in Vibrio cholerae.

    OpenAIRE

    Coyne, V E; al-Harthi, L

    1992-01-01

    Vibrio cholerae synthesized the pigment melanin in response to specific physiological conditions that were stressful to the bacterium. Pigmentation was induced when V. cholerae was subjected to hyperosmotic stress in conjunction with elevated growth temperatures (above 30 degrees C). The salt concentration tolerated by V. cholerae was lowered by additional abiotic factors such as acidic starting pH of the growth medium and limitation of organic nutrients. Although the amount of toxin detected...

  2. Cholera: a great global concern.

    Science.gov (United States)

    Mandal, Shyamapada; Mandal, Manisha Deb; Pal, Nishith Kumar

    2011-07-01

    Cholera, caused by the infection of toxigenic Vibrio cholerae (V. cholerae) to humans, is a life threatening diarrheal disease with epidemic and pandemic potential. The V. cholerae, both O1 and O139 serogroups, produce a potent enterotoxin (cholera toxin) responsible for the lethal symptoms of the disease. The O1 serogroup has two biotypes (phenotypes), classical and El Tor; each of which has two major serotypes (based on antigenic responses), Ogawa and Inaba and the extremely rare Hikojima. V. cholerae O1 strains interconvert and switch between the Ogawa and Inaba serotypes. Fluid and electrolyte replacement is the mainstay of treatment of cholera patients; the severe cases require antibiotic treatment to reduce the duration of illness and replacement of fluid intake. The antibiotic therapy currently has faced difficulties due to the rapid emergence and spread of multidrug resistant V. cholerae causing several outbreaks in the globe. Currently, cholera has been becoming endemic in an increasing number of geographical areas, reflecting a failure in implementation of control measures. However, the current safe oral vaccines lower the number of resistant infections and could thus represent an effective intervention measure to control antibiotic resistance in cholera. Overall, the priorities for cholera control remain public health interventions through improved drinking water, sanitation, surveillance and access to health care facilities, and further development of safe, effective and appropriate vaccines. Thus, this review describes the facts and phenomena related to the disease cholera, which is still a great threat mainly to the developing countries, and hence a grave global concern too. Copyright © 2011 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  3. Suppression of HIV replication in vitro by CpG and CpG conjugated to the non toxic B subunit of cholera toxin.

    Science.gov (United States)

    Nowroozalizadeh, Salma; Jansson, Marianne; Adamsson, Jenni; Lindblad, Marianne; Fenyö, Eva-Maria; Holmgren, Jan; Harandi, Ali M

    2008-05-01

    Administration of oligodeoxynucleotides (ODNs) containing CpG motifs generates a rapid and potent response of CC-chemokines, known as ligands of the HIV-1 co-receptor CCR5, in the murine female genital tract. The present study explored the potential HIV inhibitory activities of different human CpG prototypes either alone or conjugated to the non-toxic subunit of cholera toxin (CTB). Results showed that in vitro replication of both HIV-1 and HIV-2 can be suppressed by different human CpG prototypes. Importantly, the conjugation of CpG ODN to CTB (CTB-CpG) enhanced the antiviral activity of CpG against primary HIV-1 isolates of both R5 and X4 phenotypes in peripheral blood mononuclear cells (PBMC) as well as U87.CD4 co-receptor indicator cells. CTB-CpGs triggered higher amounts of MIP-1alpha, and MIP-1beta in PBMC than the corresponding CpG ODNs, which may explain the superior antiviral effect of CTB-CpG against R5 virus in PBMC. Incubation of PBMC with CpG ODN and CTB-CpG did not alter surface expression of HIV-1 receptors indicating that the observed anti-HIV-1 effect is not mediated through down regulation of HIV-1 receptors on target cells. Further, the enhanced antiviral effect of CTB-CpG was dependent on the presence of phosphorothioate backbone in the ODN, whereas the presence of CpG motif in ODNs was dispensable. These results have implications for the development of novel intervention strategies to prevent HIV infection.

  4. A unique role of the cholera toxin A1-DD adjuvant for long-term plasma and memory B cell development.

    Science.gov (United States)

    Bemark, Mats; Bergqvist, Peter; Stensson, Anneli; Holmberg, Anna; Mattsson, Johan; Lycke, Nils Y

    2011-02-01

    Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ∼36 wk, but DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.

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

    Science.gov (United States)

    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.

  6. Immune response of broiler chickens immunized orally with the recombinant proteins flagellin and the subunit B of cholera toxin associated with Lactobacillus spp.

    Science.gov (United States)

    Baptista, A A S; Donato, T C; Garcia, K C O D; Gonçalves, G A M; Coppola, M P; Okamoto, A S; Sequeira, J L; Andreatti Filho, R L

    2014-01-01

    This study investigated the immune response of broiler chickens with oral treatment of a Lactobacillus spp. pool (PL) associated with microencapsulated recombinant proteins flagellin (FliC) and the subunit B of cholera toxin (CTB). Immune responses were evaluated by measuring IgA from intestinal fluid, serum IgY, and immunostaining of CD8(+) T lymphocytes present in the cecum. The evaluations were performed on d 0, 7, 14, 21, and 28 posttreatment. A significant increase (P < 0.05) was observed in IgA levels in all immunized groups, especially 3 wk after immunization. Treatments 2 (recombinant CTB) and 3 (recombinant FliC+CTB) showed the highest concentrations. Similarly, serum concentrations IgY (μg/mL) increased along the experiment, and the means for treatments 2 and 3 showed significant differences (P < 0.05) compared with controls, reaching concentrations of 533 and 540 μg/mL, respectively. The number of CD8(+) T lymphocytes in all treatments greatly differed (P < 0.05) compared with the negative control at 21 d posttreatment. However, only treatment 2 (recombinant CTB), 4 (PL), and 5 (recombinant FliC+ recombinant CTB + PL) remained significantly (P < 0.05) different from the control at 28 d posttreatment. Thus, it is concluded that the microencapsulated recombinant proteins administered orally to broiler chickens are capable of stimulating humoral and cellular immune response, and the combinations of these antigens with Lactobacillus spp. can influence the population of CD8(+) T cells residing in the cecum.

  7. Recombinant Bacillus subtilis spores expressing cholera toxin B subunit and Helicobacter pylori urease B confer protection against H. pylori in mice.

    Science.gov (United States)

    Zhou, Zhenwen; Dong, Hui; Huang, Yanmei; Yao, Shuwen; Liang, Bingshao; Xie, Yongqiang; Long, Yan; Mai, Jialiang; Gong, Sitang

    2017-01-01

    Helicobacter pylori infection is associated with chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The limitations of current therapies for H. pylori infection include poor compliance and antibiotic resistance. Therefore, an effective anti-H. pylori vaccine would be an alternative or complement to antibiotic treatment. Urease B (UreB) is considered an ideal vaccine antigen against H. pylori infection. In this study, cholera toxin B subunit (CTB), a mucosal adjuvant, was used to enhance the immunogenicity of a novel Bacillus subtilis spore vaccine expressing CTB-UreB, along with the B. subtilis spore coat protein CotC as a fusion protein. Oral administration of B. subtilis spores expressing CotC-UreB or CotC-CTB-UreB led to increased levels of UreB-specific IgG in serum and UreB-specific IgA in faeces, as well as elevated levels of IL-10 and IFN-γ in splenocytes. In addition, oral administration of CotC-UreB or CotC-CTB-UreB spores induced significant reductions (80.0 and 90.5 %, respectively) in gastric H. pylori bacterial load (1.11±0.36×105 and 0.53±0.21×105 c.f.u., respectively) compared to that of the CotC control group (5.56±1.64×105 c.f.u., P<0.01). Moreover, CotC-CTB-UreB spores were significantly more effective at reducing the bacterial load than CotC-UreB spores (P<0.05). These results indicate that CotC-CTB-UreB-expressing B. subtilis spores are a potential vaccine candidate for the control of H. pylori infection.

  8. An important role for adenine, cholera toxin, hydrocortisone and triiodothyronine in the proliferation, self-renewal and differentiation of limbal stem cells in vitro.

    Science.gov (United States)

    Yu, Min; Bojic, Sanja; Figueiredo, Gustavo S; Rooney, Paul; de Havilland, Julian; Dickinson, Anne; Figueiredo, Francisco C; Lako, Majlinda

    2016-11-01

    The cornea is a self-renewing tissue located at the front of the eye. Its transparency is essential for allowing light to focus onto the retina for visual perception. The continuous renewal of corneal epithelium is supported by limbal stem cells (LSCs) which are located in the border region between conjunctiva and cornea known as the limbus. Ex vivo expansion of LSCs has been successfully applied in the last two decades to treat patients with limbal stem cell deficiency (LSCD). Various methods have been used for their expansion, yet the most widely used culture media contains a number of ingredients derived from animal sources which may compromise the safety profile of human LSC transplantation. In this study we sought to understand the role of these components namely adenine, cholera toxin, hydrocortisone and triiodothyronine with the aim of re-defining a safe and GMP compatible minimal media for the ex vivo expansion of LSCs on human amniotic membrane. Our data suggest that all four components play a critical role in maintaining LSC proliferation and promoting LSC self-renewal. However removal of adenine and triiodothyronine had a more profound impact and led to LSC differentiation and loss of viability respectively, suggesting their essential role for ex vivo expansion of LSCs. Replacement of each of the components with GMP-grade reagents resulted in equal growth to non-GMP grade media, however an enhanced differentiation of LSCs was observed, suggesting that additional combinations of GMP grade reagents need to be tested to achieve similar or better level of LSC maintenance in the same manner as the traditional LSC media.

  9. Comparison of Intranasal Outer Membrane Vesicles with Cholera Toxin and Injected MF59C.1 as Adjuvants for Malaria Transmission Blocking Antigens AnAPN1 and Pfs48/45

    Directory of Open Access Journals (Sweden)

    Michael Pritsch

    2016-01-01

    Full Text Available Purified protein vaccines often require adjuvants for efficient stimulation of immune responses. There is no licensed mucosal adjuvant on the market to adequately boost the immune response to purified antigens for intranasal applications in humans. Bacterial outer membrane vesicles (OMV are attractive candidates potentially combining antigenic and adjuvant properties in one substance. To more precisely characterize the potential of Escherichia coli OMV for intranasal vaccination with heterologous antigens, immune responses for AnAPN1 and Pfs48/45 as well as ovalbumin as a reference antigen were assessed in mice. The intranasal adjuvant cholera toxin (CT and parenteral adjuvant MF59C.1 were used in comparison. Vaccinations were administered intranasally or subcutaneously. Antibodies (total IgG and IgM as well as subclasses IgG1, IgG2a, IgG2b, and IgG3 were measured by ELISA. T cell responses (cytotoxic T cells, Th1, Th17, and regulatory T cells were determined by flow cytometry. When OMV were used as adjuvant for intranasal immunization, antibody and cellular responses against all three antigens could be induced, comparable to cholera toxin and MF59C.1. Antigen-specific IgG titres above 1 : 105 could be detected in all groups. This study provides the rationale for further development of OMV as a vaccination strategy in malaria and other diseases.

  10. Piperine treatment suppresses Helicobacter pylori toxin entry in to gastric epithelium and minimizes β-catenin mediated oncogenesis and IL-8 secretion in vitro.

    Science.gov (United States)

    Tharmalingam, Nagendran; Park, Min; Lee, Min Ho; Woo, Hyun Jun; Kim, Hyun Woo; Yang, Ji Yeong; Rhee, Ki-Jong; Kim, Jong-Bae

    2016-01-01

    Helicobacter pylori related gastric cancer initiation has been studied widely. The objective of our present study was to evaluate the effect of a single compound piperine on H. pylori infection and its anti-inflammatory and anti-cancer effects in vitro. Cytotoxicity was tested by Ez-cytox cell viability assay kit. Effects of piperine on H. pylori toxin gene expression and IL-8 expression in mammalian cells during infection were assessed by RT-PCR. Effects of piperine on toxin entry into host cells, E-cadherin cleavage by H. pylori, and the changes in H. pylori mediated β-catenin expression and IL-8 secretion were determined by immunoblotting. Piperine treatment restrained the entry of CagA and VacA into AGS cells. Piperine administration in H. pylori infection reduced E-cadherin cleavage in stomach epithelium. In addition, H. pylori induced β-catenin up-regulation was reduced. Piperine administration impaired IL-8 secretion in H. pylori-infected gastric epithelial cells. As we reported previously piperine restrained H. pylori motility. The possible reason behind the H. pylori inhibition mechanism of piperine could be the dwindled motility, which weakened H. pylori adhesion to gastric epithelial cells. The reduced adhesion decreased the toxin entry thereby secreting less amount of IL-8. In addition, piperine treatment suppressed H. pylori protease led to reduction of E-cadherin cleavage and β-catenin expression resulting in diminished β-catenin translocation into the nucleus thus decreasing the risk of oncogenesis. To our knowledge, this is the preliminary report of piperine mediated H. pylori infection control on gastric epithelial cells in-vitro.

  11. The Vibrio cholerae Type VI Secretion System: Evaluating Its Role in the Human Disease Cholera

    Directory of Open Access Journals (Sweden)

    Sarah T. Miyata

    2010-11-01

    Full Text Available Vibrio cholerae, the marine bacterium responsible for the diarrheal disease cholera, utilizes a multitude of virulence factors to cause disease. The importance of two of these factors, the toxin co-regulated pilus (TCP and cholera toxin (CT, has been well documented for pandemic O1 and epidemic O139 serogroups. In contrast, endemic non-O1 and non-O139 serogroups can cause localized outbreaks of cholera-like illness, often in the absence of TCP and CT. One virulence mechanism used by these strains is the type VI secretion system (T6SS to export toxins across the cell envelope and confer toxicity towards eukaryotic and prokaryotic organisms. The V. cholerae strain V52 (an O37 serogroup strain possesses a constitutively active T6SS and was responsible for an outbreak of gastroenteritis in Sudan in 1968. To evaluate a potential role of the T6SS in the disease cholera, we compared the T6SS clusters of V. cholerae strains with sequenced genomes. We found that the majority of V. cholerae strains, including one pandemic strain, contain intact T6SS gene clusters; thus, we propose that the T6SS is a conserved mechanism that allows pandemic and endemic V. cholerae to persist both in the host and in the environment.

  12. Discrimination on three related toxin genes of Vibrio Cholerae using bv multiplex real time PCR%多重荧光定量PCR甄别3种霍乱弧菌相关毒素基因

    Institute of Scientific and Technical Information of China (English)

    金大智; 张政; 罗芸; 叶菊莲; 程苏云; 吴方; 金郁

    2011-01-01

    In order to assess Vibrio Cholerae which containing related toxin genes, a rapid, sensitive and specific assay based on multiplex real time PCR was developed in this study. The cholera toxin sub-unit A gene CctxA), accessory cholera en-terotoxin gene (ace) , and zonula occludens toxin gene (zot) of Vibrio Cholerae was chosen as targets, and then the primers and TaqMan probe were designed. Furthermore, multiplex real time PCR was applied to detect 70 Vibrio Cholerae isolated from samples, and PCR products were sequenced in order to affirm the results of multiplex real time PCR. The results showed that ctxA, ace and zot gene of Vibrio Cholerae were detected by using multiplex real time PCR accurately and quickly. When other bacteria containing no all three target toxin genes were detected, no positive results appeared. The sensitivity was 102cfu/mL for ctxA gene and lOcfu/mL for ace and zot gene in pure culture. The standard plasmids according to each toxin gene were con-structed, and consequently the detection limit of ace gene and zot gene was lOcopies/μL and the detection limit of ctxA gene was 102copies/μL. The coefficient of variation of intra-assay and inter-assay was less than 5. 0%. When this assay was applied directly to identify 70 Vibrio Cholerae, the results showed that 40 strains (57. 2%) were positive to ctxA gene, 31 strains (44. 3%) were positive to ace gene, and 46 strains (65.7%) were positive to zot gene. The results above were the same to the results obtained from the sequencing assays. The coincidence was 100%. It is demonstrated that multiplex real time PCR is a simple, accurate and feasible assay for discriminating three related toxin genes of Vibrio Cholerae. The assay described here provided a reliable tool for epidemiologic survey and epidemic monitoring.%目的 为了判定霍乱弧菌是否携带相关毒力因子,研发一种快速、准确、特异检测霍乱弧菌3种毒素基因的多重荧光定量PCR方法.方法 针对霍乱弧

  13. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii

    Directory of Open Access Journals (Sweden)

    Salah Shanan

    2016-04-01

    Full Text Available Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 with A. castellanii. The interaction between A. castellanii and V. cholerae strains was studied by means of amoeba cell counts, viable counts of the bacteria in the absence or presence of amoebae, and of the intracellularly growing bacteria, visualised by electron microscopy. These results show that all V. cholerae can grow and survive outside and inside the amoebae, disclosing that V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 all can be considered as facultative intracellular bacteria.

  14. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii.

    Science.gov (United States)

    Shanan, Salah; Bayoumi, Magdi; Saeed, Amir; Sandström, Gunnar; Abd, Hadi

    2016-01-01

    Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 with A. castellanii. The interaction between A. castellanii and V. cholerae strains was studied by means of amoeba cell counts, viable counts of the bacteria in the absence or presence of amoebae, and of the intracellularly growing bacteria, visualised by electron microscopy. These results show that all V. cholerae can grow and survive outside and inside the amoebae, disclosing that V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 all can be considered as facultative intracellular bacteria.

  15. Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain

    Directory of Open Access Journals (Sweden)

    Alexey S. Ladokhin

    2017-09-01

    Full Text Available Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation—the Open-Channel State (OCS—as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo.

  16. Transmission of Infectious Vibrio cholerae through Drinking Water among the Household Contacts of Cholera Patients (CHoBI7 Trial)

    Science.gov (United States)

    Rafique, Raisa; Rashid, Mahamud-ur; Monira, Shirajum; Rahman, Zillur; Mahmud, Md. Toslim; Mustafiz, Munshi; Saif-Ur-Rahman, K. M.; Johura, Fatema-Tuz; Islam, Saiful; Parvin, Tahmina; Bhuyian, Md. Sazzadul I.; Sharif, Mohsena B.; Rahman, Sabita R.; Sack, David A.; Sack, R. Bradley; George, Christine M.; Alam, Munirul

    2016-01-01

    Recurrent cholera causes significant morbidity and mortality among the growing population of Dhaka, the capital city of Bangladesh. Previous studies have demonstrated that household contacts of cholera patients are at >100 times higher risk of cholera during the week after the presentation of the index patient. Our prospective study investigated the mode of transmission of Vibrio cholerae, the cause of cholera, in the households of cholera patients in Dhaka city. Out of the total 420 rectal swab samples analyzed from 84 household contacts and 330 water samples collected from 33 households, V. cholerae was isolated from 20%(17/84) of household contacts, 18%(6/33) of stored drinking water, and 27%(9/33) of source water samples. Phenotypic and molecular analyses results confirmed the V. cholerae isolates to be toxigenic and belonging to serogroup O1 biotype El Tor (ET) possessing cholera toxin of classical biotype (altered ET). Phylogenetic analysis by pulsed-field gel electrophoresis (PFGE) showed the V. cholerae isolates to be clonally linked, as >95% similarity was confirmed by sub-clustering patterns in the PFGE (NotI)-based dendrogram. Mapping results showed cholera patients to be widely distributed across 25 police stations. The data suggesting the transmission of infectious V. cholerae within the household contacts of cholera patients through drinking water underscores the need for safe water to prevent spread of cholera and related deaths in Dhaka city. PMID:27803695

  17. Transmission of Infectious Vibrio cholerae Through Drinking Water among the Household Contacts of Cholera Patients (CHoBI7 Trial

    Directory of Open Access Journals (Sweden)

    Raisa Rafique

    2016-10-01

    Full Text Available Recurrent cholera causes significant morbidity and mortality among the growing population of Dhaka, the capital city of Bangladesh. Previous studies have demonstrated that household contacts of cholera patients are at >100 times higher risk of cholera during the week after the presentation of the index patient. Our prospective study investigated the mode of transmission of Vibrio cholerae, the cause of cholera, in the households of cholera patients in Dhaka city. Of total 420 rectal swab samples analyzed from 84 household contacts and 330 water samples collected from 33 households, V. cholerae was isolated from 20%(17/84 of household contacts, 18%(6/33 of stored drinking water, and 27%(9/33 of source water samples. Phenotypic and molecular analyses results confirmed the V. cholerae isolates to be toxigenic and belonging to serogroup O1 biotype El Tor (ET possessing cholera toxin of classical biotype (altered ET. Phylogenetic analysis by pulsed-field gel electrophoresis (PFGE showed the V. cholerae isolates to be clonally linked, as >95% similarity was confirmed by sub-clustering patterns in the PFGE (NotI-based dendrogram. Mapping results showed cholera patients to be widely distributed across 25 police stations with the highest incidence in households near the major rivers and polluted water bodies. The data presented on the transmission of infectious V. cholerae within the household contacts of cholera patients through drinking water underscores the need for safe water to prevent spread of cholera and related deaths in Dhaka city.

  18. 霍乱毒素黏膜免疫佐剂作用研究进展%Cholera toxin as an adjuvant for mucosal immunization

    Institute of Scientific and Technical Information of China (English)

    陈晓月; 金宁一; 邹伟; 海洋

    2007-01-01

    @@ 霍乱(cholera)是由霍乱弧菌(vibrio cholerae)引起的急性传染病,属三大国际检疫传染病之一,也是我国法定管理的甲类传染病.其病理变化主要由霍乱弧菌的肠毒素所致,临床表现为不同程度的腹泻、呕吐、失水,少数患者出现肌肉痉挛、代谢性酸中毒、循环衰竭等.

  19. Oral administration of a fusion protein between the cholera toxin B subunit and the 42-amino acid isoform of amyloid-β peptide produced in silkworm pupae protects against Alzheimer's disease in mice.

    Directory of Open Access Journals (Sweden)

    Si Li

    Full Text Available A key molecule in the pathogenesis of Alzheimer's disease (AD is a 42-amino acid isoform of the amyloid-β peptide (Aβ42, which is the most toxic element of senile plaques. In this study, to develop an edible, safe, low-cost vaccine for AD, a cholera toxin B subunit (CTB-Aβ42 fusion protein was successfully expressed in silkworm pupae. We tested the silkworm pupae-derived oral vaccination containing CTB-Aβ42 in a transgenic mouse model of AD. Anti-Aβ42 antibodies were induced in these mice, leading to a decreased Aβ deposition in the brain. We also found that the oral administration of the silk worm pupae vaccine improved the memory and cognition of mice, as assessed using a water maze test. These results suggest that the new edible CTB-Aβ42 silkworm pupae-derived vaccine has potential clinical application in the prevention of AD.

  20. H/KDEL receptors mediate host cell intoxication by a viral A/B toxin in yeast

    Science.gov (United States)

    Becker, Björn; Blum, Andrea; Gießelmann, Esther; Dausend, Julia; Rammo, Domenik; Müller, Nina C.; Tschacksch, Emilia; Steimer, Miriam; Spindler, Jenny; Becherer, Ute; Rettig, Jens; Breinig, Frank; Schmitt, Manfred J.

    2016-01-01

    A/B toxins such as cholera toxin, Pseudomonas exotoxin and killer toxin K28 contain a KDEL-like amino acid motif at one of their subunits which ensures retrograde toxin transport through the secretory pathway of a target cell. As key step in host cell invasion, each toxin binds to distinct plasma membrane receptors that are utilized for cell entry. Despite intensive efforts, some of these receptors are still unknown. Here we identify the yeast H/KDEL receptor Erd2p as membrane receptor of K28, a viral A/B toxin carrying an HDEL motif at its cell binding β-subunit. While initial toxin binding to the yeast cell wall is unaffected in cells lacking Erd2p, binding to spheroplasts and in vivo toxicity strongly depend on the presence of Erd2p. Consistently, Erd2p is not restricted to membranes of the early secretory pathway but extends to the plasma membrane where it binds and internalizes HDEL-cargo such as K28 toxin, GFPHDEL and Kar2p. Since human KDEL receptors are fully functional in yeast and restore toxin sensitivity in the absence of endogenous Erd2p, toxin uptake by H/KDEL receptors at the cell surface might likewise contribute to the intoxication efficiency of A/B toxins carrying a KDEL-motif at their cytotoxic A-subunit(s). PMID:27493088

  1. Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

    Directory of Open Access Journals (Sweden)

    T. Petr

    2010-05-01

    Full Text Available A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde, which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit. Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at -20°C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0±0.3% only. The loss from dried brain homogenate was 9.5±1.1%. Thus, anhydrous conditions (dry tissue samples and anhydrous acetone are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion, and removal of cholesterol, which can act as a hydrophobic blocking barrier.

  2. 霍乱毒素A基因内部翻译调控元件具有翻译起始功能%Translation Initiation Function of the Regulation Element in the Operon of Cholera Toxin A

    Institute of Scientific and Technical Information of China (English)

    曹诚; 李平; 李杰之; 石成华; 马清钧

    2000-01-01

    To demonstrate that there existed translation coupling between cholera toxin A subunit gene and B subunit gene, and give the answer why the expression level of B gene is five times more than that of A gene, α report system for the investigation of translation coupling was constructed by using lacZ gene as reporter. Frame-shift mutation was introduced near the C terminal of ctxA gene, and the ribosome would read through its normal stop codon. The report plasmid was constructed and it was found that the expression level of lacZ gene decreased five times after the frame-shift mutation. The translation of cholera toxin B subunit gene was translational coupled with A subunit gene, and was responsible for the differential expression level of the two genes.%通过大肠杆菌体外转录-体外翻译系统,证明霍乱毒素A基因内部的翻译调控元件具有翻译起始功能,且其翻译起始效率较ctxA基因高得多.当ctxA的起始密码突变时,从该元件起始的翻译效率下降,说明基因内翻译起始效率受ctxA翻译起始的调控。结果进一步证实了霍乱毒素A、B亚基比例表达调控的翻译弱化-翻译偶联机理。

  3. Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein.

    Science.gov (United States)

    Gabig, T G; English, D; Akard, L P; Schell, M J

    1987-02-05

    Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.

  4. Drinking cholera

    DEFF Research Database (Denmark)

    Grant, Stephen Lawrence; Tamason, Charlotte Crim; Hoque, Bilqis Amin

    2015-01-01

    Objectives: To measure the salinity levels of common water sources in coastal Bangladesh andexplore perceptions of water palatability among the local population to investigate the plausibility oflinking cholera outbreaks in Bangladesh with ingestion of saline-rich cholera-infected river water...... beconducive to V. cholerae survival. Furthermore, salinity levels of participant’s drinking water sourceswere all well below the levels required for optimal survival of V. cholerae. Respondents explainedthat they preferred less salty and more aesthetically pleasing drinking water. Conclusion: Theoretically, V....... cholerae can survive in the river systems in Bangladesh; however,water sources which have been contaminated with river water are avoided as potential drinkingwater sources. Furthermore, there are no physical connecting points between the river system anddrinking water sources among the study population...

  5. IFN-gamma-induced chemokines synergize with pertussis toxin to promote T cell entry to the central nervous system

    DEFF Research Database (Denmark)

    Millward, Jason M; Caruso, Maria; Campbell, Iain L

    2007-01-01

    was predominantly localized to meningeal and ependymal cells, and was also seen in astrocytes and microglia. IFN-gamma-induced chemokine expression did not lead to inflammation. However, when pertussis toxin was given i.p. to mice infected with the IFN-gamma vector, there was a dramatic increase in the number of T...

  6. Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria-pertussis-tetanus (DPT) combination vaccine.

    Science.gov (United States)

    Isaka, Masanori; Komiya, Takako; Takahashi, Motohide; Yasuda, Yoko; Taniguchi, Tooru; Zhao, Yanqiu; Matano, Keiko; Matsui, Hideyuki; Maeyama, Jun-Ichi; Morokuma, Kazunori; Ohkuma, Kunio; Goto, Norihisa; Tochikubo, Kunio

    2004-08-13

    Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens. When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not. Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths. DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge. These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.

  7. Survival of Vibrio cholerae in industrially polluted water, with ...

    African Journals Online (AJOL)

    DRINIE

    2004-01-01

    Jan 1, 2004 ... prepared in this manner for all the experiments. Baseline counts of ..... showed that sunlight induces propagation of this toxin-encoding lysogenic ... toxigenic Vibrio cholerae O1 with a common duckweed, Lemma minor, in ...

  8. Vibrio cholerae as a predator: lessons from evolutionary principles

    Directory of Open Access Journals (Sweden)

    Stefan ePukatzki

    2013-12-01

    Full Text Available Diarrheal diseases are the second-most common cause of death among children under the age of five worldwide. Cholera alone, caused by the marine bacterium Vibrio cholerae, is responsible for several million cases and over 120,000 deaths annually. When contaminated water is ingested, V. cholerae passes through the gastric acid barrier, penetrates the mucin layer of the small intestine, and adheres to the underlying epithelial lining. V. cholerae multiplies rapidly, secretes cholera toxin, and exits the human host in vast numbers during diarrheal purges. How V. cholerae rapidly reaches such high numbers during each purge is not clearly understood. We propose that V. cholerae employs its bactericidal type VI secretion system to engage in intraspecies and intraguild predation for nutrient acquisition to support rapid growth and multiplication.

  9. Quantifying Vibrio cholerae enterotoxicity in a zebrafish infection model.

    Science.gov (United States)

    Mitchell, Kristie C; Breen, Paul; Britton, Sarah; Neely, Melody N; Withey, Jeffrey H

    2017-06-16

    Vibrio cholerae is the etiological agent of cholera, an acute intestinal infection in humans characterized by voluminous watery diarrhea. Cholera is spread through ingestion of contaminated food or water, primarily in developing countries that lack the proper infrastructure for proper water and sewage treatment. Vibrio cholerae is an aquatic bacterium that inhabits coastal and estuarine areas, and is known to have several environmental reservoirs, including fish. Our laboratory has recently described the use of the zebrafish as a new animal model for the study of V. cholerae intestinal colonization, pathogenesis, and transmission.As early as six hours after exposure to V. cholerae, zebrafish develop diarrhea. Prior work in our laboratory has shown that this is not due to the action of cholera toxin. We hypothesize that accessory toxins produced by V. cholerae are the cause of diarrhea in infected zebrafish. In order to assess the effects of accessory toxins in the zebrafish, it was necessary to develop a method of quantifying diarrheal volume as a measure of pathogenesis. Here, we have adapted cell density, protein, and mucin assays, along with enumeration of V. cholerae in the zebrafish intestinal tract and in the infection water, to achieve this goal. Combined, these assays should help us determine which toxins have the greatest diarrheagenic effect in fish, and consequently, which toxins may play a role in environmental transmission.Importance Identification of the accessory toxins that cause diarrhea in zebrafish can help us understand more about the role of fish in the wild as aquatic reservoirs for V. cholerae It is plausible that accessory toxins can act to prolong colonization and subsequent shedding of V. cholerae back into the environment, thus perpetuating and facilitating transmission during an outbreak. It is also possible that accessory toxins help to maintain low levels of intestinal colonization in fish, giving V. cholerae an advantage when

  10. Antibody-based biological toxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Menking, D.E.; Goode, M.T. [Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD (United States)

    1995-12-01

    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a direct competition assay, antibodies against Cholera toxin, Staphylococcus Enterotoxin B or ricin were noncovalently immobilized on quartz fibers and probed with fluorescein isothiocyanate (FITC) - labeled toxins. In the indirect competition assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified anti-toxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or anti-toxin IgG in a dose dependent manner and the detection of the toxins was in the nanomolar range.

  11. MucoRice-cholera toxin B-subunit, a rice-based oral cholera vaccine, down-regulates the expression of α-amylase/trypsin inhibitor-like protein family as major rice allergens.

    Science.gov (United States)

    Kurokawa, Shiho; Nakamura, Rika; Mejima, Mio; Kozuka-Hata, Hiroko; Kuroda, Masaharu; Takeyama, Natsumi; Oyama, Masaaki; Satoh, Shigeru; Kiyono, Hiroshi; Masumura, Takehiro; Teshima, Reiko; Yuki, Yoshikazu

    2013-07-05

    To develop a cold chain- and needle/syringe-free rice-based cholera vaccine (MucoRice-CTB) for human use, we previously advanced the MucoRice system by introducing antisense genes specific for endogenous rice storage proteins and produced a molecularly uniform, human-applicable, high-yield MucoRice-CTB devoid of plant-associated sugar. To maintain the cold chain-free property of this vaccine for clinical application, we wanted to use a polished rice powder preparation of MucoRice-CTB without further purification but wondered whether this might cause an unexpected increase in rice allergen protein expression levels in MucoRice-CTB and prompt safety concerns. Therefore, we used two-dimensional fluorescence difference gel electrophoresis and shotgun MS/MS proteomics to compare rice allergen protein expression levels in MucoRice-CTB and wild-type (WT) rice. Both proteomics analyses showed that the only notable change in the expression levels of rice allergen protein in MucoRice-CTB, compared with those in WT rice, was a decrease in the expression levels of α-amylase/trypsin inhibitor-like protein family such as the seed allergen protein RAG2. Real-time PCR analysis showed mRNA of RAG2 reduced in MucoRice-CTB seed. These results demonstrate that no known rice allergens appear to be up-reregulated by genetic modification of MucoRice-CTB, suggesting that MucoRice-CTB has potential as a safe oral cholera vaccine for clinical application.

  12. Using common spatial distributions of atoms to relate functionally divergent influenza virus N10 and N11 protein structures to functionally characterized neuraminidase structures, toxin cell entry domains, and non-influenza virus cell entry domains.

    Directory of Open Access Journals (Sweden)

    Arthur Weininger

    Full Text Available The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase

  13. Using Common Spatial Distributions of Atoms to Relate Functionally Divergent Influenza Virus N10 and N11 Protein Structures to Functionally Characterized Neuraminidase Structures, Toxin Cell Entry Domains, and Non-Influenza Virus Cell Entry Domains

    Science.gov (United States)

    Weininger, Arthur; Weininger, Susan

    2015-01-01

    The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase-facilitated cell

  14. Second-pandemic strain of Vibrio cholerae from the Philadelphia cholera outbreak of 1849.

    Science.gov (United States)

    Devault, Alison M; Golding, G Brian; Waglechner, Nicholas; Enk, Jacob M; Kuch, Melanie; Tien, Joseph H; Shi, Mang; Fisman, David N; Dhody, Anna N; Forrest, Stephen; Bos, Kirsten I; Earn, David J D; Holmes, Edward C; Poinar, Hendrik N

    2014-01-23

    In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europe, and North America. The causes of these outbreaks and the genomic strain identities remain a mystery. We used targeted high-throughput sequencing to reconstruct the Vibrio cholerae genome from the preserved intestine of a victim of the 1849 cholera outbreak in Philadelphia, part of the second cholera pandemic. This O1 biotype strain has 95 to 97% similarity with the classical O395 genome, differing by 203 single-nucleotide polymorphisms (SNPs), lacking three genomic islands, and probably having one or more tandem cholera toxin prophage (CTX) arrays, which potentially affected its virulence. This result highlights archived medical remains as a potential resource for investigations into the genomic origins of past pandemics.

  15. Methods to assess the impact of mass oral cholera vaccination campaigns under real field conditions.

    Directory of Open Access Journals (Sweden)

    Jacqueline Deen

    Full Text Available There is increasing interest to use oral cholera vaccination as an additional strategy to water and sanitation interventions against endemic and epidemic cholera. There are two internationally-available and WHO-prequalified oral cholera vaccines: an inactivated vaccine containing killed whole-cells of V. cholerae O1 with recombinant cholera toxin B-subunit (WC/rBS and a bivalent inactivated vaccine containing killed whole cells of V. cholerae O1 and V. cholerae O139 (BivWC. The efficacy, effectiveness, direct and indirect (herd protection conferred by WC/rBS and BivWC are well established. Yet governments may need local evidence of vaccine impact to justify and scale-up mass oral cholera vaccination campaigns. We discuss various approaches to assess oral cholera vaccine protection, which may be useful to policymakers and public health workers considering deployment and evaluation of the vaccine.

  16. Characterization of Aeromonas trota strains that cross-react with Vibrio cholerae O139 Bengal.

    Science.gov (United States)

    Albert, M J; Ansaruzzaman, M; Shimada, T; Rahman, A; Bhuiyan, N A; Nahar, S; Qadri, F; Islam, M S

    1995-12-01

    It has previously been shown that Vibrio cholerae O139 Bengal shares antigens with V. cholerae serogroups O22 and O155. We detected six surface water isolates of Aeromonas trota that agglutinated in polyclonal antisera to V. cholerae O139 and V. cholerae O22 but not in antiserum to V. cholerae O155. On the basis of agglutinin-absorption studies, the antigenic relationship between the cross-reacting bacteria were found to be in an a,b-a,c fashion, where a is the common antigenic epitope and b and c are unique epitopes. The antigen sharing between A. trota strains and V. cholerae O139 was confirmed in immunoblot studies. However, A. trota strains did not react with two monoclonal antibodies specific for V. cholerae O139 and, consequently, tested negative in the Bengal SMART rapid diagnostic test for V. cholerae O139 which uses one of the monoclonal antibodies. A polyclonal antiserum to a cross-reacting A. trota strain cross-protected infant mice against cholera on challenge with virulent V. cholerae O139. All A. trota strains were cytotoxic for HeLa cells, positive for adherence to HEp-2 cells, and weakly invasive for HEp-2 cells; one strain was heat-stable toxin positive in the suckling mouse assay; however, all strains were negative for cholera toxin-like enterotoxin. Studies on bacteria that share somatic antigen with V. cholerae O139 may shed further light on the genesis of V. cholerae O139.

  17. Plasma and Mucosal Immunoglobulin M, Immunoglobulin A, and Immunoglobulin G Responses to the Vibrio cholerae O1 Protein Immunome in Adults With Cholera in Bangladesh.

    Science.gov (United States)

    Charles, Richelle C; Nakajima, Rie; Liang, Li; Jasinskas, Al; Berger, Amanda; Leung, Daniel T; Kelly, Meagan; Xu, Peng; Kovác, Pavol; Giffen, Samantha R; Harbison, James D; Chowdhury, Fahima; Khan, Ashraful I; Calderwood, Stephen B; Bhuiyan, Taufiqur Rahman; Harris, Jason B; Felgner, Philip L; Qadri, Firdausi; Ryan, Edward T

    2017-07-01

    Cholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera. We probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay. Overall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O-specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate-2-oxoglutarate aminotransferase. This study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine.

  18. Effectiveness of the WC/rBS oral cholera vaccine in the prevention of traveler's diarrhea: a prospective cohort study.

    Science.gov (United States)

    López-Gigosos, Rosa; Campins, Magda; Calvo, María J; Pérez-Hoyos, Santiago; Díez-Domingo, Javier; Salleras, Luis; Azuara, María T; Martínez, Xavier; Bayas, José M; Ramón Torrell, Josep M; Pérez-Cobaleda, María A; Núñez-Torrón, María E; Gorgojo, Lydia; García-Rodríguez, Magdalena; Díez-Díaz, Rosa; Armadans, Luis; Sánchez-Fernández, Concepción; Mejías, Teresa; Masuet, Cristina; Pinilla, Rafael; Antón, Nieves; Segarra, Pilar

    2013-03-01

    Traveler's diarrhea (TD) is the most frequent disease among people from industrialized countries who travel to less developed ones, especially sub-Saharan Africa, Southern Asia and South America. The most common bacteria causing TD is enterotoxigenic Escherichia coli (ETEC). The WC/rBS cholera vaccine (Dukoral) has been shown to induce cross-protection against ETEC by means of the B subunit of the cholera toxin. The aim of the study was to evaluate the effectiveness of the WC/rBS cholera vaccine in preventing TD. Between May 1 and September 30 (2007), people seeking pre-travel advice in ten Spanish international vaccination centers were included in a prospective cohort study of travelers to cholera risk countries. The incidence rates of TD were adjusted for variables whose frequencies were statistically different (entry point 0.10) between the vaccinated and non-vaccinated cohorts. The vaccinated cohort (n = 544 travelers) included people vaccinated with the WC/rBS cholera vaccine, and the non-vaccinated cohort (n = 530 travelers) by people not vaccinated. The cumulative incidence rate of TD was 1.69 in vaccinated and 2.14 in non-vaccinated subjects. The adjusted relative risk of TD in vaccinated travelers was 0.72 (95% CI: 0.58-0.88) and the adjusted vaccination effectiveness was 28% (95% CI: 12-42). The WC/rBS cholera vaccine prevents TD in 2 out of 7 travelers (preventive fraction: 28%). The number needed to vaccinate (NNV) to prevent 1 case of TD is 10.

  19. Non-toxigenic environmental Vibrio cholerae O1 strain from Haiti provides evidence of pre-pandemic cholera in Hispaniola

    Science.gov (United States)

    Azarian, Taj; Ali, Afsar; Johnson, Judith A.; Jubair, Mohammad; Cella, Eleonora; Ciccozzi, Massimo; Nolan, David J.; Farmerie, William; Rashid, Mohammad H.; Sinha-Ray, Shrestha; Alam, Meer T.; Morris, J. Glenn; Salemi, Marco

    2016-01-01

    Vibrio cholerae is ubiquitous in aquatic environments, with environmental toxigenic V. cholerae O1 strains serving as a source for recurrent cholera epidemics and pandemic disease. However, a number of questions remain about long-term survival and evolution of V. cholerae strains within these aquatic environmental reservoirs. Through monitoring of the Haitian aquatic environment following the 2010 cholera epidemic, we isolated two novel non-toxigenic (ctxA/B-negative) Vibrio cholerae O1. These two isolates underwent whole-genome sequencing and were investigated through comparative genomics and Bayesian coalescent analysis. These isolates cluster in the evolutionary tree with strains responsible for clinical cholera, possessing genomic components of 6th and 7th pandemic lineages, and diverge from “modern” cholera strains around 1548 C.E. [95% HPD: 1532–1555]. Vibrio Pathogenicity Island (VPI)-1 was present; however, SXT/R391-family ICE and VPI-2 were absent. Rugose phenotype conversion and vibriophage resistance evidenced adaption for persistence in aquatic environments. The identification of V. cholerae O1 strains in the Haitian environment, which predate the first reported cholera pandemic in 1817, broadens our understanding of the history of pandemics. It also raises the possibility that these and similar environmental strains could acquire virulence genes from the 2010 Haitian epidemic clone, including the cholera toxin producing CTXϕ. PMID:27786291

  20. Non-toxigenic environmental Vibrio cholerae O1 strain from Haiti provides evidence of pre-pandemic cholera in Hispaniola.

    Science.gov (United States)

    Azarian, Taj; Ali, Afsar; Johnson, Judith A; Jubair, Mohammad; Cella, Eleonora; Ciccozzi, Massimo; Nolan, David J; Farmerie, William; Rashid, Mohammad H; Sinha-Ray, Shrestha; Alam, Meer T; Morris, J Glenn; Salemi, Marco

    2016-10-27

    Vibrio cholerae is ubiquitous in aquatic environments, with environmental toxigenic V. cholerae O1 strains serving as a source for recurrent cholera epidemics and pandemic disease. However, a number of questions remain about long-term survival and evolution of V. cholerae strains within these aquatic environmental reservoirs. Through monitoring of the Haitian aquatic environment following the 2010 cholera epidemic, we isolated two novel non-toxigenic (ctxA/B-negative) Vibrio cholerae O1. These two isolates underwent whole-genome sequencing and were investigated through comparative genomics and Bayesian coalescent analysis. These isolates cluster in the evolutionary tree with strains responsible for clinical cholera, possessing genomic components of 6(th) and 7(th) pandemic lineages, and diverge from "modern" cholera strains around 1548 C.E. [95% HPD: 1532-1555]. Vibrio Pathogenicity Island (VPI)-1 was present; however, SXT/R391-family ICE and VPI-2 were absent. Rugose phenotype conversion and vibriophage resistance evidenced adaption for persistence in aquatic environments. The identification of V. cholerae O1 strains in the Haitian environment, which predate the first reported cholera pandemic in 1817, broadens our understanding of the history of pandemics. It also raises the possibility that these and similar environmental strains could acquire virulence genes from the 2010 Haitian epidemic clone, including the cholera toxin producing CTXϕ.

  1. OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay

    NARCIS (Netherlands)

    Paauw, A.; Trip, H.; Niemcewicz, M.; Sellek, R.; Heng, J.M.E.; Mars-Groenendijk, R.H.; Jong, A.L. de; Majchrzykiewicz-Koehorst, J.A.; Olsen, J.S.; Tsivtsivadze, E.

    2014-01-01

    Background Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effecti

  2. OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay

    NARCIS (Netherlands)

    Paauw, A.; Trip, H.; Niemcewicz, M.; Sellek, R.; Heng, J.M.E.; Mars-Groenendijk, R.H.; Jong, A.L. de; Majchrzykiewicz-Koehorst, J.A.; Olsen, J.S.; Tsivtsivadze, E.

    2014-01-01

    Background Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an

  3. OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay

    NARCIS (Netherlands)

    Paauw, A.; Trip, H.; Niemcewicz, M.; Sellek, R.; Heng, J.M.E.; Mars-Groenendijk, R.H.; Jong, A.L. de; Majchrzykiewicz-Koehorst, J.A.; Olsen, J.S.; Tsivtsivadze, E.

    2014-01-01

    Background Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effecti

  4. B subunits of cholera toxin and thermolabile enterotoxin of Escherichia coli have similar adjuvant effect as whole molecules on rotavirus 2/6-VLP specific antibody responses and induce a Th17-like response after intrarectal immunization.

    Science.gov (United States)

    Thiam, Fatou; Charpilienne, Annie; Poncet, Didier; Kohli, Evelyne; Basset, Christelle

    2015-12-01

    The purpose of this study was to evaluate the adjuvant effect of the B subunits of cholera toxin (CT) and the thermolabile enterotoxin of Escherichia coli (LT) by the intrarectal route of immunization and compare them to the whole molecules CT and LT-R192G, a non toxic mutant of LT, using 2/6-VLP as an antigen, in mice. All molecules induced similar antigen specific antibody titers in serum and feces, whereas different T cell profiles were observed. CTB and LTB, conversely to CT and LT-R192G, did not induce detectable production of IL-2 by antigen specific T cells. Moreover, CTB, conversely to LT-R192G, CT and LTB, did not induce antigen specific CD4+CD25+Foxp3- and Foxp3+ T cells, thus showing different effects between the B subunits themselves. However, all molecules induced an antigen specific Th17 response. In conclusion, B subunits are potent adjuvants on B cell responses by the intrarectal route. Although their impact on T cell responses are different, all molecules induce a 2/6-VLP-specific Th17 T cell response that may play a major role in helping B cell responses and thus in adjuvanticity and protection.

  5. Expression of cholera toxin B subunit-lumbrokinase in edible sunflower seeds-the use of transmucosal carrier to enhance its fusion protein's effect on protection of rats and mice against thrombosis.

    Science.gov (United States)

    Guan, Chunfeng; Ji, Jing; Jin, Chao; Wang, Gang; Li, Xiaozhou; Guan, Wenzhu

    2014-01-01

    Lumbrokinase (LK) is a group of serine proteases with strong fibrinolytic and thrombolytic activities and is useful for treating diseases caused by thrombus. Cholera toxin B subunit (CTB) has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for the induction of oral tolerance. We investigate here the application of CTB as a transmucosal carrier in enhancing its fusion protein-LKs effect to protect rats against thrombosis. Thus, in this study, CTB-LK fusion gene separated by a furin cleavage site was expressed in seeds of Helianthus annuus L. The activity of recombinant protein in seeds of transgenic sunflower was confirmed by Western blot analysis, fibrin plate assays and GM1 -ganglioside ELISA. The thrombosis model of rats and mice revealed that the oral administration of peeled seeds of sunflower expressing CTB-LK had a more significant anti-thrombotic effect on animals compared with that administration of peeled seeds of sunflower expressing LK. It is possible to conclude that CTB can successfully enhance its fusion protein to be absorbed in rats or mice thrombosis model. The use of CTB as a transmucosal carrier in the delivery of transgenic plant-derived oral therapeutic proteins was supported. In addition, for the purpose of that recombinant CTB-LK was designed for oral administration, thus the expression of CTB-LK in edible sunflower seeds eliminated the need for downstream processing of proteins.

  6. Biological feature of cholera toxin and heat-labile enterotoxin%霍乱毒素及大肠杆菌不耐热肠毒素生物学特性的研究

    Institute of Scientific and Technical Information of China (English)

    郭学青; 邹全明

    2000-01-01

    @@ 霍乱毒素(cholera toxin,CT)和大肠杆菌不耐热肠毒素(heat-labile enterotoxin,LT)随着传染病和流行病学的发展得以关注,近年由于疫苗的进展再次得到重视.它们是细菌蛋白毒素家族成员,通过G蛋白的ADP-核糖基化而发挥其毒性[1].两者关系密切,氨基酸序列同源性约80%,均包括一个240个残基的A链和5个103个残基的B亚单位[2],即AB5六聚体.B五聚体与GM1神经节苷脂受体结合,决定毒素对宿主细胞的选择亲和性.A亚单位有使上皮细胞G蛋白Gsα的一个精氨酸残基ADP-核糖基化的活性.两者具有较强的粘膜免疫原性和粘膜免疫佐剂效应.了解其结构、粘膜免疫机制,对于粘膜疫苗的发展有重要指导意义.

  7. Low-dose oral tolerance due to antigen in the diet suppresses differentially the cholera toxin-adjuvantized IgE, IgA and IgG response

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Kjær, Tanja; Frøkiær, Hanne

    2003-01-01

    Background: Cholera toxin (CT) is used as a mucosal adjuvant amongst other applications for studying food allergy because oral administration of antigen with CT induces an antigen-specific type 2 response, including IgE and IgA production. Priorly established oral tolerance due to antigen...... soy-trypsin inhibitor (KSTI) (F0 mice) and mice fed a soy-free diet (F2 mice) were orally immunized with KSTI and CT. KSTI-specific serum IgG1, IgG2a, IgA and IgE and fecal IgA were monitored. KSTI-stimulated cell proliferation and interleukin (IL)-6 production were determined. Results: The anti......-KSTI IgE and IgA responses in the F0 mice were substantially suppressed, while the IgG1 and IgG2a responses were not suppressed after five oral immunizations. The response suppression tended to decline with increasing numbers of immunizations suggesting that the suppression could be overcome by multiple...

  8. Cholera Treatment

    Science.gov (United States)

    ... Public Health in Haiti Haiti Pre-decision Brief Cholera in Haiti: One Year Later Related Links Healthy Water Global Water, Sanitation, & Hygiene (WASH) The Safe Water System Division of Foodborne, Waterborne, and Environmental Diseases Get Email Updates To receive email updates about ...

  9. A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine.

    Directory of Open Access Journals (Sweden)

    Alex T Nielsen

    Full Text Available A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP and cholera toxin (CT were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a

  10. 口服重组B亚单位O1/O139霍乱疫苗的制备及检定%Preparation and quality control of oral bivalent recombinant cholera toxin B subunit-O1/O139 whole cell cholera vaccine

    Institute of Scientific and Technical Information of China (English)

    张静飞; 陈磊; 董思国; 曹天成; 何金娜; 曹欣; 胡鹏; 郝倩; 魏文进

    2016-01-01

    目的 制备口服重组B亚单位O1/O139霍乱疫苗,并进行全面检定.方法 制备O1、O139群霍乱弧菌灭活菌体原液及重组霍乱毒素B亚单位(recombinant cholera toxin B subunit,rCTB)原液,按比例混合制成口服重组B亚单位O1/O139霍乱疫苗,为保护rCTB免受胃酸的破坏,制备了碳酸氢钠抗酸泡腾颗粒,并按照制定的新制品原液及成品的质控标准,对原液及成品进行全面检定.结果 制备的口服重组B亚单位O1/O139霍乱疫苗工艺稳定,各项指标均达到质控标准.结论 口服重组B亚单位O1/O139霍乱疫苗安全、有效,且制备工艺可行,符合规模化生产的要求.

  11. Antibody-secreting cell responses after Vibrio cholerae O1 infection and oral cholera vaccination in adults in Bangladesh.

    Science.gov (United States)

    Rahman, Atiqur; Rashu, Rasheduzzaman; Bhuiyan, Taufiqur Rahman; Chowdhury, Fahima; Khan, Ashraful Islam; Islam, Kamrul; LaRocque, Regina C; Ryan, Edward T; Wrammert, Jens; Calderwood, Stephen B; Qadri, Firdausi; Harris, Jason B

    2013-10-01

    Infection with Vibrio cholerae and oral cholera vaccines (OCVs) induce transient circulating plasmablast responses that peak within approximately 7 days after infection or vaccination. We previously demonstrated that plasmablast responses strongly correlate with subsequent levels of V. cholerae-specific duodenal antibodies up to 6 months after V. cholerae infection. Hence, plasmablast responses provide an early window into the immunologic memory at the mucosal surface. In this study, we characterized plasmablast responses following V. cholerae infection using a flow cytometrically defined population and compared V. cholerae-specific responses in adult patients with V. cholerae O1 infection and vaccinees who received the OCV Dukoral (Crucell Vaccines Canada). Among flow cytometrically sorted populations of gut-homing plasmablasts, almost 50% of the cells recognized either cholera toxin B subunit (CtxB) or V. cholerae O1 lipopolysaccharide (LPS). Using a traditional enzyme-linked immunosorbent spot assay (ELISPOT), we found that infection with V. cholerae O1 and OCVs induce similar responses to the protein antigen CtxB, but responses to LPS were diminished after OCV compared to those after natural V. cholerae infection. A second dose of OCV on day 14 failed to boost circulating V. cholerae-specific plasmablast responses in Bangladeshi adults. Our results differ from those in studies from areas where cholera is not endemic, in which a second vaccination on day 14 significantly boosts plasmablast responses. Given these results, it is likely that the optimal boosting strategies for OCVs differ significantly between areas where V. cholerae infection is endemic and those where it is not.

  12. Plasma Leptin Levels in Children Hospitalized with Cholera in Bangladesh.

    Science.gov (United States)

    Falkard, Brie; Uddin, Taher; Rahman, M Arifur; Franke, Molly F; Aktar, Amena; Uddin, Muhammad Ikhtear; Bhuiyan, Taufiqur Rahman; Leung, Daniel T; Charles, Richelle C; Larocque, Regina C; Harris, Jason B; Calderwood, Stephen B; Qadri, Firdausi; Ryan, Edward T

    2015-08-01

    Vibrio cholerae, the cause of cholera, induces both innate and adaptive immune responses in infected humans. Leptin is a hormone that plays a role in both metabolism and mediating immune responses. We characterized leptin levels in 11 children with cholera in Bangladesh, assessing leptin levels on days 2, 7, 30, and 180 following cholera. We found that patients at the acute stage of cholera had significantly lower plasma leptin levels than matched controls, and compared with levels in late convalescence. We then assessed immune responses to V. cholerae antigens in 74 children with cholera, correlating these responses to plasma leptin levels on day 2 of illness. In multivariate analysis, we found an association between day 2 leptin levels and development of later anti-cholera toxin B subunit (CtxB) responses. This finding appeared to be limited to children with better nutritional status. Interestingly, we found no association between leptin levels and antibody responses to V. cholerae lipopolysaccharide, a T cell-independent antigen. Our results suggest that leptin levels may be associated with cholera, including the development of immune responses to T cell-dependent antigens. © The American Society of Tropical Medicine and Hygiene.

  13. The hows and whys of constructing a native recombinant cholera vaccine

    Science.gov (United States)

    Boustanshenas, Mina; Bakhshi, Bita

    2014-01-01

    Emergence of different ctxB genotypes within virulent Vibrio cholerae populations accentuates the need to develop a vaccine that has the potential to protect against all cholera toxin genotypes. Oral administration of rCTB—alone and in combination with 2 dominant domestic killed whole cells of V. cholerae (O1 Ogawa El Tor and O1 Inaba El Tor) plus one standard V. cholerae (O1 Ogawa classic ATCC 14035)—has shown satisfactory protection as a potent vaccine candidate against toxigenic V. cholerae. PMID:24165439

  14. Genome assortment, not serogroup, defines Vibrio cholerae pandemic strains

    Energy Technology Data Exchange (ETDEWEB)

    Brettin, Thomas S [Los Alamos National Laboratory; Bruce, David C [Los Alamos National Laboratory; Challacombe, Jean F [Los Alamos National Laboratory; Detter, John C [Los Alamos National Laboratory; Han, Cliff S [Los Alamos National Laboratory; Munik, A C [Los Alamos National Laboratory; Chertkov, Olga [Los Alamos National Laboratory; Meincke, Linda [Los Alamos National Laboratory; Saunders, Elizabeth [Los Alamos National Laboratory; Choi, Seon Y [SEOUL NATL. UNIV.; Haley, Bradd J [U. MARYLAND; Taviani, Elisa [U. MARYLAND; Jeon, Yoon - Seong [INTL. VACCINE INST. SEOUL; Kim, Dong Wook [INTL. VACCINE INST. SEOUL; Lee, Jae - Hak [SEOUL NATL. UNIV.; Walters, Ronald A [PNNL; Hug, Anwar [NATL. INST. CHOLERIC ENTERIC DIS.; Colwell, Rita R [U. MARYLAND

    2009-01-01

    Vibrio cholerae, the causative agent of cholera, is a bacterium autochthonous to the aquatic environment, and a serious public health threat. V. cholerae serogroup O1 is responsible for the previous two cholera pandemics, in which classical and El Tor biotypes were dominant in the 6th and the current 7th pandemics, respectively. Cholera researchers continually face newly emerging and re-emerging pathogenic clones carrying combinations of new serogroups as well as of phenotypic and genotypic properties. These genotype and phenotype changes have hampered control of the disease. Here we compare the complete genome sequences of 23 strains of V. cholerae isolated from a variety of sources and geographical locations over the past 98 years in an effort to elucidate the evolutionary mechanisms governing genetic diversity and genesis of new pathogenic clones. The genome-based phylogeny revealed 12 distinct V. cholerae phyletic lineages, of which one, designated the V. cholerae core genome (CG), comprises both O1 classical and EI Tor biotypes. All 7th pandemic clones share nearly identical gene content, i.e., the same genome backbone. The transition from 6th to 7th pandemic strains is defined here as a 'shift' between pathogenic clones belonging to the same O1 serogroup, but from significantly different phyletic lineages within the CG clade. In contrast, transition among clones during the present 7th pandemic period can be characterized as a 'drift' between clones, differentiated mainly by varying composition of laterally transferred genomic islands, resulting in emergence of variants, exemplified by V.cholerae serogroup O139 and V.cholerae O1 El Tor hybrid clones that produce cholera toxin of classical biotype. Based on the comprehensive comparative genomics presented in this study it is concluded that V. cholerae undergoes extensive genetic recombination via lateral gene transfer, and, therefore, genome assortment, not serogroup, should be used to

  15. What is cholera?

    DEFF Research Database (Denmark)

    Tamason, Charlotte Crim; Tulsiani, Suhella; Siddique, A.;

    2016-01-01

    a third ofthe respondents did not associate diarrhea with cholera or mentioned symptoms that could not be caused by cholera (29%). Approximately half of the respondents associated water with the cause of cholera (56%) and only 8% associated cholera with sanitation or hygiene. Shame and stigma (54%) were...

  16. Modulation of the humoral and cellular immune response in Abeta immunotherapy by the adjuvants monophosphoryl lipid A (MPL), cholera toxin B subunit (CTB) and E. coli enterotoxin LT(R192G).

    Science.gov (United States)

    Maier, Marcel; Seabrook, Timothy J; Lemere, Cynthia A

    2005-10-25

    Abeta vaccination or passive transfer of human-specific anti-Abeta antibodies are approaches under investigation to prevent and/or treat Alzheimer's disease (AD). Successful active Abeta vaccination requires a strong and safe adjuvant to induce anti-Abeta antibody formation. We compared the adjuvants monophosphoryl lipid A (MPL)/trehalose dicorynomycolate (TDM), cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin LT(R192G) for their ability to induce a humoral and cellular immune reaction, using fibrillar Abeta1-40/42 as a common immunogen in wildtype B6D2F1 mice. Subcutaneous (s.c.) administration with MPL/TDM resulted in anti-Abeta antibodies levels up to four times higher compared to s.c. LT(R192G). Using MPL/TDM, the anti-Abeta antibodies induced were mainly IgG2b, IgG1 and lower levels of IgG2a and IgM, with a moderate splenocyte proliferation and IFN-gamma production in vitro upon stimulation with Abeta1-40/42. LT(R192G), previously shown by us to induce robust titers of anti-Abeta antibodies, generated predominantly IgG2b and IgG1 anti-Abeta antibodies with very low splenocyte proliferation and IFN-gamma production. Weekly intranasal (i.n.) administration over 11 weeks of Abeta40/42 with CTB induced only moderate levels of antibodies. All immunogens generated antibodies that recognized mainly the Abeta1-7 epitope and specifically detected amyloid plaques on AD brain sections. In conclusion, MPL/TDM, in addition to LT(R192G), is an effective adjuvant when combined with Abeta40/42 and may aid in the design of Abeta immunotherapy.

  17. Release from Th1-type immune tolerance in spleen and enhanced production of IL-5 in Peyer's patch by cholera toxin B induce the glomerular deposition of IgA.

    Science.gov (United States)

    Yamanaka, Takahiro; Tamauchi, Hidekazu; Suzuki, Yusuke; Suzuki, Hitoshi; Horikoshi, Satoshi; Terashima, Masazumi; Iwabuchi, Kazuya; Habu, Sonoko; Okumura, Ko; Tomino, Yasuhiko

    2016-04-01

    We examined the pathogenesis of glomerular damage in Th2 type-dependent GATA-3 transgenic (GATA-3 Tg) mice with IgA nephropathy (IgAN). GATA-3 Tg mice were immunized orally using OVA plus cholera toxin B (CTB), and measurement of the serum IgA antibody level and histopathological examination were performed. Marked increases in the serum levels of OVA-specific IgA antibody, IgA and IgG, C3 deposits analogous to those seen in IgAN, and expansion of the matrix in association with mesangial cell proliferation were observed. Furthermore, glomerular IgA deposits were co-localized with mannan-binding lectin (MBL) deposits, which might actually have been abnormal IgA deposits. In GATA-3/TCR-Tg mice that had been orally sensitized with CTB plus OVA and were re-stimulated with OVA in vitro, cultured Peyer's patch cells showed the enhanced production of IL-5 and supernatants from cultures of spleen cells showed a reduction of TGF-β production with a simultaneous increase in IL-2 production and the recovery of IFN-γ formation. The amount of TGF-β produced by the spleen cells was found to be correlated with the amount of IFN-γ and IL-IL-2 produced by the cells. Also, the percentage of regulatory T cells (Treg) in the spleens of mice sensitized with OVA plus CTB was lower than that in mice orally sensitized with OVA alone. These results suggest that the increased production of IL-5 from Peyer's patch cells (PPc) and the restored Th1-type immune response might cause the production of abnormal IgA and might induce the deposition of IgA in glomeruli.

  18. Accessory cholera enterotoxin, Ace, from Vibrio cholerae: structure, unfolding, and virstatin binding.

    Science.gov (United States)

    Chatterjee, Tanaya; Mukherjee, Debadrita; Dey, Sucharita; Pal, Aritrika; Hoque, Kazi Mirajul; Chakrabarti, Pinak

    2011-04-12

    Vibrio cholerae accessory cholera enterotoxin (Ace) is the third toxin, along with cholera toxin (CT) and zonula occludens toxin (Zot), that causes the endemic disease cholera. Structural characterization of Ace has been restricted because of the limited production of this toxic protein by V. cholerae. We have cloned, overexpressed, and purified Ace from V. cholerae strain O395 in Escherichia coli to homogeneity and determined its biological activity. The unfolding of the purified protein was investigated using circular dichroism and intrinsic tryptophan fluorescence. Because Ace is predominantly a hydrophobic protein, the degree of exposure of hydrophobic regions was identified from the spectral changes of the environment-sensitive fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) that quenches the fluorescence of tryptophan residues of Ace in a concentration-dependent manner. Results showed that bis-ANS binds one monomeric unit of Ace with a 1:1 stoichiometry and a K' of 0.72 μM. Ace exists as a dimer, with higher oligomeric forms appearing upon glutaraldehyde cross-linking. This study also reports the binding of virstatin, a small molecule that inhibits virulence regulation in V. cholerae, to Ace. The binding constant (K=9×10(4) M(-1)) and the standard free energy change (ΔG°=-12 kcal mol(-1)) of Ace-virstatin interaction have been evaluated by the fluorescence quenching method. The binding does not affect the oligomeric status of Ace. A cell viability assay of the antibacterial activity of Ace has been performed using various microbial strains. A homology model of Ace, consistent with the experimental results, has been constructed.

  19. Phenotypic and Genetic Heterogeneity in Vibrio cholerae O139 Isolated from Cholera Cases in Delhi, India during 2001-2006

    Directory of Open Access Journals (Sweden)

    Raikamal Ghosh

    2016-08-01

    Full Text Available Incidence of epidemic Vibrio cholerae serogroup O139 has declined in cholera endemic countries. However, sporadic cholera caused by V. cholerae O139 with notable genetic changes is still reported from many regions. In the present study, 42 V. cholerae O139 strains isolated from 2001 to 2006 in Delhi, India, were retrospectively analyzed to understand their phenotype and molecular characteristics. The majority of isolates were resistant to ampicillin, furazolidone and nalidixic acid. Though the integrative conjugative element was detected in all the O139 isolates, the 2004-2006 isolates remained susceptible to co-trimoxazole, chloramphenicol, and streptomycin. Cholera toxin genotype 1 was present in the majority of the O139 isolates while few had type 3 or a novel type 4. In the cholera toxin encoding gene (ctx restriction fragment length polymorphism, the majority of the isolates harbored three copies of CTX element, of which one was truncated. In this study, the ctx was detected for the first time in the small chromosome of V. cholerae O139 and one isolate harbored 5 copies of CTX element, of which 3 were truncated. The ribotype BII pattern was found in most of the O139 isolates. Three V. cholerae O139 isolated in 2001 had a new ribotype BVIII. Pulsed-field gel electrophoresis analysis revealed clonal variation in 2001 isolates compared to the 2004-2006 isolates. Molecular changes in V. cholerae O139 have to be closely monitored as this information may help in understanding the changing genetic features of this pathogen in relation to the epidemiology of cholera.

  20. Phenotypic and Genetic Heterogeneity in Vibrio cholerae O139 Isolated from Cholera Cases in Delhi, India during 2001-2006.

    Science.gov (United States)

    Ghosh, Raikamal; Sharma, Naresh C; Halder, Kalpataru; Bhadra, Rupak K; Chowdhury, Goutam; Pazhani, Gururaja P; Shinoda, Sumio; Mukhopadhyay, Asish K; Nair, G Balakrish; Ramamurthy, Thadavarayan

    2016-01-01

    Incidence of epidemic Vibrio cholerae serogroup O139 has declined in cholera endemic countries. However, sporadic cholera caused by V. cholerae O139 with notable genetic changes is still reported from many regions. In the present study, 42 V. cholerae O139 strains isolated from 2001 to 2006 in Delhi, India, were retrospectively analyzed to understand their phenotype and molecular characteristics. The majority of isolates were resistant to ampicillin, furazolidone and nalidixic acid. Though the integrative conjugative element was detected in all the O139 isolates, the 2004-2006 isolates remained susceptible to co-trimoxazole, chloramphenicol, and streptomycin. Cholera toxin genotype 1 was present in the majority of the O139 isolates while few had type 3 or a novel type 4. In the cholera toxin encoding gene (ctx) restriction fragment length polymorphism, the majority of the isolates harbored three copies of CTX element, of which one was truncated. In this study, the ctx was detected for the first time in the small chromosome of V. cholerae O139 and one isolate harbored 5 copies of CTX element, of which 3 were truncated. The ribotype BII pattern was found in most of the O139 isolates. Three V. cholerae O139 isolated in 2001 had a new ribotype BVIII. Pulsed-field gel electrophoresis analysis revealed clonal variation in 2001 isolates compared to the 2004-2006 isolates. Molecular changes in V. cholerae O139 have to be closely monitored as this information may help in understanding the changing genetic features of this pathogen in relation to the epidemiology of cholera.

  1. Great cormorants (Phalacrocorax carbo) as potential vectors for the dispersal of Vibrio cholerae.

    Science.gov (United States)

    Laviad-Shitrit, Sivan; Lev-Ari, Tidhar; Katzir, Gadi; Sharaby, Yehonatan; Izhaki, Ido; Halpern, Malka

    2017-08-11

    Vibrio cholerae is the cause of cholera, a devastating epidemic and pandemic disease. Despite its importance, the way of its global dissemination is unknown. V. cholerae is abundant in aquatic habitats and is known to be borne by copepods, chironomids and fishes. Our aim was to determine if fish-eating birds act as vectors in the spread of V. cholerae by consuming infected fish. We determined the existence of V. cholerae in the microbiome of 5/7 wild cormorants' intestine. In three of these V. cholerae-positive wild cormorants, the presence of a gene for cholera toxin (ctxA) was detected. We subsequently tested eight captive, hand-reared cormorants, divided into two equal groups. Prior to the experiment, the feces of the cormorants were V. cholerae-negative. One group was fed exclusively on tilapias, which are naturally infected with V. cholerae, and the other was fed exclusively on goldfish or on koi that were V. cholerae-negative. We detected V. cholerae in the feces of the tilapia-fed, but not in the goldfish/koi-fed, cormorants. Hence, we demonstrate that fish-eating birds can be infected with V. cholerae from their fish prey. The large-scale movements of many fish-eating birds provide a potential mechanism for the global distribution of V. cholerae.

  2. Cholera outbreaks (2012) in three districts of Nepal reveal clonal transmission of multi-drug resistant Vibrio cholerae O1.

    Science.gov (United States)

    Dixit, Sameer M; Johura, Fatema-Tuz; Manandhar, Sulochana; Sadique, Abdus; Rajbhandari, Rajesh M; Mannan, Shahnewaj B; Rashid, Mahamud-Ur; Islam, Saiful; Karmacharya, Dibesh; Watanabe, Haruo; Sack, R Bradley; Cravioto, Alejandro; Alam, Munirul

    2014-07-15

    Although endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission of Vibrio cholerae associated with 2012 cholera outbreaks in Nepal. V. cholerae (n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE). All V. cholerae strains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed the V. cholerae strains to possess the B-7 allele of ctx subunit B. DNA sequencing of tcpA revealed a point mutation at amino acid position 64 (N → S) while the ctxAB promoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'. V. cholerae possessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498-511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence of gyrA and parC of the NAR strains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the Nepalese V. cholerae to be clonal, and related closely with V. cholerae associated with cholera in

  3. Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1.

    Science.gov (United States)

    Kenner, J R; Coster, T S; Taylor, D N; Trofa, A F; Barrera-Oro, M; Hyman, T; Adams, J M; Beattie, D T; Killeen, K P; Spriggs, D R

    1995-10-01

    Cholera vaccine candidate Peru-15 was derived from a Vibrio cholerae O1 El Tor Inaba strain by deleting the cholera toxin genetic element, introducing the gene encoding cholera toxin B subunit into recA, and screening for nonmotility. In a controlled study, Peru-15 (2 x 10(8) cfu) was administered to 11 volunteers. No vaccinee developed diarrhea, and 10 of 11 had > 4-fold rises in vibriocidal antibody titers. One month later, 5 vaccinees and 5 control volunteers were challenged with wild type V. cholerae O1. Four of 5 controls developed diarrhea (mean, 1.9 L). Two Peru-15 vaccinees developed diarrhea, 1 with volunteer had not developed a significant vibriocidal immune response to vaccination. Peru-15 shows promise as a single-dose, oral cholera vaccine that is safe, immunogenic, and protective.

  4. The Vibrio cholerae type VI secretion system employs diverse effector modules for intraspecific competition.

    Science.gov (United States)

    Unterweger, Daniel; Miyata, Sarah T; Bachmann, Verena; Brooks, Teresa M; Mullins, Travis; Kostiuk, Benjamin; Provenzano, Daniele; Pukatzki, Stefan

    2014-04-01

    Vibrio cholerae is a Gram-negative bacterial pathogen that consists of over 200 serogroups with differing pathogenic potential. Only strains that express the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) are capable of pandemic spread of cholera diarrhoea. Regardless, all V. cholerae strains sequenced to date harbour genes for the type VI secretion system (T6SS) that translocates effectors into neighbouring eukaryotic and prokaryotic cells. Here we report that the effectors encoded within these conserved gene clusters differ widely among V. cholerae strains, and that immunity proteins encoded immediately downstream from the effector genes protect their host from neighbouring bacteria producing corresponding effectors. As a consequence, strains with matching effector-immunity gene sets can coexist, while strains with different sets compete against each other. Thus, the V. cholerae T6SS contributes to the competitive behaviour of this species.

  5. High-Resolution Crystal Structures Elucidate the Molecular Basis of Cholera Blood Group Dependence.

    Science.gov (United States)

    Heggelund, Julie Elisabeth; Burschowsky, Daniel; Bjørnestad, Victoria Ariel; Hodnik, Vesna; Anderluh, Gregor; Krengel, Ute

    2016-04-01

    Cholera is the prime example of blood-group-dependent diseases, with individuals of blood group O experiencing the most severe symptoms. The cholera toxin is the main suspect to cause this relationship. We report the high-resolution crystal structures (1.1-1.6 Å) of the native cholera toxin B-pentamer for both classical and El Tor biotypes, in complexes with relevant blood group determinants and a fragment of its primary receptor, the GM1 ganglioside. The blood group A determinant binds in the opposite orientation compared to previously published structures of the cholera toxin, whereas the blood group H determinant, characteristic of blood group O, binds in both orientations. H-determinants bind with higher affinity than A-determinants, as shown by surface plasmon resonance. Together, these findings suggest why blood group O is a risk factor for severe cholera.

  6. Ca2+ influx and tyrosine kinases trigger Bordetella adenylate cyclase toxin (ACT endocytosis. Cell physiology and expression of the CD11b/CD18 integrin major determinants of the entry route.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3, its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca(2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

  7. Cholera: something old, something new.

    Science.gov (United States)

    Sigman, Michael; Luchette, Fred A

    2012-08-01

    In the aftermath of a devastating earthquake in early 2011, Haiti fell victim to an outbreak of cholera that claimed thousands of lives and affected populations in nearby Dominican Republic, Venezuela, and even the United States. This was the first time cholera had been reported in Haiti in more than 100 years. The sudden appearance of cholera, a pathogen with no known non-human host, raised the question of how it was introduced to an island that has long been spared this disease. The purpose of this review is to provide an overview of the history of cholera, its pathophysiology and virulence factors, and current recommendations for treatment. Articles published in the past 10 years were identified by a search of the medical literature using PUBMED and reviewed. Bibliographies of each article also were reviewed for additional pertinent articles. The recent epidemic was caused by a strain that has been responsible for disease in South Asia since 1961, the seventh and most recent strain identified since 1900. It is transmitted by the fecal-oral route. Once infected, the patient develops a rapidly dehydrating diarrheal illness caused by the cholera toxin, which activates cytoplasmic adenylate cyclase of the intestinal epithelial cells by adenosine diphosphate (ADP)-ribosylation of the stimulatory G protein. The high cyclic adenosine monophosphate (cAMP) concentrations activate the cystic fibrosis transmembrane conductance regulator, causing a dramatic efflux of ions and water from infected enterocytes and leading to watery diarrhea. The first line of therapy is oral hydration with intravenous fluids; antibiotics are reserved for patients with severe dehydration. Spread of cholera is preventable with simple modifications of hygiene and water preparation. Cholera has re-emerged as a major infectious disease in the recent past, with a global increase in its incidence. Vaccination should be considered as an adjunct for controlling the epidemics and also for volunteer

  8. Bacterial toxins as immunomodulators.

    Science.gov (United States)

    Donaldson, David S; Williams, Neil A

    2009-01-01

    Bacterial toxins are the causative agent at pathology in a variety of diseases. Although not always the primary target of these toxins, many have been shown to have potent immunomodulatory effects, for example, inducing immune responses to co-administered antigens and suppressing activation of immune cells. These abilities of bacterial toxins can be harnessed and used in a therapeutic manner, such as in vaccination or the treatment of autoimmune diseases. Furthermore, the ability of toxins to gain entry to cells can be used in novel bacterial toxin based immuno-therapies in order to deliver antigens into MHC Class I processing pathways. Whether the immunomodulatory properties of these toxins arose in order to enhance bacterial survival within hosts, to aid spread within the population or is pure serendipity, it is interesting to think that these same toxins potentially hold the key to preventing or treating human disease.

  9. Vibrio cholerae Colonization of Soft-Shelled Turtles.

    Science.gov (United States)

    Wang, Jiazheng; Yan, Meiying; Gao, He; Lu, Xin; Kan, Biao

    2017-07-15

    Vibrio cholerae is an important human pathogen and environmental microflora species that can both propagate in the human intestine and proliferate in zooplankton and aquatic organisms. Cholera is transmitted through food and water. In recent years, outbreaks caused by V. cholerae-contaminated soft-shelled turtles, contaminated mainly with toxigenic serogroup O139, have been frequently reported, posing a new foodborne disease public health problem. In this study, the colonization by toxigenic V. cholerae on the body surfaces and intestines of soft-shelled turtles was explored. Preferred colonization sites on the turtle body surfaces, mainly the carapace and calipash of the dorsal side, were observed for the O139 and O1 strains. Intestinal colonization was also found. The colonization factors of V. cholerae played different roles in the colonization of the soft-shelled turtle's body surface and intestine. Mannose-sensitive hemagglutinin (MSHA) of V. cholerae was necessary for body surface colonization, but no roles were found for toxin-coregulated pili (TCP) or N-acetylglucosamine-binding protein A (GBPA). Both TCP and GBPA play important roles for colonization in the intestine, whereas the deletion of MSHA revealed only a minor colonization-promoting role for this factor. Our study demonstrated that V. cholerae can colonize the surfaces and the intestines of soft-shelled turtles and indicated that the soft-shelled turtles played a role in the transmission of cholera. In addition, this study showed that the soft-shelled turtle has potential value as an animal model in studies of the colonization and environmental adaption mechanisms of V. cholerae in aquatic organisms.IMPORTANCE Cholera is transmitted through water and food. Soft-shelled turtles contaminated with Vibrio cholerae (commonly the serogroup O139 strains) have caused many foodborne infections and outbreaks in recent years, and they have become a foodborne disease problem. Except for epidemiological

  10. Treatment of cholera-like diarrhoea with oral rehydration.

    Science.gov (United States)

    Atia, A; Buchman, A L

    2010-09-01

    Cholera diarrhoea remains a major global health problem that has caused seven pandemics. The pathogenesis of cholera is attributable to the production of cholera toxin by the causative pathogen, Vibrio cholerae. The toxin causes increased production of cyclic adenosine monophosphate and this results in massive water and electrolyte secretion into the intestinal lumen. These changes manifest clinically as the painless defecation of voluminous stools that resemble 'rice water', leading to severe dehydration. The cornerstone in the management of cholera diarrhoea is the use of oral rehydration solutions (ORS) to replace the water and electrolytes lost as stools. The World Health Organization recommends the use of ORS of 'reduced osmolarity' for the treatment of acute non-cholera diarrhoea and the use of rice-based ORS for the management of cholera diarrhoea. Although several attempts have been made to improve ORS, studies to evaluate some of the modifications, which include the addition of amylase-resistant starch, the use of amino acids (such as glycine, alanine and glutamine) as sodium cotransporters, and zinc-supplemented ORS, are still needed.

  11. Whole-genome sequence comparisons reveal the evolution of Vibrio cholerae O1.

    Science.gov (United States)

    Kim, Eun Jin; Lee, Chan Hee; Nair, G Balakrish; Kim, Dong Wook

    2015-08-01

    The analysis of the whole-genome sequences of Vibrio cholerae strains from previous and current cholera pandemics has demonstrated that genomic changes and alterations in phage CTX (particularly in the gene encoding the B subunit of cholera toxin) were major features in the evolution of V. cholerae. Recent studies have revealed the genetic mechanisms in these bacteria by which new variants of V. cholerae are generated from type-specific strains; these mechanisms suggest that certain strains are selected by environmental or human factors over time. By understanding the mechanisms and driving forces of historical and current changes in the V. cholerae population, it would be possible to predict the direction of such changes and the evolution of new variants; this has implications for the battle against cholera. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Environmental surveillance for toxigenic Vibrio cholerae in surface waters of Haiti.

    Science.gov (United States)

    Kahler, Amy M; Haley, Bradd J; Chen, Arlene; Mull, Bonnie J; Tarr, Cheryl L; Turnsek, Maryann; Katz, Lee S; Humphrys, Michael S; Derado, Gordana; Freeman, Nicole; Boncy, Jacques; Colwell, Rita R; Huq, Anwar; Hill, Vincent R

    2015-01-01

    Epidemic cholera was reported in Haiti in 2010, with no information available on the occurrence or geographic distribution of toxigenic Vibrio cholerae in Haitian waters. In a series of field visits conducted in Haiti between 2011 and 2013, water and plankton samples were collected at 19 sites. Vibrio cholerae was detected using culture, polymerase chain reaction, and direct viable count methods (DFA-DVC). Cholera toxin genes were detected by polymerase chain reaction in broth enrichments of samples collected in all visits except March 2012. Toxigenic V. cholerae was isolated from river water in 2011 and 2013. Whole genome sequencing revealed that these isolates were a match to the outbreak strain. The DFA-DVC tests were positive for V. cholerae O1 in plankton samples collected from multiple sites. Results of this survey show that toxigenic V. cholerae could be recovered from surface waters in Haiti more than 2 years after the onset of the epidemic.

  13. Environmental Surveillance for Toxigenic Vibrio cholerae in Surface Waters of Haiti

    Science.gov (United States)

    Kahler, Amy M.; Haley, Bradd J.; Chen, Arlene; Mull, Bonnie J.; Tarr, Cheryl L.; Turnsek, Maryann; Katz, Lee S.; Humphrys, Michael S.; Derado, Gordana; Freeman, Nicole; Boncy, Jacques; Colwell, Rita R.; Huq, Anwar; Hill, Vincent R.

    2015-01-01

    Epidemic cholera was reported in Haiti in 2010, with no information available on the occurrence or geographic distribution of toxigenic Vibrio cholerae in Haitian waters. In a series of field visits conducted in Haiti between 2011 and 2013, water and plankton samples were collected at 19 sites. Vibrio cholerae was detected using culture, polymerase chain reaction, and direct viable count methods (DFA-DVC). Cholera toxin genes were detected by polymerase chain reaction in broth enrichments of samples collected in all visits except March 2012. Toxigenic V. cholerae was isolated from river water in 2011 and 2013. Whole genome sequencing revealed that these isolates were a match to the outbreak strain. The DFA-DVC tests were positive for V. cholerae O1 in plankton samples collected from multiple sites. Results of this survey show that toxigenic V. cholerae could be recovered from surface waters in Haiti more than 2 years after the onset of the epidemic. PMID:25385860

  14. Cholera in pregnancy: Clinical and immunological aspects.

    Science.gov (United States)

    Khan, Ashraful I; Chowdhury, Fahima; Leung, Daniel T; Larocque, Regina C; Harris, Jason B; Ryan, Edward T; Calderwood, Stephen B; Qadri, Firdausi

    2015-10-01

    The objective of this study was to examine the clinical and immunological features of cholera in pregnancy. Women of reproductive age presenting to the icddr,b Dhaka hospital with cholera, and enrolled as part of a larger cohort study, were tested for pregnancy on admission. We compared initial clinical features and immune responses of pregnant patients with non-pregnant female patients at days 2, 7 and 21 after infection. Among reproductive age women enrolled between January 2001 and May 2006, 9.7% (14/144) were pregnant. The duration of diarrhoea prior to admission tended to be higher in pregnant compared to non-pregnant patients (p=0.08), but other clinical characteristics did not differ. Antibody responses to cholera toxin B subunit (CtxB), toxin-coregulated pilus A (TcpA), Vibrio cholerae lipopolysaccharide (LPS), and serum vibriocidal antibody responses, were comparable between pregnant and non-pregnant patients. There were no deaths among the pregnant cases or non-pregnant controls, and no adverse foetal outcomes, including stillbirths, during 21 days of follow up of pregnant cases. To our knowledge, this is the first report of immune responses in pregnant women with cholera. We found that pregnant woman early in pregnancy has comparable clinical illness and subsequent immune responses compared to non-pregnant women. These findings suggest that the evaluation of safety and immunogenicity of oral cholera vaccines in pregnancy should be an area of future investigations. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. 霍乱毒素A亚单位基因的原核表达及单克隆抗体的制备%Prokaryotic expression of cholera toxin subunit A gene and preparation of monoclonal antibody

    Institute of Scientific and Technical Information of China (English)

    陈健; 韦雨杏; 解晓燕; 朱召芹; 万延民; 徐建青

    2014-01-01

    目的 原核表达并纯化霍乱毒素A亚单位(cholera toxin subunitA,CTA)基因,建立分泌CTA单克隆抗体的杂交瘤细胞系,并制备单克隆抗体.方法 构建原核表达质粒pET30a-CTA,转化大肠埃希菌(E.coli)BL21(DE3),IPTG诱导表达6h,表达产物经12% SDS-PAGE分析后,用Ni2+亲和层析柱纯化;将纯化的重组CTA蛋白免疫C57BL/6小鼠,运用杂交瘤细胞技术制备抗CTA的单抗,采用Western blot法及体外阻断实验检测抗CTA单抗的特异性及对CT毒性作用的体外阻断.结果 原核表达质粒pET30a-CTA经PCR及双酶切鉴定,证明构建正确;表达的重组CTA蛋白在相对分子质量25 000 ~ 35 000之间可见明显的目的蛋白条带,纯化后的重组CTA蛋白纯度达90%以上;获得1株可分泌抗CTA单抗的杂交瘤细胞株G6C3-D10,可与CTA蛋白特异性结合,在相对分子质量25 000 ~ 35 000之间可见明显的特异条带;杂交瘤培养上清中CTA特异性抗体滴度为54,与CT混合后,可明显抑制CT所诱导的细胞内环腺苷酸(cyclic adenosine monophosphate,cAMP)水平的升高(P<0.01).结论 原核表达并纯化了CTA基因,获得了能够分泌具有阻断CT毒性作用的单抗的细胞株,为CTA毒性作用和佐剂机制的进一步研究提供了参考.

  16. Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC from V. cholerae strains.

    Directory of Open Access Journals (Sweden)

    Sridhar Elluri

    Full Text Available Outer membrane vesicles (OMVs released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC, is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and

  17. A Natural Vaccine Candidate Strain Against Cholera

    Institute of Scientific and Technical Information of China (English)

    LIUYAN-QING; QIGUO-MING; 等

    1995-01-01

    El Tor Vibrio cholerae(EVC)strains may be classified into two kinds-epidemigenic(EEVC)strains and non-epidemigenic(NEEVC)strains-based on a phage-biotyping system.A large number of EEVC strains have been screened for toxigenic and putative colonization attributes.One such naturally occurring strain(designated IEM101)has been found which is devoid of genes encoding cholera toxin(CT),accessory cholera enterotoxin(ACE),zonula occludens toxin(ZOT),but possesses RS1 sequences and toixn-coregulated pilus A gene(tcpA)although tcpA is poorly expressed.It expresses type B pili but does not posses type C pili.It is an El Tor Ogawa strain and does not cause fluid accumulation in rabbit ileal loop tests.Active immunization of rabbits with strain IEM101 elicited good protection against challenge with virulent strains of V.cholerae Ol.Oral administration cased no side effects in 15 human volunteers.colonized the gut for four to ten days and elicited good immune responses.

  18. Cholera Prevention and Control

    Science.gov (United States)

    ... name="commit" type="submit" value="Submit" /> Prevention & Control Recommend on Facebook Tweet Share Compartir Prevention of ... of cholera and other diarrheal disease prevention. Prevention Control Topics Six Basic Cholera Prevention Messages I nfection ...

  19. What is cholera?

    DEFF Research Database (Denmark)

    Tamason, Charlotte Crim; Tulsiani, Suhella; Siddique, A.;

    2016-01-01

    Background: Cholera has afflicted the Indian sub-continent for centuries, predominantly in West Bengal and modern-day Bangladesh. This preliminary study aims to understand the current level of knowledge of cholera in female Bangladeshi caretakers, which is important in the outcome of the disease...... and its spread. A pilot study was conducted among 85 women in Bangladesh using qualitative questionnaires to explore the ability of female caretakers in identifying cholera and its transmission. Findings: The survey revealed that though all the female caretakers were aware of the term “cholera,” nearly...... a third ofthe respondents did not associate diarrhea with cholera or mentioned symptoms that could not be caused by cholera (29%). Approximately half of the respondents associated water with the cause of cholera (56%) and only 8% associated cholera with sanitation or hygiene. Shame and stigma (54%) were...

  20. Molecular characterization of the circulating strains of Vibrio cholerae during 2010 cholera outbreak in Nigeria.

    Science.gov (United States)

    Oyedeji, Kolawole S; Niemogha, Mary-Theresa; Nwaokorie, Francisca O; Bamidele, Tajudeen A; Ochoga, Michael; Akinsinde, Kehinde A; Brai, Bartholomew I; Oladele, David; Omonigbehin, Emmanuel A; Bamidele, Moses; Fesobi, Toun W; Musa, Adesola Z; Adeneye, Adeniyi K; Smith, Stella I; Ujah, Innocent A

    2013-06-01

    This study aimed at characterizing the phenotypic and toxigenic status of circulating strains of cholera during outbreaks in Nigeria, employing molecular typing techniques. Two hundred and one samples of rectal swabs, stool, vomitus, water (from the well, borehole, sachet, stream, and tap) and disinfectants (sodium hypochlorite) were collected from three states in the country. The samples were inoculated on thiosulphate-citrate bile salt-sucrose (TCBS), Cary-Blair transport medium and smeared on glass slides for direct examination. The Vibrio cholerae isolates were serotyped, biotyped, and characterized using PCR of the cytotoxin gene A (ctxA), wbeO1, and wbfO139 gene primer. Of the 201 samples screened, 96 were positive for V cholerae O1 (48%), with 69 (72%) positive for ctxA gene. The results from this study showed that the circulating strains of cholera in Nigeria were of Ogawa serotype, also observed in other outbreaks in Nigeria (1991, 1992, and 1996). However, the strains were of the Classical biotype and were mainly (72%) ctxA gene-positive. This current investigation has confirmed the production of cholera toxin by the circulating strains, and this could be harnessed for possible cholera vaccine production in Nigeria.

  1. Malonate inhibits virulence gene expression in Vibrio cholerae.

    Science.gov (United States)

    Minato, Yusuke; Fassio, Sara R; Häse, Claudia C

    2013-01-01

    We previously found that inhibition of the TCA cycle, either through mutations or chemical inhibition, increased toxT transcription in Vibrio cholerae. In this study, we found that the addition of malonate, an inhibitor of succinate dehydrogenase (SDH), decreased toxT transcription in V. cholerae, an observation inconsistent with the previous pattern observed. Unlike another SDH inhibitor, 2-thenoyltrifluoroacetone (TTFA), which increased toxT transcription and slightly inhibited V. cholerae growth, malonate inhibited toxT transcription in both the wild-type strain and TCA cycle mutants, suggesting malonate-mediated inhibition of virulence gene expression is independent to TCA cycle activity. Addition of malonate also inhibited ctxB and tcpA expressions but did not affect aphA, aphB, tcpP and toxR expressions. Malonate inhibited cholera toxin (CT) production in both V. cholerae classical biotype strains O395N1 and CA401, and El Tor biotype strain, N16961. Consistent with previous reports, we confirmed that these strains of V. cholerae did not utilize malonate as a primary carbon source. However, we found that the addition of malonate to the growth medium stimulated V. cholerae growth. All together, these results suggest that metabolizing malonate as a nutrient source negatively affects virulence gene expression in V. cholerae.

  2. Molecular insights into the evolutionary pathway of Vibrio cholerae O1 atypical El Tor variants.

    Science.gov (United States)

    Kim, Eun Jin; Lee, Dokyung; Moon, Se Hoon; Lee, Chan Hee; Kim, Sang Jun; Lee, Jae Hyun; Kim, Jae Ouk; Song, Manki; Das, Bhabatosh; Clemens, John D; Pape, Jean William; Nair, G Balakrish; Kim, Dong Wook

    2014-09-01

    Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor.

  3. Cholera in Zimbabwe

    NARCIS (Netherlands)

    Pruyt, E.

    2009-01-01

    By the end of December 2008, alarming reports and articles concerning the cholera outbreak in Zimbabwe received plenty of international media coverage. By that time nearly 30000 cases of cholera infections and 1600 cholera deaths had been reported. In the first week of January 2009, a System Dynamic

  4. Cholera in Zimbabwe

    NARCIS (Netherlands)

    Pruyt, E.

    2009-01-01

    By the end of December 2008, alarming reports and articles concerning the cholera outbreak in Zimbabwe received plenty of international media coverage. By that time nearly 30000 cases of cholera infections and 1600 cholera deaths had been reported. In the first week of January 2009, a System

  5. Cholera in Zimbabwe

    NARCIS (Netherlands)

    Pruyt, E.

    2009-01-01

    By the end of December 2008, alarming reports and articles concerning the cholera outbreak in Zimbabwe received plenty of international media coverage. By that time nearly 30000 cases of cholera infections and 1600 cholera deaths had been reported. In the first week of January 2009, a System Dynamic

  6. Survival and proliferation of the lysogenic bacteriophage CTXΦ in Vibrio cholerae

    Institute of Scientific and Technical Information of China (English)

    Fenxia; Fan; Biao; Kan

    2015-01-01

    The lysogenic phage CTXΦ of Vibrio cholerae can transfer the cholera toxin gene both horizontally(inter-strain) and vertically(cell proliferation). Due to its diversity in form and species, the complexity of regulatory mechanisms, and the important role of the infection mechanism in the production of new virulent strains of V.cholerae, the study of the lysogenic phage CTXΦ has attracted much attention. Based on the progress of current research, the genomic features and their arrangement, the host-dependent regulatory mechanisms of CTXΦ phage survival, proliferation and propagation were reviewed to further understand the phage’s role in the evolutionary and epidemiological mechanisms of V. cholerae.

  7. Detection of ctx gene positive non-O1/non-O139 V. cholerae in shrimp aquaculture environments.

    Science.gov (United States)

    Madhusudana, Rao B; Surendran, P K

    2013-06-01

    Water and post-larvae samples from black tiger (Penaeus monodon) shrimp hatcheries; pond water, pond sediment and shrimp from aquaculture farms were screened for the presence of V. cholerae. A V. cholerae-duplex PCR method was developed by utilizing V. cholerae species specific sodB primers and ctxAB genes specific primers. Incidence of V. cholerae was not observed in shrimp hatchery samples but was noticed in aquaculture samples. The incidence of V. cholerae was higher in pond water (7.6%) than in pond sediment (5.2%). Shrimp head (3.6%) portion had relatively higher incidence than shrimp muscle (1.6%). All the V. cholerae isolates (n = 42) belonged to non-O1/non-O139 serogroup, of which 7% of the V. cholerae isolates were potentially cholera-toxigenic (ctx positive). All the ctx positive V. cholerae (n = 3) were isolated from the pond water. Since, cholera toxin (CT) is the major contributing factor for cholera gravis, it is proposed that the mere presence of non-O1/non-O139 V. cholerae need not be the biohazard criterion in cultured black tiger shrimp but only the presence of ctx carrying non-O1/non-O139 V. cholerae may be considered as potential public health risk.

  8. A Bistable Switch and Anatomical Site Control Vibrio cholerae Virulence Gene Expression in the Intestine

    DEFF Research Database (Denmark)

    Nielsen, Alex Toftgaard; Dolganov, N. A.; Rasmussen, Thomas

    2010-01-01

    A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene...... expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co...... into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients....

  9. Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes

    Energy Technology Data Exchange (ETDEWEB)

    Lowden, Michael J.; Skorupski, Karen; Pellegrini, Maria; Chiorazzo, Michael G.; Taylor, Ronald K.; Kull, F. Jon (Dartmouth)

    2010-03-04

    Cholera is an acute intestinal infection caused by the bacterium Vibrio cholerae. In order for V. cholerae to cause disease, it must produce two virulence factors, the toxin-coregulated pilus (TCP) and cholera toxin (CT), whose expression is controlled by a transcriptional cascade culminating with the expression of the AraC-family regulator, ToxT. We have solved the 1.9 {angstrom} resolution crystal structure of ToxT, which reveals folds in the N- and C-terminal domains that share a number of features in common with AraC, MarA, and Rob as well as the unexpected presence of a buried 16-carbon fatty acid, cis-palmitoleate. The finding that cis-palmitoleic acid reduces TCP and CT expression in V. cholerae and prevents ToxT from binding to DNA in vitro provides a direct link between the host environment of V. cholerae and regulation of virulence gene expression.

  10. Molecular Epidemiology of Cholera Outbreaks during the Rainy Season in Mandalay, Myanmar.

    Science.gov (United States)

    Roobthaisong, Amonrattana; Okada, Kazuhisa; Htun, Nilar; Aung, Wah Wah; Wongboot, Warawan; Kamjumphol, Watcharaporn; Han, Aye Aye; Yi, Yi; Hamada, Shigeyuki

    2017-08-14

    Cholera, caused by Vibrio cholerae, remains a global threat to public health. In Myanmar, the availability of published information on the occurrence of the disease is scarce. We report here that cholera incidence in Mandalay generally exhibited a single annual peak, with an annual average of 312 patients with severe dehydration over the past 5 years (since 2011) and was closely associated with the rainy season. We analyzed cholera outbreaks, characterized 67 isolates of V. cholerae serogroup O1 in 2015 from patients from Mandalay, and compared them with 22 V. cholerae O1 isolates (12 from Mandalay and 10 from Yangon) in 2014. The isolates carried the classical cholera toxin B subunit (ctxB), the toxin-co-regulated pilus A (tcpA) of Haitian type, and repeat sequence transcriptional regulator (rstR) of El Tor type. Two molecular typing methods, pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis (MLVA), differentiated the 89 isolates into seven pulsotypes and 15 MLVA profiles. Pulsotype Y15 and one MLVA profile (11, 7, 7, 16, 7) were predominantly found in the isolates from cholera outbreaks in Mandalay, 2015. Pulsotypes Y11, Y12, and Y15 with some MLVA profiles were detected in the isolates from two remote areas, Mandalay and Yangon, with temporal changes. These data suggested that cholera spread from the seaside to the inland area in Myanmar.

  11. Single-Cell Analysis of the Plasmablast Response to Vibrio cholerae Demonstrates Expansion of Cross-Reactive Memory B Cells

    OpenAIRE

    Kauffman, Robert C.; Bhuiyan, Taufiqur R.; Nakajima, Rie; Mayo-Smith, Leslie M.; Rashu, Rasheduzzaman; Hoq, Mohammad Rubel; Chowdhury, Fahima; Khan, Ashraful Islam; Rahman, Atiqur; Bhaumik, Siddhartha K.; Harris, Levelle; O'Neal, Justin T.; Trost, Jessica F.; Alam, Nur Haq; Jasinskas, Algis

    2016-01-01

    ABSTRACT We characterized the acute B cell response in adults with cholera by analyzing the repertoire, specificity, and functional characteristics of 138 monoclonal antibodies (MAbs) generated from single-cell-sorted plasmablasts. We found that the cholera-induced responses were characterized by high levels of somatic hypermutation and large clonal expansions. A majority of the expansions targeted cholera toxin (CT) or lipopolysaccharide (LPS). Using a novel proteomics approach, we were able...

  12. Drug response and genetic properties of Vibrio cholerae associated with endemic cholera in north-eastern Thailand, 2003-2011.

    Science.gov (United States)

    Chomvarin, Chariya; Johura, Fatema-Tuz; Mannan, Shahnewaj B; Jumroenjit, Warin; Kanoktippornchai, Boonnapa; Tangkanakul, Waraluk; Tantisuwichwong, Napaporn; Huttayananont, Sriwanna; Watanabe, Haruo; Hasan, Nur A; Huq, Anwar; Cravioto, Alejandro; Colwell, Rita R; Alam, Munirul

    2013-04-01

    Cholera, caused by Vibrio cholerae, results in significant morbidity and mortality worldwide, including Thailand. Representative V. cholerae strains associated with endemic cholera (n = 32), including strains (n = 3) from surface water sources, in Khon Kaen, Thailand (2003-2011), were subjected to microbiological, molecular and phylogenetic analyses. According to phenotypic and related genetic data, all tested V. cholerae strains belonged to serogroup O1, biotype El Tor (ET), Inaba (IN) or Ogawa (OG). All of the strains were sensitive to gentamicin and ciprofloxacin, while multidrug-resistant (MDR) strains showing resistance to erythromycin, tetracycline, trimethoprim/sulfamethoxazole and ampicillin were predominant in 2007. V. cholerae strains isolated before and after 2007 were non-MDR. All except six diarrhoeal strains possessed ctxA and ctxB genes and were toxigenic altered ET, confirmed by MAMA-PCR and DNA sequencing. Year-wise data revealed that V. cholerae INET strains isolated between 2003 and 2004, plus one strain isolated in 2007, lacked the RS1 sequence (rstC) and toxin-linked cryptic plasmid (TLC)-specific genetic marker, but possessed CTX(CL) prophage genes ctxB(CL) and rstR(CL). A sharp genetic transition was noted, namely the majority of V. cholerae strains in 2007 and all in 2010 and 2011 were not repressor genotype rstR(CL) but instead were rstR(ET), and all ctx(+) strains possessed RS1 and TLC-specific genetic markers. DNA sequencing data revealed that strains isolated since 2007 had a mutation in the tcpA gene at amino acid position 64 (N→S). Four clonal types, mostly of environmental origin, including subtypes, reflected genetic diversity, while distinct signatures were observed for clonally related, altered ET from Thailand, Vietnam and Bangladesh, confirmed by distinct subclustering patterns observed in the PFGE (NotI)-based dendrogram, suggesting that endemic cholera is caused by V. cholerae indigenous to Khon Kaen.

  13. Drug response and genetic properties of Vibrio cholerae associated with endemic cholera in north-eastern Thailand, 2003–2011

    Science.gov (United States)

    Chomvarin, Chariya; Johura, Fatema-Tuz; Mannan, Shahnewaj B.; Jumroenjit, Warin; Kanoktippornchai, Boonnapa; Tangkanakul, Waraluk; Tantisuwichwong, Napaporn; Huttayananont, Sriwanna; Watanabe, Haruo; Hasan, Nur A.; Huq, Anwar; Cravioto, Alejandro; Colwell, Rita R.

    2013-01-01

    Cholera, caused by Vibrio cholerae, results in significant morbidity and mortality worldwide, including Thailand. Representative V. cholerae strains associated with endemic cholera (n = 32), including strains (n = 3) from surface water sources, in Khon Kaen, Thailand (2003–2011), were subjected to microbiological, molecular and phylogenetic analyses. According to phenotypic and related genetic data, all tested V. cholerae strains belonged to serogroup O1, biotype El Tor (ET), Inaba (IN) or Ogawa (OG). All of the strains were sensitive to gentamicin and ciprofloxacin, while multidrug-resistant (MDR) strains showing resistance to erythromycin, tetracycline, trimethoprim/sulfamethoxazole and ampicillin were predominant in 2007. V. cholerae strains isolated before and after 2007 were non-MDR. All except six diarrhoeal strains possessed ctxA and ctxB genes and were toxigenic altered ET, confirmed by MAMA-PCR and DNA sequencing. Year-wise data revealed that V. cholerae INET strains isolated between 2003 and 2004, plus one strain isolated in 2007, lacked the RS1 sequence (rstC) and toxin-linked cryptic plasmid (TLC)-specific genetic marker, but possessed CTXCL prophage genes ctxBCL and rstRCL. A sharp genetic transition was noted, namely the majority of V. cholerae strains in 2007 and all in 2010 and 2011 were not repressor genotype rstRCL but instead were rstRET, and all ctx+ strains possessed RS1 and TLC-specific genetic markers. DNA sequencing data revealed that strains isolated since 2007 had a mutation in the tcpA gene at amino acid position 64 (N→S). Four clonal types, mostly of environmental origin, including subtypes, reflected genetic diversity, while distinct signatures were observed for clonally related, altered ET from Thailand, Vietnam and Bangladesh, confirmed by distinct subclustering patterns observed in the PFGE (NotI)-based dendrogram, suggesting that endemic cholera is caused by V. cholerae indigenous to Khon Kaen. PMID:23319310

  14. 霍乱毒素B亚基与人胰岛素B链融合蛋白的安全性评价%Safety evaluation of cholera toxin B subunit-insulin B chain fusion protein

    Institute of Scientific and Technical Information of China (English)

    李磊; 潘伟; 陈国灿; 邓军; 陈云祥; 宣尧仙

    2015-01-01

    目的 评价霍乱毒素B亚基与人胰岛素B链融合蛋白(cholera toxin B subunit-insulin B chain,CTB-INS)在恒河猴体内的安全性.方法 将恒河猴随机分为4组,每组10只,雌雄各半,高、中、低剂量组分别经口给予4.0、2.0和1.0 g/(kg·d)CTB-INS,对照组经口给予10 ml/(kg·d)0.5%羧甲基纤维素钠(carboxymethyl cellulose sodium,CMC-Na),每天上午同一时间给药1次,每周给药6d,连续给药9.0个月.每天上午观察、记录恒河猴的一般体征、摄食量,每周称体重、测体温1次.给药前、给药1.5、3.0、4.5、6.0、7.5、9.0个月(停药次日)及恢复期结束(停药4周),分别检测心电图、眼科、免疫学、血液学、血液生化学、尿常规等指标.给药3.0、6.0、9.0个月及恢复期结束,每组随机解剖2只猴(9.0个月为4只),雌雄各半,观察脏器变化,计算脏器系数,并进行组织病理学观察.结果 各剂量CTB-INS对恒河猴各指标检测结果均无明显影响,与对照组相似.各剂量组恒河猴脏器及其组织病理学检查均未发现异常,部分剂量组恒河猴的胸腺系数平均值高于对照组,肺脏系数、甲状腺系数平均值低于对照组.结论 经口给予CTB-INS对恒河猴无明显毒副作用,未见中毒靶器官,无毒(影响)剂量为4.0 g/(kg·d).

  15. Cholera toxin and pregnancy promote regeneration of the retinal ganglion cells in golden hamster%霍乱毒素及妊娠对金黄地鼠视网膜节细胞再生的作用

    Institute of Scientific and Technical Information of China (English)

    李飞; 黄锦桃; 李海标

    2011-01-01

    目的:观察霍乱毒素(CTx)及妊娠对成年金黄地鼠视神经扎断后视网膜节细胞(RGCs)轴突再生的促进作用.方法:确定成年金黄地鼠交配3d后,扎断视神经(ON)近端,玻璃体内注射CTx.动物随机分为实验组和对照组:对照组为单纯扎断ON为损伤组、损伤PBS组;实验组分为CTx组与妊娠后扎断ON妊娠损伤组与妊娠CTx组.术后动物存活3周.用荧光金逆行标记再生的视网膜节细胞( RGCs),在荧光镜下观察视网膜平铺片再生RGCs的数量变化,并比较实验组各组再生RGCs的周长.结果:CTx组、妊娠损伤组、妊娠CTx组视网膜再生RGCs平均数比损伤组及PBS组增加,差异具有统计学意义.妊娠CTx组比CTx组、妊娠损伤组视网膜再生RGCs平均数增加.实验组各组再生的RGCs周长相比差异无统计学意义.结论:妊娠及玻璃体注入CTx具有促进视神经扎断后视网膜节细胞轴突再生的作用,两者有协同作用,各组再生的RGCs大小无明显差别.%Objective: To investigate the effects of cholera toxin (CTx) and pregnancy on promoting the axon regeneration of retinal ganglion cells (RGCs) in hamster retina. Methods: In day 3 after golden hamster mating, optic nerve (ON) micro-crushed, CTx was injected intravitrously. The rats were separated into a regenerating control group (injuried group and in-juried+PBS group) and an experiment group (injuried+CTx group, pregnancy + injuried group, pregnancy+injuried+CTx group). The rats in each group were allowed to survive for 3 weeks. The regenerating RGCs were labeled retrogradely with fluorogold, and changes in number of regenerating RGCs in each retina were observed under a fluorescence microscope. The circumference of regenerating RGCs in each experimental group was compared. Results: The mean numbers of regenerating RGCs in the injuried+CTx group, pregnancy + injuried group and pregnancy+injuried+CTx group were increased and significantly higher than those in the

  16. Cholera toxin%霍乱毒素

    Institute of Scientific and Technical Information of China (English)

    杨淑静; 马清钧

    2000-01-01

    霍乱毒素是一种很强的粘膜免疫佐剂,本文介绍了霍乱毒素的结构、霍乱毒素毒性的分子机理及近年来在霍乱毒素佐剂活性方面的研究进展,并探讨了霍乱毒素佐剂活性的分子机理.

  17. The population structure of Vibrio cholerae from the Chandigarh Region of Northern India.

    Directory of Open Access Journals (Sweden)

    Moataz Abd El Ghany

    2014-07-01

    Full Text Available Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century.Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA identified 16 distinct clusters.The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.

  18. The Population Structure of Vibrio cholerae from the Chandigarh Region of Northern India

    KAUST Repository

    Abd El Ghany, Moataz

    2014-07-24

    Background:Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century.Methodology/Principal Findings:Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII) and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST) distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA) identified 16 distinct clusters.Conclusions/Significance:The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.

  19. Vibrio cholerae Biofilms and Cholera Pathogenesis

    Science.gov (United States)

    Silva, Anisia J.; Benitez, Jorge A.

    2016-01-01

    Vibrio cholerae can switch between motile and biofilm lifestyles. The last decades have been marked by a remarkable increase in our knowledge of the structure, regulation, and function of biofilms formed under laboratory conditions. Evidence has grown suggesting that V. cholerae can form biofilm-like aggregates during infection that could play a critical role in pathogenesis and disease transmission. However, the structure and regulation of biofilms formed during infection, as well as their role in intestinal colonization and virulence, remains poorly understood. Here, we review (i) the evidence for biofilm formation during infection, (ii) the coordinate regulation of biofilm and virulence gene expression, and (iii) the host signals that favor V. cholerae transitions between alternative lifestyles during intestinal colonization, and (iv) we discuss a model for the role of V. cholerae biofilms in pathogenicity. PMID:26845681

  20. Vibrio cholerae Biofilms and Cholera Pathogenesis.

    Directory of Open Access Journals (Sweden)

    Anisia J Silva

    2016-02-01

    Full Text Available Vibrio cholerae can switch between motile and biofilm lifestyles. The last decades have been marked by a remarkable increase in our knowledge of the structure, regulation, and function of biofilms formed under laboratory conditions. Evidence has grown suggesting that V. cholerae can form biofilm-like aggregates during infection that could play a critical role in pathogenesis and disease transmission. However, the structure and regulation of biofilms formed during infection, as well as their role in intestinal colonization and virulence, remains poorly understood. Here, we review (i the evidence for biofilm formation during infection, (ii the coordinate regulation of biofilm and virulence gene expression, and (iii the host signals that favor V. cholerae transitions between alternative lifestyles during intestinal colonization, and (iv we discuss a model for the role of V. cholerae biofilms in pathogenicity.

  1. Vibrio cholerae Biofilms and Cholera Pathogenesis.

    Science.gov (United States)

    Silva, Anisia J; Benitez, Jorge A

    2016-02-01

    Vibrio cholerae can switch between motile and biofilm lifestyles. The last decades have been marked by a remarkable increase in our knowledge of the structure, regulation, and function of biofilms formed under laboratory conditions. Evidence has grown suggesting that V. cholerae can form biofilm-like aggregates during infection that could play a critical role in pathogenesis and disease transmission. However, the structure and regulation of biofilms formed during infection, as well as their role in intestinal colonization and virulence, remains poorly understood. Here, we review (i) the evidence for biofilm formation during infection, (ii) the coordinate regulation of biofilm and virulence gene expression, and (iii) the host signals that favor V. cholerae transitions between alternative lifestyles during intestinal colonization, and (iv) we discuss a model for the role of V. cholerae biofilms in pathogenicity.

  2. Modeling cholera outbreaks.

    Science.gov (United States)

    Chao, Dennis L; Longini, Ira M; Morris, J Glenn

    2014-01-01

    Mathematical modeling can be a valuable tool for studying infectious disease outbreak dynamics and simulating the effects of possible interventions. Here, we describe approaches to modeling cholera outbreaks and how models have been applied to explore intervention strategies, particularly in Haiti. Mathematical models can play an important role in formulating and evaluating complex cholera outbreak response options. Major challenges to cholera modeling are insufficient data for calibrating models and the need to tailor models for different outbreak scenarios.

  3. Modeling cholera outbreaks

    OpenAIRE

    Chao, Dennis L.; Ira M Longini; Morris, J. Glenn

    2014-01-01

    Mathematical modeling can be a valuable tool for studying infectious disease outbreak dynamics and simulating the effects of possible interventions. Here, we describe approaches to modeling cholera outbreaks and how models have been applied to explore intervention strategies, particularly in Haiti. Mathematical models can play an important role in formulating and evaluating complex cholera outbreak response options. Major challenges to cholera modeling are insufficient data for calibrating mo...

  4. Modeling cholera outbreaks

    Science.gov (United States)

    Longini, Ira M.; Morris, J. Glenn

    2014-01-01

    Mathematical modeling can be a valuable tool for studying infectious disease outbreak dynamics and simulating the effects of possible interventions. Here, we describe approaches to modeling cholera outbreaks and how models have been applied to explore intervention strategies, particularly in Haiti. Mathematical models can play an important role in formulating and evaluating complex cholera outbreak response options. Major challenges to cholera modeling are insufficient data for calibrating models and the need to tailor models for different outbreak scenarios. PMID:23412687

  5. Modeling cholera outbreaks

    OpenAIRE

    Dennis L Chao; Longini, Ira M.; Morris, J. Glenn

    2014-01-01

    Mathematical modeling can be a valuable tool for studying infectious disease outbreak dynamics and simulating the effects of possible interventions. Here, we describe approaches to modeling cholera outbreaks and how models have been applied to explore intervention strategies, particularly in Haiti. Mathematical models can play an important role in formulating and evaluating complex cholera outbreak response options. Major challenges to cholera modeling are insufficient data for calibrating mo...

  6. 霍乱毒素B亚单位与志贺毒素2B亚单位融合表达及抗原性检测%Clone and express ctb-stx2 b fusion gene in Enterohemrrhagic escherichia coli O157:H7 Shigeai toxin 2B subunit and V cholera toxin B subunit and the detection of their immunogenicity

    Institute of Scientific and Technical Information of China (English)

    李振军; 孙强正; 景怀琦; 徐建国

    2008-01-01

    Objective To clone and express the fusion gene encoding Enterohemrrhagic escherichia coli O157:H7(EHEC O157:H7)Shigela toxin 2B subunit(Stx2B)and vibrio cholera toxin B subunit (CTB)as well as to detect the immunogenicity and GM1-binding ability of fusion protein.Methods To design a primer to amplify stx2b gene and ctb-stx2b fusion gene encoding Stx2B and CTB-Stx2B respectively and to clone the genes into express plasmid pET30a(+)C in order to construct pET30a-ctb-stx2b after T-A sequencing was varified,then to transform constructed plasmid into E.coliBL21(DE3)induced by IPTG and purified by a purify kit and to detect molecular weight and immunogenicity by SDSPAGE and Western-blot.Results The amplified ctb-stx2b fragments appeared to he 750 bp and gene sequence was identical to designed sequence.The prokaryotic expression system pET30a-ctb-stx2b/BL21 could express protein weight about Mr20×103and the expressed protein could react to CTB monoclone anti-body.The fusion protein CTB-Stx2B could bind GM1.Conclusion CTB-Stx2B had successfully been expressed in prokaryotic while the expressed protein had good immunogenicity and GM1-Binding ability.This study provided information on further EHEC O157:H7 vaccine research.%目的 克隆表达出血性大肠埃希菌(EHEC)O157:H7志贺毒素2B亚单位(Stx2B)与霍乱毒素B亚单位(CTB)的融合蛋白(CTB-Stx2B),并检测其抗原性和与神经节苷脂(GM1)结合能力.方法 设计引物扩增融合蛋白CTB-Stx2B的编码基因ctb-stx2b,T-A克隆测序验证后克隆入原核表达质粒pET30a(+)C,构建表达质粒pET30a(+)-ctb-stx2b,转化E.coliBL21(DE3),IPTG诱导表达、纯化,获得目的蛋白CTB-Stx2B,SDS-PAGE和Western-blot检测其抗原性和形成五聚体的能力;GM1-ELISA法检测其与GM1结合能力.结果 扩增出约750 bp的目的片段,测序鉴定与设计序列一致;原核表达质粒pET30a(+)-ctb-stx2b转化E.coliBL21(DE3)后,经酶切和PCR扩增鉴定正确;IPTG

  7. Toxigenic Vibrio cholerae identified in estuaries of Tanzania using PCR techniques.

    Science.gov (United States)

    Dalusi, Lucy; Lyimo, Thomas J; Lugomela, Charles; Hosea, Ken M M; Sjöling, Sara

    2015-03-01

    The current study assessed the occurrence of the Vibrio cholerae serogroups O1 and O139 in environmental samples along salinity gradients in three selected estuaries of Tanzania both through culture independent methods and by cultured bacteria. Occurrence of V. cholerae was determined by PCR targeting the V. cholerae outer membrane protein gene ompW. Furthermore, the presence of toxigenic strains and serogroups O1 and O139 was determined using multiplex PCR with specific primers targeting the cholera toxin gene subunit A, ctxA, and serotype specific primers, O1-rfb and O139-rfb, respectively. Results showed that V. cholerae occurred in approximately 10% (n = 185) of both the environmental samples and isolated bacteria. Eight of the bacteria isolates (n = 43) were confirmed as serogroup O1 while one belonged to serogroup O139, the first reported identification of this epidemic strain in East African coastal waters. All samples identified as serogroup O1 or O139 and a number of non-O1/O139 strains were ctxA positive. This study provides in situ evidence of the presence of pathogenic V. cholerae O1 and O139 and a number of V. cholerae non-O1/O139 that carry the cholera toxin gene in estuaries along the coast of Tanzania. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Haitian variant ctxB producing Vibrio cholerae O1 with reduced susceptibility to ciprofloxacin is persistent in Yavatmal, Maharashtra, India, after causing a cholera outbreak.

    Science.gov (United States)

    Kumar, P; Mishra, D K; Deshmukh, D G; Jain, M; Zade, A M; Ingole, K V; Yadava, P K

    2014-05-01

    Vibrio cholerae O1 biotype El Tor producing Haitian variant Cholera Toxin (HCT) and showing reduced susceptibility to ciprofloxacin caused a cholera outbreak associated with a high case fatality rate (4.5) in India. HCT-secreting strains responsible for severe cholera epidemics in Orissa (India), Western Africa and Haiti were associated with increased mortality. There is a pressing need for an integrated multidisciplinary approach to combat further spread of newly emerging variant strains. The therapeutic effect of ciprofloxacin was diminished whereas use of doxycycline in moderate to severe cholera patients was found to be effective in outbreak management. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

  9. Cholera outbreaks in India.

    Science.gov (United States)

    Ramamurthy, Thandavarayan; Sharma, Naresh C

    2014-01-01

    Cholera is a global health problem as several thousands of cases and deaths occur each year. The unique epidemiologic attribute of the disease is its propensity to occur as outbreaks that may flare-up into epidemics, if not controlled. The causative bacterial pathogen Vibrio cholerae prevails in the environment and infects humans whenever there is a breakdown in the public health component. The Indian subcontinent is vulnerable to this disease due its vast coastlines with areas of poor sanitation, unsafe drinking water, and overcrowding. Recently, it was shown that climatic conditions also play a major role in the persistence and spread of cholera. Constant change in the biotypes and serotypes of V. cholerae are also important aspects that changes virulence and survival of the pathogen. Such continuous changes increase the infection ability of the pathogen affecting the susceptible population including the children. The short-term carrier status of V. cholerae has been studied well at community level and this facet significantly contributes to the recurrence of cholera. Several molecular tools recognized altering clonality of V. cholerae in relation with the advent of a serogroup or serotype. Rapid identification systems were formulated for the timely detection of the pathogen so as to identify and control the outbreak and institute proper treatment of the patients. The antimicrobials used in the past are no longer useful in the treatment of cholera as V. cholerae has acquired several mechanisms for multiple antimicrobial resistance. This upsurge in antimicrobial resistance directly influences the management of the disease. This chapter provides an overview of cholera prevalence in India, possible sources of infection, and molecular epidemiology along with antimicrobial resistance of V. cholerae.

  10. Stool C difficile toxin

    Science.gov (United States)

    ... toxin; Colitis - toxin; Pseudomembranous - toxin; Necrotizing colitis - toxin; C difficile - toxin ... be analyzed. There are several ways to detect C difficile toxin in the stool sample. Enzyme immunoassay ( ...

  11. Construction and evaluation of V. cholerae O139 mutant, VCUSM21P, as a safe live attenuated cholera vaccine.

    Science.gov (United States)

    Murugaiah, Chandrika; Nik Mohd Noor, Nik Zuraina; Mustafa, Shyamoli; Manickam, Ravichandran; Pattabhiraman, Lalitha

    2014-01-01

    Cholera is a major infectious disease, affecting millions of lives annually. In endemic areas, implementation of vaccination strategy against cholera is vital. As the use of safer live vaccine that can induce protective immunity against Vibrio cholerae O139 infection is a promising approach for immunization, we have designed VCUSM21P, an oral cholera vaccine candidate, which has ctxA that encodes A subunit of ctx and mutated rtxA/C, ace and zot mutations. VCUSM21P was found not to disassemble the actin of HEp2 cells. It colonized the mice intestine approximately 1 log lower than that of the Wild Type (WT) strain obtained from Hospital Universiti Sains Malaysia. In the ileal loop assay, unlike WT challenge, 1×10⁶ and 1×10⁸ colony forming unit (CFU) of VCUSM21P was not reactogenic in non-immunized rabbits. Whereas, the reactogenicity caused by the WT in rabbits immunized with 1×10¹⁰ CFU of VCUSM21P was found to be reduced as evidenced by absence of fluid in loops administered with 1×10²-1×10⁷ CFU of WT. Oral immunization using 1×10¹⁰ CFU of VCUSM21P induced both IgA and IgG against Cholera Toxin (CT) and O139 lipopolysaccharides (LPS). The serum vibriocidal antibody titer had a peak rise of 2560 fold on week 4. Following Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) experiment, the non-immunized rabbits were found not to be protected against lethal challenge with 1×10⁹ CFU WT, but 100% of immunized rabbits survived the challenge. In the past eleven years, V. cholerae O139 induced cholera has not been observed. However, attenuated VCUSM21P vaccine could be used for vaccination program against potentially fatal endemic or emerging cholera caused by V. cholerae O139.

  12. Genetic diversity of environmental Vibrio cholerae O1 strains isolated in Northern Vietnam.

    Science.gov (United States)

    Takemura, Taichiro; Murase, Kazunori; Maruyama, Fumito; Tran, Thi Luong; Ota, Atsushi; Nakagawa, Ichiro; Nguyen, Dong Tu; Ngo, Tu Cuong; Nguyen, Thi Hang; Tokizawa, Asako; Morita, Masatomo; Ohnishi, Makoto; Nguyen, Binh Minh; Yamashiro, Tetsu

    2017-10-01

    Cholera epidemics have been recorded periodically in Vietnam during the seventh cholera pandemic. Since cholera is a water-borne disease, systematic monitoring of environmental waters for Vibrio cholerae presence is important for predicting and preventing cholera epidemics. We conducted monitoring, isolation, and genetic characterization of V. cholerae strains in Nam Dinh province of Northern Vietnam from Jul 2013 to Feb 2015. In this study, four V. cholerae O1 strains were detected and isolated from 110 analyzed water samples (3.6%); however, none of them carried the cholera toxin gene, ctxA, in their genomes. Whole genome sequencing and phylogenetic analysis revealed that the four O1 isolates were separated into two independent clusters, and one of them diverged from a common ancestor with pandemic strains. The analysis of pathogenicity islands (CTX prophage, VPI-I, VPI-II, VSP-I, and VSP-II) indicated that one strain (VNND_2014Jun_6SS) harbored an unknown prophage-like sequence with high homology to vibriophage KSF-1 phi and VCY phi, identified from Bangladesh and the USA, respectively, while the other three strains carried tcpA gene with a distinct sequence demonstrating a separate clonal lineage. These results suggest that the aquatic environment can harbor highly divergent V. cholera strains and serve as a reservoir for multiple V. cholerae virulence-associated genes which may be exchanged via mobile genetic elements. Therefore, continuous monitoring and genetic characterization of V. cholerae strains in the environment should contribute to the early detection of the sources of infection and prevention of cholera outbreaks as well as to understanding the natural ecology and evolution of V. cholerae. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Quadruplex PCR for simultaneous detection of serotype, biotype, toxigenic potential, and central regulating factor of Vibrio cholerae.

    Science.gov (United States)

    Khuntia, Hemant Kumar; Pal, Bibhuti Bhusan; Chhotray, Guru Prasada

    2008-07-01

    A quadruplex PCR was developed for the simultaneous detection of genes specific for Vibrio cholerae O1 and/or O139 serogroup (wbe and/or wbf), cholera toxin A subunit (ctxA), toxin-coregulated pilus (tcpA), and central regulating protein ToxR (toxR) in a single tube reaction. This is a simple, rapid, and accurate approach for the detection of toxigenic V. cholerae O1 and/or O139 and can prevent the rapid spread of the disease by early detection.

  14. The caveolae-mediated sv40 entry pathway bypasses the golgi complex en route to the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Kuksin Dmitry

    2005-04-01

    Full Text Available Abstract Background Simian virus 40 (SV40 enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi. Results We confirmed our earlier finding that SV40 co localizes with the Golgi marker β-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide. Conclusion The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with β-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi.

  15. The caveolae-mediated sv40 entry pathway bypasses the golgi complex en route to the endoplasmic reticulum.

    Science.gov (United States)

    Norkin, Leonard C; Kuksin, Dmitry

    2005-04-19

    Simian virus 40 (SV40) enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi. We confirmed our earlier finding that SV40 co localizes with the Golgi marker beta-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide. The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with beta-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi.

  16. The repertoire of glycosphingolipids recognized by Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    John Benktander

    Full Text Available The binding of cholera toxin to the ganglioside GM1 as the initial step in the process leading to diarrhea is nowadays textbook knowledge. In contrast, the knowledge about the mechanisms for attachment of Vibrio cholerae bacterial cells to the intestinal epithelium is limited. In order to clarify this issue, a large number of glycosphingolipid mixtures were screened for binding of El Tor V. cholerae. Several specific interactions with minor complex non-acid glycosphingolipids were thereby detected. After isolation of binding-active glycosphingolipids, characterization by mass spectrometry and proton NMR, and comparative binding studies, three distinct glycosphingolipid binding patterns were defined. Firstly, V. cholerae bound to complex lacto/neolacto glycosphingolipids with the GlcNAcβ3Galβ4GlcNAc sequence as the minimal binding epitope. Secondly, glycosphingolipids with a terminal Galα3Galα3Gal moiety were recognized, and the third specificity was the binding to lactosylceramide and related compounds. V. cholerae binding to lacto/neolacto glycosphingolipids, and to the other classes of binding-active compounds, remained after deletion of the chitin binding protein GbpA. Thus, the binding of V. cholerae to chitin and to lacto/neolacto containing glycosphingolipids represents two separate binding specificities.

  17. Occurrence in Mexico, 1998-2008, of Vibrio cholerae CTX+ El Tor carrying an additional truncated CTX prophage.

    Science.gov (United States)

    Alam, Munirul; Rashed, Shah Manzur; Mannan, Shahnewaj Bin; Islam, Tarequl; Lizarraga-Partida, Marcial Leonardo; Delgado, Gabriela; Morales-Espinosa, Rosario; Mendez, Jose Luis; Navarro, Armando; Watanabe, Haruo; Ohnishi, Makoto; Hasan, Nur A; Huq, Anwar; Sack, R Bradley; Colwell, Rita R; Cravioto, Alejandro

    2014-07-08

    The seventh cholera pandemic caused by Vibrio cholerae O1 El Tor (ET) has been superseded in Asia and Africa by altered ET possessing the cholera toxin (CTX) gene of classical (CL) biotype. The CL biotype of V. cholerae was isolated, along with prototypic and altered ET, during the 1991 cholera epidemic in Mexico and subsequently remained endemic until 1997. Microbiological, molecular, and phylogenetic analyses of clinical and environmental V. cholerae isolated in Mexico between 1998 and 2008 revealed important genetic events favoring predominance of ET over CL and altered ET. V. cholerae altered ET was predominant after 1991 but not after 2000. V. cholerae strains isolated between 2001 and 2003 and a majority isolated in 2004 lacked CTX prophage (Φ) genes encoding CTX subunits A and B and repeat sequence transcriptional regulators of ET and CL biotypes: i.e., CTXΦ(-). Most CTXΦ(-) V. cholerae isolated in Mexico between 2001 and 2003 also lacked toxin coregulated pili tcpA whereas some carried either tcpA(ET) or a variant tcpA with noticeable sequence dissimilarity from tcpA(CL). The tcpA variants were not detected in 2005 after CTXΦ(+) ET became dominant. All clinical and environmental V. cholerae O1 strains isolated during 2005-2008 in Mexico were CTXΦ(+) ET, carrying an additional truncated CTXΦ instead of RS1 satellite phage. Despite V. cholerae CTXΦ(-) ET exhibiting heterogeneity in pulsed-field gel electrophoresis patterns, CTXΦ(+) ET isolated during 2004-2008 displayed homogeneity and clonal relationship with V. cholerae ET N16961 and V. cholerae ET isolated in Peru.

  18. Occurrence in Mexico, 1998–2008, of Vibrio cholerae CTX+ El Tor carrying an additional truncated CTX prophage

    Science.gov (United States)

    Alam, Munirul; Rashed, Shah Manzur; Mannan, Shahnewaj Bin; Islam, Tarequl; Lizarraga-Partida, Marcial Leonardo; Delgado, Gabriela; Morales-Espinosa, Rosario; Mendez, Jose Luis; Navarro, Armando; Watanabe, Haruo; Ohnishi, Makoto; Hasan, Nur A.; Huq, Anwar; Sack, R. Bradley; Colwell, Rita R.; Cravioto, Alejandro

    2014-01-01

    The seventh cholera pandemic caused by Vibrio cholerae O1 El Tor (ET) has been superseded in Asia and Africa by altered ET possessing the cholera toxin (CTX) gene of classical (CL) biotype. The CL biotype of V. cholerae was isolated, along with prototypic and altered ET, during the 1991 cholera epidemic in Mexico and subsequently remained endemic until 1997. Microbiological, molecular, and phylogenetic analyses of clinical and environmental V. cholerae isolated in Mexico between 1998 and 2008 revealed important genetic events favoring predominance of ET over CL and altered ET. V. cholerae altered ET was predominant after 1991 but not after 2000. V. cholerae strains isolated between 2001 and 2003 and a majority isolated in 2004 lacked CTX prophage (Φ) genes encoding CTX subunits A and B and repeat sequence transcriptional regulators of ET and CL biotypes: i.e., CTXΦ−. Most CTXΦ− V. cholerae isolated in Mexico between 2001 and 2003 also lacked toxin coregulated pili tcpA whereas some carried either tcpAET or a variant tcpA with noticeable sequence dissimilarity from tcpACL. The tcpA variants were not detected in 2005 after CTXΦ+ ET became dominant. All clinical and environmental V. cholerae O1 strains isolated during 2005–2008 in Mexico were CTXΦ+ ET, carrying an additional truncated CTXΦ instead of RS1 satellite phage. Despite V. cholerae CTXΦ− ET exhibiting heterogeneity in pulsed-field gel electrophoresis patterns, CTXΦ+ ET isolated during 2004–2008 displayed homogeneity and clonal relationship with V. cholerae ET N16961 and V. cholerae ET isolated in Peru. PMID:24958870

  19. Staphylococcus aureus α-toxin-mediated cation entry depolarizes membrane potential and activates p38 MAP kinase in airway epithelial cells.

    Science.gov (United States)

    Eiffler, Ina; Behnke, Jane; Ziesemer, Sabine; Müller, Christian; Hildebrandt, Jan-Peter

    2016-09-01

    Membrane potential (Vm)-, Na(+)-, or Ca(2+)-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with Staphylococcus aureus α-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel-forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla pore, were used to investigate the permeability of the pore. Na(+) as well as Ca(2+) ions were able to pass the Hla pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by α-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense.

  20. Regulation of virulence in Vibrio cholerae: the ToxR regulon.

    Science.gov (United States)

    Childers, Brandon M; Klose, Karl E

    2007-06-01

    Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera. This disease consists of enormous fluid loss through stools, which can be fatal. Cholera epidemics appear in explosive outbreaks that have occurred repeatedly throughout history. The virulence factors toxin coregulated pilus (TCP) and cholera toxin (CT) are essential for colonization of the host and enterotoxicity, respectively. These virulence factors are under the control of ToxT, an AraC/XylS family protein that activates transcription of the genes encoding TCP and CT. ToxT is under the control of a virulence regulatory cascade known as the ToxR regulon, which responds to environmental stimuli to ensure maximal virulence-factor induction within the human intestine. An understanding of this intricate signaling pathway is essential for the development of methods to treat and prevent this devastating disease.

  1. Prevalence of Vibrio cholerae O1 El Tor variant in a cholera-endemic zone of Kenya.

    Science.gov (United States)

    Saidi, Suleiman M; Chowdhury, Nityananda; Awasthi, Sharda P; Asakura, Masahiro; Hinenoya, Atsushi; Iijima, Yoshio; Yamasaki, Shinji

    2014-03-01

    Since 2007, Kenya has experienced an increase in cholera outbreaks characterized by a high fatality rate. In this study, we characterized 81 Vibrio cholerae isolates from diarrhoeal stool samples in Nyanza, a cholera-endemic lake region of Kenya, for virulence properties, clonality and antibiotic susceptibility. Eighty of these isolates were V. cholerae O1 El Tor variants carrying the classical ctxB gene sequence, while one isolate was V. cholerae non-O1/O139. All of the El Tor variants were of clonal origin, as revealed by PFGE, and were susceptible to ampicillin, tetracycline, ciprofloxacin, fosfomycin, kanamycin and norfloxacin. However, the isolates showed resistance to sulfamethoxazole/trimethoprim and streptomycin, and intermediate resistance to nalidixic acid, chloramphenicol and imipenem. The non-O1/O139 isolate carried the cholix toxin II gene (chxA II) and was susceptible to all antimicrobials tested except ampicillin. We propose that an El Tor variant clone caused the Nyanza cholera outbreak of 2007-2008.

  2. Molecular evidence of cholera outbreak caused by a toxigenic Vibrio cholerae O1 El tor variant strain in Kelantan, Malaysia.

    Science.gov (United States)

    Ang, Geik Yong; Yu, Choo Yee; Balqis, Kamarudin; Elina, Husni Tan; Azura, Hussin; Hani, Mat Hussin; Yean, Chan Yean

    2010-11-01

    A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype.

  3. EPIDEMIOLOGY OF CHOLERA OUTBREAK IN KAMPALA ...

    African Journals Online (AJOL)

    hi-tech

    Between December 1997 and March 1998, 6228 cases of cholera were reported, of which 1091. (17.5%) were ... The extension of the epidemic of cholera that has been observed in .... cholera vaccines in the prevention and control of cholera.

  4. The Role of Cyanobacteria Blooms in Cholera Epidemic in Bangladesh

    Science.gov (United States)

    Sagir Ahmed, Md.; Raknuzzaman, Md.; Akther, Hafeza; Ahmed, Sumaiya

    A study was conducted on association of Vibrio cholerae with plankton specially emphasis on cyanobacteria in relation to some physico-chemical parameters in the River Buriganga, Dhaka, from January to December 2002. Monthly abundance of phytoplankton and zooplankton varied from 457 to 14166 and from 169 to 1055 individual L-1, respectively. Monthly average of faecal coliform in water, zooplankton and phytoplankton samples were 3.99x109, 4.54x103 and 4.28x102 (CFU L-1), respectively. During epidemics, toxigenic V. cholerae 01 and 0139 were isolated from the patients as well as from the surface water. V. cholerae 01 and 0139 were also isolated from plankton samples. More over, it was observed that ctx (cholera toxic) positive in water and phytoplankton samples of the river. A bloom of Oscillatoria sp. (1.6x104 individual L-1) occurred in the upper reaches of the River Buriganga in May 2002. Methanol-water extract of bloom sample was analyzed by high performance liquid chromatography with UV detection and Mass Spectrum (MS) detected microcystin-RR. Cyanobacteria are abundant in the aquatic environment of Bangladesh and it was established that V. cholerae maintain a symbiotic relationship with these algae particularly mucilaginous cyanobacteria. During epidemics, patients symptoms included diarrhea, vomiting and hemorrhagic enteritis and in severe cases hemorrhagic diarrhea. So, question has arisen that which is responsible, microcystins or cholera for death of cholera/diarrhea patients in Bangladesh. Future research should be directed to isolate microcystins and cholera toxins from the epidemic areas to clarify the fact.

  5. Riverbed Sediments as Reservoirs of Multiple Vibrio cholerae Virulence-Associated Genes: A Potential Trigger for Cholera Outbreaks in Developing Countries

    Directory of Open Access Journals (Sweden)

    Akebe Luther King Abia

    2017-01-01

    Full Text Available Africa remains the most cholera stricken continent in the world as many people lacking access to safe drinking water rely mostly on polluted rivers as their main water sources. However, studies in these countries investigating the presence of Vibrio cholerae in aquatic environments have paid little attention to bed sediments. Also, information on the presence of virulence-associated genes (VAGs in environmental ctx-negative V. cholerae strains in this region is lacking. Thus, we investigated the presence of V. cholerae VAGs in water and riverbed sediment of the Apies River, South Africa. Altogether, 120 samples (60 water and 60 sediment samples collected from ten sites on the river (January and February 2014 were analysed using PCR. Of the 120 samples, 37 sediment and 31 water samples were positive for at least one of the genes investigated. The haemolysin gene (hlyA was the most isolated gene. The cholera toxin (ctxAB and non-O1 heat-stable (stn/sto genes were not detected. Genes were frequently detected at sites influenced by human activities. Thus, identification of V. cholerae VAGs in sediments suggests the possible presence of V. cholerae and identifies sediments of the Apies River as a reservoir for potentially pathogenic V. cholerae with possible public health implications.

  6. Prevalence and characterization of Vibrio cholerae isolated from shrimp products imported into Denmark

    DEFF Research Database (Denmark)

    Dalsgaard, A.; Bjergskov, T.; Jeppesen, V.F.

    1996-01-01

    A total of 3,555 metric tonnes of warm water shrimp were imported into Denmark from December 1994 to July 1995. V. cholerae O1 was not detected in any of the 748 samples analyzed. Non-Ol V. cholerae was found in a single (0.1%) cooked frozen shrimp product and in five (0.7%) raw frozen products......, all originating from shrimp produced in aquaculture. Six isolated strains agglutinated in polyvalent O antisera, but did not agglutinate in Ogawa or Inaba antisera. The six strains were resistant to colistin and sulfisoxazole; three strains also showed resistance to ampicillin. None of the strains...... contained plasmids or genes encoding cholera toxin (CT) or heat-stable enterotoxin (NAG-ST), The absence of V. cholerae O1 and the low number of samples containing CT and NAG-ST negative non-Ol strains in imported shrimp suggest that I! cholerae in such products may not constitute a public health problem....

  7. Prevalence and characterization of Vibrio cholerae isolated from shrimp products imported into Denmark

    DEFF Research Database (Denmark)

    Dalsgaard, A.; Bjergskov, T.; Jeppesen, V.F.

    1996-01-01

    A total of 3,555 metric tonnes of warm water shrimp were imported into Denmark from December 1994 to July 1995. V. cholerae O1 was not detected in any of the 748 samples analyzed. Non-Ol V. cholerae was found in a single (0.1%) cooked frozen shrimp product and in five (0.7%) raw frozen products...... contained plasmids or genes encoding cholera toxin (CT) or heat-stable enterotoxin (NAG-ST), The absence of V. cholerae O1 and the low number of samples containing CT and NAG-ST negative non-Ol strains in imported shrimp suggest that I! cholerae in such products may not constitute a public health problem....

  8. Molecular Analysis and Toxigenic Potential of Vibrio cholerae Isolated from Hilsha fish (Tenualosa ilisha), Bangladesh

    DEFF Research Database (Denmark)

    Hossain, Zenat Zebin; Farhana, Israt; Tulsiani, Suhella

    Exposure to contaminated fish may upsurge the virulent strains of Vibrio cholerae, the deadly human pathogen in the households of rural and urban Bangladesh. Since V. cholerae spreading was reported from the Bay of Bengal, this study hypothesized that Hilsha (Tenualosa ilisha), a marine and fresh...... water fish may serve as a transmission vehicle of potential emerging epidemic causing strains. For this, we studied 9 toxigenic V. cholerae strains isolated from Hilsha fish including 6 V. cholerae O1 and 3 non O1/O139 serogroups for virulence associated genotype and their pathogenic potential on animal...... model and human cancer cell line . The V. cholerae O1 and non- O1/O139 strains possessed diverse virulence genes but lacked some major toxin genes like ctxA, tcp etc. Eight of the nine strains showed survivability up to 1% sodium chloride in broth culture which indicates their coastal origin. All nine...

  9. Cholera outbreak in South Africa, 2008-2009: laboratory analysis of Vibrio cholerae O1 strains.

    Science.gov (United States)

    Ismail, Husna; Smith, Anthony M; Tau, Nomsa P; Sooka, Arvinda; Keddy, Karen H

    2013-11-01

    A total of 720 Vibrio cholerae O1 strains were recovered for investigation from an outbreak of cholera in South Africa between November 2008 and April 2009. Strains were characterized by serotype testing. Antimicrobial susceptibility testing. Genetic diversity of 248 strains was investigated using pulsed-field gel electrophoresis (PFGE) analysis. Extended characterization was performed on 90 strains. Molecular analysis included: polymerase chain reaction (PCR) identification of ctxA and tcpA genes, sequencing the ctxAB gene, and investigation of molecular mechanisms conferring antimicrobial resistance. The majority of strains were characterized as serotype Ogawa. Strains showed multidrug resistance. Approximately 1.0% of strains displayed extended-spectrum β-lactamase (ESBL) activity. Strains showed very similar PFGE patterns. Ninety strains selected for extended characterization showed the following results: Strains possessed the cholera toxin (CT) and all were PCR positive for the tcpA-El Tor variant. Sequencing of the ctxB gene matched the B-1 allele. Strains harbored the SXT element. Strains that displayed ESBL activity possessed a 140-kilobase-pair plasmid that produced the TEM-63 β-lactamase. Nalidixic acid-resistant strains harbored mutations in GyrA (Ser83-Ile) and ParC (Ser85-Leu). These data highlight the rapid development of antimicrobial resistance and spread of V. cholerae O1 El Tor variants expressing the classical CT within South Africa.

  10. Antibody microarrays for native toxin detection.

    Science.gov (United States)

    Rucker, Victor C; Havenstrite, Karen L; Herr, Amy E

    2005-04-15

    We have developed antibody-based microarray techniques for the multiplexed detection of cholera toxin beta-subunit, diphtheria toxin, anthrax lethal factor and protective antigen, Staphylococcus aureus enterotoxin B, and tetanus toxin C fragment in spiked samples. Two detection schemes were investigated: (i) a direct assay in which fluorescently labeled toxins were captured directly by the antibody array and (ii) a competition assay that employed unlabeled toxins as reporters for the quantification of native toxin in solution. In the direct assay, fluorescence measured at each array element is correlated with labeled toxin concentration to yield baseline binding information (Langmuir isotherms and affinity constants). Extending from the direct assay, the competition assay yields information on the presence, identity, and concentration of toxins. A significant advantage of the competition assay over reported profiling assays is the minimal sample preparation required prior to analysis because the competition assay obviates the need to fluorescently label native proteins in the sample of interest. Sigmoidal calibration curves and detection limits were established for both assay formats. Although the sensitivity of the direct assay is superior to that of the competition assay, detection limits for unmodified toxins in the competition assay are comparable to values reported previously for sandwich-format immunoassays of antibodies arrayed on planar substrates. As a demonstration of the potential of the competition assay for unlabeled toxin detection, we conclude with a straightforward multiplexed assay for the differentiation and identification of both native S. aureus enterotoxin B and tetanus toxin C fragment in spiked dilute serum samples.

  11. Cholera in Azov area

    OpenAIRE

    O. N. Domashenko; T. A. Belomerya; N. V. Martynova; G. N. Daragan; Demkovich, O.O.; U. V. Malakhova; G. I. Zemlyanskaya; Popova, D.M.

    2015-01-01

    The purpose of research is analysis of clinical course and treatment results of patients with cholera in the Azov area. Materials and methods. During the period from 29.05.2011 to 19.08.2011 33 cases of cholera (32 adults and 1 child) and 25 vibrio carriers (22 adults and 3 children), which were caused by toxigenic strains of Vibrio cholera El Tor serogroup O1 Ogawa. Results. Likely factors of disease transmission in Mariupol are sea and river water, and the fish that were caught in the water...

  12. Molecular Epidemiology and Antibiotic Susceptibility of Vibrio cholerae Associated with a Large Cholera Outbreak in Ghana in 2014.

    Science.gov (United States)

    Eibach, Daniel; Herrera-León, Silvia; Gil, Horacio; Hogan, Benedikt; Ehlkes, Lutz; Adjabeng, Michael; Kreuels, Benno; Nagel, Michael; Opare, David; Fobil, Julius N; May, Jürgen

    2016-05-01

    Ghana is affected by regular cholera epidemics and an annual average of 3,066 cases since 2000. In 2014, Ghana experienced one of its largest cholera outbreaks within a decade with more than 20,000 notified infections. In order to attribute this rise in cases to a newly emerging strain or to multiple simultaneous outbreaks involving multi-clonal strains, outbreak isolates were characterized, subtyped and compared to previous epidemics in 2011 and 2012. Serotypes, biotypes, antibiotic susceptibilities were determined for 92 Vibrio cholerae isolates collected in 2011, 2012 and 2014 from Southern Ghana. For a subgroup of 45 isolates pulsed-field gel electrophoresis, multilocus sequence typing and multilocus-variable tandem repeat analysis (MLVA) were performed. Eighty-nine isolates (97%) were identified as ctxB (classical type) positive V. cholerae O1 biotype El Tor and three (3%) isolates were cholera toxin negative non-O1/non-O139 V. cholerae. Among the selected isolates only sulfamethoxazole/trimethoprim resistance was detectable in 2011, while 95% of all 2014 isolates showed resistance towards sulfamethoxazole/trimethoprim, ampicillin and reduced susceptibility to ciprofloxacin. MLVA achieved the highest subtype discrimination, revealing 22 genotypes with one major outbreak cluster in each of the three outbreak years. Apart from those clusters genetically distant genotypes circulate during each annual epidemic. This analysis suggests different endemic reservoirs of V. cholerae in Ghana with distinct annual outbreak clusters accompanied by the occurrence of genetically distant genotypes. Preventive measures for cholera transmission should focus on aquatic reservoirs. Rapidly emerging multidrug resistance must be monitored closely.

  13. Molecular Epidemiology and Antibiotic Susceptibility of Vibrio cholerae Associated with a Large Cholera Outbreak in Ghana in 2014.

    Directory of Open Access Journals (Sweden)

    Daniel Eibach

    2016-05-01

    Full Text Available Ghana is affected by regular cholera epidemics and an annual average of 3,066 cases since 2000. In 2014, Ghana experienced one of its largest cholera outbreaks within a decade with more than 20,000 notified infections. In order to attribute this rise in cases to a newly emerging strain or to multiple simultaneous outbreaks involving multi-clonal strains, outbreak isolates were characterized, subtyped and compared to previous epidemics in 2011 and 2012.Serotypes, biotypes, antibiotic susceptibilities were determined for 92 Vibrio cholerae isolates collected in 2011, 2012 and 2014 from Southern Ghana. For a subgroup of 45 isolates pulsed-field gel electrophoresis, multilocus sequence typing and multilocus-variable tandem repeat analysis (MLVA were performed. Eighty-nine isolates (97% were identified as ctxB (classical type positive V. cholerae O1 biotype El Tor and three (3% isolates were cholera toxin negative non-O1/non-O139 V. cholerae. Among the selected isolates only sulfamethoxazole/trimethoprim resistance was detectable in 2011, while 95% of all 2014 isolates showed resistance towards sulfamethoxazole/trimethoprim, ampicillin and reduced susceptibility to ciprofloxacin. MLVA achieved the highest subtype discrimination, revealing 22 genotypes with one major outbreak cluster in each of the three outbreak years. Apart from those clusters genetically distant genotypes circulate during each annual epidemic.This analysis suggests different endemic reservoirs of V. cholerae in Ghana with distinct annual outbreak clusters accompanied by the occurrence of genetically distant genotypes. Preventive measures for cholera transmission should focus on aquatic reservoirs. Rapidly emerging multidrug resistance must be monitored closely.

  14. Seroepidemiologic survey of epidemic cholera in Haiti to assess spectrum of illness and risk factors for severe disease.

    Science.gov (United States)

    Jackson, Brendan R; Talkington, Deborah F; Pruckler, James M; Fouché, M D Bernadette; Lafosse, Elsie; Nygren, Benjamin; Gómez, Gerardo A; Dahourou, Georges A; Archer, W Roodly; Payne, Amanda B; Hooper, W Craig; Tappero, Jordan W; Derado, Gordana; Magloire, Roc; Gerner-Smidt, Peter; Freeman, Nicole; Boncy, Jacques; Mintz, Eric D

    2013-10-01

    To assess the spectrum of illness from toxigenic Vibrio cholerae O1 and risk factors for severe cholera in Haiti, we conducted a cross-sectional survey in a rural commune with more than 21,000 residents. During March 22-April 6, 2011, we interviewed 2,622 residents ≥ 2 years of age and tested serum specimens from 2,527 (96%) participants for vibriocidal and antibodies against cholera toxin; 18% of participants reported a cholera diagnosis, 39% had vibriocidal titers ≥ 320, and 64% had vibriocidal titers ≥ 80, suggesting widespread infection. Among seropositive participants (vibriocidal titers ≥ 320), 74.5% reported no diarrhea and 9.0% had severe cholera (reported receiving intravenous fluids and overnight hospitalization). This high burden of severe cholera is likely explained by the lack of pre-existing immunity in this population, although the virulence of the atypical El Tor strain causing the epidemic and other factors might also play a role.

  15. Extraintestinal Infections Caused by Non-toxigenic Vibrio cholerae non-O1/non-O139.

    Science.gov (United States)

    Chowdhury, Goutam; Joshi, Sangeeta; Bhattacharya, Sanjay; Sekar, Uma; Birajdar, Balaji; Bhattacharyya, Arpita; Shinoda, Sumio; Ramamurthy, Thandavarayan

    2016-01-01

    Vibrio cholerae is an aerobic, sucrose fermentative Gram-negative bacterium that generally prevails in the environment. Pathogenic V. cholerae is well-known as causative agent of acute diarrhea. Apart from enteric infections, V. cholerae may also cause other diseases. However, their role in causing extraintestinal infections is not fully known as it needs proper identification and evaluation. Four cases of extraintestinal infections due to V. cholerae non-O1/non-O139 have been investigated. The isolates were screened for phenotypic and genetic characteristics with reference to their major virulence genes. Serologically distinct isolates harbored rtx, msh, and hly but lacked enteric toxin encoding genes that are generally present in toxigenic V. cholerae. Timely detection of this organism can prevent fatalities in hospital settings. The underlying virulence potential of V. cholerae needs appropriate testing and intervention.

  16. Genomic analysis of immune response against Vibrio cholerae hemolysin in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Surasri N Sahu

    Full Text Available Vibrio cholerae cytolysin (VCC is among the accessory V. cholerae virulence factors that may contribute to disease pathogenesis in humans. VCC, encoded by hlyA gene, belongs to the most common class of bacterial toxins, known as pore-forming toxins (PFTs. V. cholerae infects and kills Caenorhabditis elegans via cholerae toxin independent manner. VCC is required for the lethality, growth retardation and intestinal cell vacuolation during the infection. However, little is known about the host gene expression responses against VCC. To address this question we performed a microarray study in C. elegans exposed to V. cholerae strains with intact and deleted hlyA genes.Many of the VCC regulated genes identified, including C-type lectins, Prion-like (glutamine [Q]/asparagine [N]-rich-domain containing genes, genes regulated by insulin/IGF-1-mediated signaling (IIS pathway, were previously reported as mediators of innate immune response against other bacteria in C. elegans. Protective function of the subset of the genes up-regulated by VCC was confirmed using RNAi. By means of a machine learning algorithm called FastMEDUSA, we identified several putative VCC induced immune regulatory transcriptional factors and transcription factor binding motifs. Our results suggest that VCC is a major virulence factor, which induces a wide variety of immune response- related genes during V. cholerae infection in C. elegans.

  17. Intracellular trafficking of bacterial toxins.

    Science.gov (United States)

    Williams, Jeffrey M; Tsai, Billy

    2016-08-01

    Bacterial toxins often translocate across a cellular membrane to gain access into the host cytosol, modifying cellular components in order to exert their toxic effects. To accomplish this feat, these toxins traffic to a membrane penetration site where they undergo conformational changes essential to eject the toxin's catalytic subunit into the cytosol. In this brief review, we highlight recent findings that elucidate both the trafficking pathways and membrane translocation mechanisms of toxins that cross the plasma, endosomal, or endoplasmic reticulum (ER) membrane. These findings not only illuminate the specific nature of the host-toxin interactions during entry, but should also provide additional therapeutic strategies to prevent or alleviate the bacterial toxin-induced diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Vibrio cholerae hemolysin is required for lethality, developmental delay, and intestinal vacuolation in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Hediye Nese Cinar

    Full Text Available BACKGROUND: Cholera toxin (CT and toxin-co-regulated pili (TCP are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations. CONCLUSIONS/SIGNIFICANCE: Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.

  19. Induction of Protective Immune Response in Mice and Rhesus Monkeys by Immunization with Fusion Protein of Cholera Toxin B Subunit and Multiples of Plasmodium falciparum%重组霍乱毒素B亚基与疟原虫多表位融合蛋白 的免疫保护作用研究

    Institute of Scientific and Technical Information of China (English)

    曹诚; 李平; 石成华; 钟辉; 时运林; 李杰之; 马清钧

    2000-01-01

    Recombinant fusion protein of cholera toxin B subunit (CTB) and poly-valent protective epitopes of plasmodium falciparum was given to i. m to C57BL/6j mice and rhesus monkeys three times. In rhesus monkeys, high level of antibodies for CTB (1:6400) and malaria epitopes (1:3200) amtobpdoes were elicited as well as the specific CTL activity for P. plasmodium. After the mice were challenged with sporozoites of P. yeolli, about 50 % of them were protected from the patent infection. A blood-stage challenge with 108 of P. cynomolgi parasite were given to rhesus monkeys, which showed that two animals in control group were patent infection for at least 30 days, in contrast, the two animals immunized were recovered respectively at the day of 11 and 15 after challenges. The results suggested that cholera toxin acts as an effective adjuvent in the development of malaria vaccine.%对以霍乱毒素B亚基为载体蛋白的重组疟疾多价抗原在小鼠及恒河猴中的免疫原性及对相应疟原虫感染的免疫保护作用进行了研究。结果表明:该抗原免疫小鼠后,对约氏疟子孢子攻击的保护率在50%左右;恒河猴免疫后用1×108食蟹疟裂殖子攻击,对照组2只动物在攻击后4d感染,感染持续30d以上;免疫组2只动物中,两只动物在感染6~7d后完全恢复,且1只推迟3d感染,说明该抗原具有免疫保护作用。

  20. Detection of toxigenic vibrio cholera from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

    CSIR Research Space (South Africa)

    Theron, J

    2000-09-01

    Full Text Available A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic...

  1. Cholera Fact Sheet

    Science.gov (United States)

    ... facilities (chlorination) interventions at the household level (water filtration, chemical or solar disinfection of water, safe water ... spread of cholera and contributes to increasing antimicrobial resistance. Rapid access to treatment is essential during a ...

  2. Cholera outbreaks in Africa.

    Science.gov (United States)

    Mengel, Martin A; Delrieu, Isabelle; Heyerdahl, Leonard; Gessner, Bradford D

    2014-01-01

    During the current seventh cholera pandemic, Africa bore the major brunt of global disease burden. More than 40 years after its resurgence in Africa in 1970, cholera remains a grave public health problem, characterized by large disease burden, frequent outbreaks, persistent endemicity, and high CFRs, particularly in the region of the central African Great Lakes which might act as reservoirs for cholera. There, cases occur year round with a rise in incidence during the rainy season. Elsewhere in sub-Saharan Africa, cholera occurs mostly in outbreaks of varying size with a constant threat of widespread epidemics. Between 1970 and 2011, African countries reported 3,221,050 suspected cholera cases to the World Health Organization, representing 46 % of all cases reported globally. Excluding the Haitian epidemic, sub-Saharan Africa accounted for 86 % of reported cases and 99 % of deaths worldwide in 2011. The number of cholera cases is possibly much higher than what is reported to the WHO due to the variation in modalities, completeness, and case definition of national cholera data. One source on country specific incidence rates for Africa, adjusting for underreporting, estimates 1,341,080 cases and 160,930 deaths (52.6 % of 2,548,227 estimated cases and 79.6 % of 209,216 estimated deaths worldwide). Another estimates 1,411,453 cases and 53,632 deaths per year, respectively (50 % of 2,836,669 estimated cases and 58.6 % of 91,490 estimated deaths worldwide). Within Africa, half of all cases between 1970 and 2011 were notified from only seven countries: Angola, Democratic Republic of the Congo, Mozambique, Nigeria, Somalia, Tanzania, and South Africa. In contrast to a global trend of decreasing case fatality ratios (CFRs), CFRs have remained stable in Africa at approximately 2 %. Early propagation of cholera outbreaks depends largely on the extent of individual bacterial shedding, host and organism characteristics, the likelihood of people coming into contact with

  3. Regional cholera response discussion

    CSIR Research Space (South Africa)

    Funke, Nicola S

    2009-09-30

    Full Text Available research questions identified by the CSIR on cholera as a trans- or cross-border communicable disease that requires complex interventions in terms of preparedness and response, management, prevention and mitigation. The workshop commenced with a...

  4. Cholera in Azov area

    Directory of Open Access Journals (Sweden)

    O. N. Domashenko

    2015-01-01

    Full Text Available The purpose of research is analysis of clinical course and treatment results of patients with cholera in the Azov area. Materials and methods. During the period from 29.05.2011 to 19.08.2011 33 cases of cholera (32 adults and 1 child and 25 vibrio carriers (22 adults and 3 children, which were caused by toxigenic strains of Vibrio cholera El Tor serogroup O1 Ogawa. Results. Likely factors of disease transmission in Mariupol are sea and river water, and the fish that were caught in the waters of the city. Typical and watery diarrhoea, vomiting, abdominal pain and lack of normal body temperature, dehydration syndrome, characterized clinical cholera for adults in most cases. The mean duration of diarrhoea was 6,6 days. At 46.9% observed atypical symptoms in 10 (31,3% – abdominal pain (1 patient cramping in 7 cases, localized in the epigastria region, at 2-over stomach. In 5 patients (15,6% had an increase in body temperature to 37,2–37,7 degrees Celsius. In 15 (46,9% patients had severe nausea accompanied by vomiting. Easy for cholera was observed in 1 (3.1%, moderate – in 14 (43,8%, heavy – in 17 (53,1% patients. Dehydration I level is set at 4 (12,5%, II – from 6 (18,7%, III – in 18 (56,3%, IV – 4 (12,5% patients. Cholera outbreak was characterized by a predominance of severe disease and severe dehydration (III and IV, which was observed in 68.8% of patients. The decisive factor in the treatment of cholera patients was initiated in a timely manner rehydration therapy, in particular the introduction of the solution «Trisol». Against the background of rehydration therapy hyperkalaemia was observed in 9,4% of cases, vascular rehydration at 9,4%, the cell rehydration in 3,1% of patients. Fatal accidents cholera outbreaks have not been observed. Conclusion. Clinical diagnosis of cholera and the provision of medical care in the prehospital phase were poor, indicating the need for systematic conducting training seminars among experts

  5. Retrograde trafficking of AB₅ toxins: mechanisms to therapeutics.

    Science.gov (United States)

    Mukhopadhyay, Somshuvra; Linstedt, Adam D

    2013-10-01

    Bacterial AB5 toxins are a clinically relevant class of exotoxins that include several well-known members such as Shiga, cholera, and pertussis toxins. Infections with toxin-producing bacteria cause devastating human diseases that affect millions of individuals each year and have no definitive medical treatment. The molecular targets of AB5 toxins reside in the cytosol of infected cells, and the toxins reach the cytosol by trafficking through the retrograde membrane transport pathway that avoids degradative late endosomes and lysosomes. Focusing on Shiga toxin as the archetype member, we review recent advances in understanding the molecular mechanisms involved in the retrograde trafficking of AB5 toxins and highlight how these basic science advances are leading to the development of a promising new therapeutic approach based on inhibiting toxin transport.

  6. Promoter activities in Vibrio cholerae ctx phi prophage.

    Science.gov (United States)

    Fando, R; Pérez, J L; Rodriguez, B L; Campos, J; Robert, A; García, L; Silva, A; Benitez, J A

    1997-04-01

    Comparison of cholera toxin (CT) production directed by different gene constructs and S1 nuclease mapping revealed the presence of a ctxB-specific promoter within the ctxA coding sequence. Initiation of transcription in this region occurred in wild-type El Tor and classical biotype choleragenic vibrios. We propose that transcription from the ctxB-specific promoter and a stronger ribosomal binding site on the ctxB mRNA synergistically contribute to achieve the correct (5B:1A) subunit stoichiometry. Plasmid pB, a CT promoterless vector expressing only CTB, was used to detect promoter activity by restoration of A-subunit synthesis. Promoter activity expressed in vitro and in vivo was detected upstream of the zonula occludens toxin gene, suggesting that this factor could be produced in vivo to contribute to fluid accumulation. No promoter activity was detected in vitro and in vivo upstream from the accessory cholera enterotoxin gene.

  7. Grape extracts inhibit multiple events in the cell biology of cholera intoxication.

    Directory of Open Access Journals (Sweden)

    Srikar Reddy

    Full Text Available Vibrio cholerae produces cholera toxin (CT, an AB5 protein toxin that is primarily responsible for the profuse watery diarrhea of cholera. CT is secreted into the extracellular milieu, but the toxin attacks its Gsα target within the cytosol of a host cell. Thus, CT must cross a cellular membrane barrier in order to function. This event only occurs after the toxin travels by retrograde vesicular transport from the cell surface to the endoplasmic reticulum (ER. The catalytic A1 polypeptide then dissociates from the rest of the toxin and assumes an unfolded conformation that facilitates its transfer to the cytosol by a process involving the quality control system of ER-associated degradation. Productive intoxication is blocked by alterations to the vesicular transport of CT and/or the ER-to-cytosol translocation of CTA1. Various plant compounds have been reported to inhibit the cytopathic activity of CT, so in this work we evaluated the potential anti-CT properties of grape extract. Two grape extracts currently sold as nutritional supplements inhibited CT and Escherichia coli heat-labile toxin activity against cultured cells and intestinal loops. CT intoxication was blocked even when the extracts were added an hour after the initial toxin exposure. A specific subset of host-toxin interactions involving both the catalytic CTA1 subunit and the cell-binding CTB pentamer were affected. The extracts blocked toxin binding to the cell surface, prevented unfolding of the isolated CTA1 subunit, inhibited CTA1 translocation to the cytosol, and disrupted the catalytic activity of CTA1. Grape extract could thus potentially serve as a novel therapeutic to prevent or possibly treat cholera.

  8. Origins of the current seventh cholera pandemic.

    Science.gov (United States)

    Hu, Dalong; Liu, Bin; Feng, Lu; Ding, Peng; Guo, Xi; Wang, Min; Cao, Boyang; Reeves, Peter R; Wang, Lei

    2016-11-29

    Vibrio cholerae has caused seven cholera pandemics since 1817, imposing terror on much of the world, but bacterial strains are currently only available for the sixth and seventh pandemics. The El Tor biotype seventh pandemic began in 1961 in Indonesia, but did not originate directly from the classical biotype sixth-pandemic strain. Previous studies focused mainly on the spread of the seventh pandemic after 1970. Here, we analyze in unprecedented detail the origin, evolution, and transition to pandemicity of the seventh-pandemic strain. We used high-resolution comparative genomic analysis of strains collected from 1930 to 1964, covering the evolution from the first available El Tor biotype strain to the start of the seventh pandemic. We define six stages leading to the pandemic strain and reveal all key events. The seventh pandemic originated from a nonpathogenic strain in the Middle East, first observed in 1897. It subsequently underwent explosive diversification, including the spawning of the pandemic lineage. This rapid diversification suggests that, when first observed, the strain had only recently arrived in the Middle East, possibly from the Asian homeland of cholera. The lineage migrated to Makassar, Indonesia, where it gained the important virulence-associated elements Vibrio seventh pandemic island I (VSP-I), VSP-II, and El Tor type cholera toxin prophage by 1954, and it then became pandemic in 1961 after only 12 additional mutations. Our data indicate that specific niches in the Middle East and Makassar were important in generating the pandemic strain by providing gene sources and the driving forces for genetic events.

  9. Genome sequencing reveals unique mutations in characteristic metabolic pathways and the transfer of virulence genes between V. mimicus and V. cholerae.

    Science.gov (United States)

    Wang, Duochun; Wang, Haiyin; Zhou, Yanyan; Zhang, Qiuxiang; Zhang, Fanfei; Du, Pengcheng; Wang, Shujing; Chen, Chen; Kan, Biao

    2011-01-01

    Vibrio mimicus, the species most similar to V. cholerae, is a microbe present in the natural environmental and sometimes causes diarrhea and internal infections in humans. It shows similar phenotypes to V. cholerae but differs in some biochemical characteristics. The molecular mechanisms underlying the differences in biochemical metabolism between V. mimicus and V. cholerae are currently unclear. Several V. mimicus isolates have been found that carry cholera toxin genes (ctxAB) and cause cholera-like diarrhea in humans. Here, the genome of the V. mimicus isolate SX-4, which carries an intact CTX element, was sequenced and annotated. Analysis of its genome, together with those of other Vibrio species, revealed extensive differences within the Vibrionaceae. Common mutations in gene clusters involved in three biochemical metabolism pathways that are used for discrimination between V. mimicus and V. cholerae were found in V. mimicus strains. We also constructed detailed genomic structures and evolution maps for the general types of genomic drift associated with pathogenic characters in polysaccharides, CTX elements and toxin co-regulated pilus (TCP) gene clusters. Overall, the whole-genome sequencing of the V. mimicus strain carrying the cholera toxin gene provides detailed information for understanding genomic differences among Vibrio spp. V. mimicus has a large number of diverse gene and nucleotide differences from its nearest neighbor, V. cholerae. The observed mutations in the characteristic metabolism pathways may indicate different adaptations to different niches for these species and may be caused by ancient events in evolution before the divergence of V. cholerae and V. mimicus. Horizontal transfers of virulence-related genes from an uncommon clone of V. cholerae, rather than the seventh pandemic strains, have generated the pathogenic V. mimicus strain carrying cholera toxin genes.

  10. A组轮状病毒VP6与霍乱毒素B亚基融合蛋白在大肠杆菌中的表达及生物活性分析%A Fusion Protein of Rotavirus VP6 and Cholera Toxin B Subunit:Expression in Escherichia coli and Analysis of Biological Activities

    Institute of Scientific and Technical Information of China (English)

    郭婷夏; 方荣祥; 李国华; 钱渊

    2001-01-01

    利用霍乱毒素B亚基(Cholera toxin B subunit,CTB)的免疫载体作用,将轮状病毒相关抗原引入口服免疫体系,可激起有效的粘膜免疫反应,这里报道了 CTB基因与A组轮状病毒地方株T114 VP6全基因的融合,并在大肠杆菌BI21(DE3)中进行了融合蛋白的表达.在IPTG诱导下得到分子量为56 kD的融合蛋白,表达量占菌体蛋白的15%.分别用抗CT的抗体和抗A组轮状病毒的高价免疫血清进行Western Blot检测,结果证明融合蛋白CTB-VP6保留了天然霍乱毒素R亚基及轮状病毒VP6的抗原性.GMi-ELISA检测表明,复性后的融合蛋白具有与神经节苷脂GMI结合的能力.

  11. The Vibrio cholerae cytolysin promotes chloride secretion from intact human intestinal mucosa.

    Directory of Open Access Journals (Sweden)

    Lucantonio Debellis

    Full Text Available BACKGROUND: The pathogenicity of the Vibrio cholerae strains belonging to serogroup O1 and O139 is due to the production of virulence factors such as cholera toxin (CT and the toxin-coregulated pilus (TCP. The remaining serogroups, which mostly lack CT and TCP, are more frequently isolated from aquatic environmental sources than from clinical samples; nevertheless, these strains have been reported to cause human disease, such as sporadic outbreaks of watery diarrhoea and inflammatory enterocolitis. This evidence suggested the possibility that other virulence factor(s than cholera toxin might be crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea, but their nature remains unknown. VCC, the hemolysin produced by virtually all Vibrio cholerae strains, has been proposed as a possible candidate, though a clear-cut demonstration attesting VCC as crucial in the pathogenesis of Vibrio cholerae-induced diarrhoea is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: Electrophysiological parameters and paracellular permeability of stripped human healthy colon tissues, obtained at subtotal colectomy, mounted in Ussing chamber were studied in the presence or absence of VCC purified from culture supernatants of V. cholerae O1 El Tor strain. Short circuit current (I(SC and transepithelial resistance (R(T were measured by a computerized voltage clamp system. The exposure of sigmoid colon specimens to 1 nM VCC resulted in an increase of I(SC by 20.7%, with respect to the basal values, while R(T was reduced by 12.3%. Moreover, increase in I(SC was abolished by bilateral Cl(- reduction. CONCLUSION/SIGNIFICANCE: Our results demonstrate that VCC, by forming anion channels on the apical membrane of enterocytes, triggers an outward transcellular flux of chloride. Such an ion movement, associated with the outward movement of Na(+ and water, might be responsible for the diarrhoea caused by the non-toxigenic strains of Vibrio cholerae.

  12. A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles.

    Science.gov (United States)

    Leitner, Deborah R; Lichtenegger, Sabine; Temel, Philipp; Zingl, Franz G; Ratzberger, Desiree; Roier, Sandro; Schild-Prüfert, Kristina; Feichter, Sandra; Reidl, Joachim; Schild, Stefan

    2015-01-01

    Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs) derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS)-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB) independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I, and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo.

  13. A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles

    Directory of Open Access Journals (Sweden)

    Deborah Raphaela Leitner

    2015-08-01

    Full Text Available Enteric infections induced by pathogens like Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC remain a massive burden in developing countries with increasing morbidity and mortality rates. Previously, we showed that the immunization with genetically detoxified outer membrane vesicles (OMVs derived from V. cholerae elicits a protective immune response based on the generation of O antigen antibodies, which effectively block the motility by binding to the sheathed flagellum. In this study, we investigated the potential of lipopolysaccharide (LPS-modified and toxin negative OMVs isolated from V. cholerae and ETEC as a combined OMV vaccine candidate. Our results indicate that the immunization with V. cholerae or ETEC OMVs induced a species-specific immune response, whereas the combination of both OMV species resulted in a high-titer, protective immune response against both pathogens. Interestingly, the immunization with V. cholerae OMVs alone resulted in a so far uncharacterized and cholera toxin B-subunit (CTB independent protection mechanism against an ETEC colonization. Furthermore, we investigated the potential use of V. cholerae OMVs as delivery vehicles for the heterologously expression of the ETEC surface antigens, CFA/I and FliC. Although we induced a detectable immune response against both heterologously expressed antigens, none of these approaches resulted in an improved protection compared to a simple combination of V. cholerae and ETEC OMVs. Finally, we expanded the current protection model from V. cholerae to ETEC by demonstrating that the inhibition of motility via anti-FliC antibodies represents a relevant protection mechanism of an OMV-based ETEC vaccine candidate in vivo.

  14. Evaluation in mice of a conjugate vaccine for cholera made from Vibrio cholerae O1 (Ogawa) O-specific polysaccharide.

    Science.gov (United States)

    Alam, Mohammad Murshid; Bufano, Megan Kelly; Xu, Peng; Kalsy, Anuj; Yu, Y; Freeman, Y Wu; Sultana, Tania; Rashu, Md Rasheduzzaman; Desai, Ishaan; Eckhoff, Grace; Leung, Daniel T; Charles, Richelle C; LaRocque, Regina C; Harris, Jason B; Clements, John D; Calderwood, Stephen B; Qadri, Firdausi; Vann, W F; Kováč, Pavol; Ryan, Edward T

    2014-02-01

    Protective immunity against cholera is serogroup specific. Serogroup specificity in Vibrio cholerae is determined by the O-specific polysaccharide (OSP) of lipopolysaccharide (LPS). Generally, polysaccharides are poorly immunogenic, especially in young children. Here we report the evaluation in mice of a conjugate vaccine for cholera (OSP:TThc) made from V. cholerae O1 Ogawa O-Specific Polysaccharide-core (OSP) and recombinant tetanus toxoid heavy chain fragment (TThc). We immunized mice intramuscularly on days 0, 21, and 42 with OSP:TThc or OSP only, with or without dmLT, a non-toxigenic immunoadjuvant derived from heat labile toxin of Escherichia coli. We detected significant serum IgG antibody responses targeting OSP following a single immunization in mice receiving OSP:TThc with or without adjuvant. Anti-LPS IgG responses were detected following a second immunization in these cohorts. No anti-OSP or anti-LPS IgG responses were detected at any time in animals receiving un-conjugated OSP with or without immunoadjuvant, and in animals receiving immunoadjuvant alone. Responses were highest following immunization with adjuvant. Serum anti-OSP IgM responses were detected in mice receiving OSP:TThc with or without immunoadjuvant, and in mice receiving unconjugated OSP. Serum anti-LPS IgM and vibriocidal responses were detected in all vaccine cohorts except in mice receiving immunoadjuvant alone. No significant IgA anti-OSP or anti-LPS responses developed in any group. Administration of OSP:TThc and adjuvant also induced memory B cell responses targeting OSP and resulted in 95% protective efficacy in a mouse lethality cholera challenge model. We describe a protectively immunogenic cholera conjugate in mice. Development of a cholera conjugate vaccine could assist in inducing long-term protective immunity, especially in young children who respond poorly to polysaccharide antigens.

  15. The global burden of cholera

    Science.gov (United States)

    Lopez, Anna Lena; You, Young Ae; Kim, Young Eun; Sah, Binod; Maskery, Brian; Clemens, John

    2012-01-01

    Abstract Objective To estimate the global burden of cholera using population-based incidence data and reports. Methods Countries with a recent history of cholera were classified as endemic or non-endemic, depending on whether they had reported cholera cases in at least three of the five most recent years. The percentages of the population in each country that lacked access to improved sanitation were used to compute the populations at risk for cholera, and incidence rates from published studies were applied to groups of countries to estimate the annual number of cholera cases in endemic countries. The estimates of cholera cases in non-endemic countries were based on the average numbers of cases reported from 2000 to 2008. Literature-based estimates of cholera case-fatality rates (CFRs) were used to compute the variance-weighted average cholera CFRs for estimating the number of cholera deaths. Findings About 1.4 billion people are at risk for cholera in endemic countries. An estimated 2.8 million cholera cases occur annually in such countries (uncertainty range: 1.4–4.3) and an estimated 87 000 cholera cases occur in non-endemic countries. The incidence is estimated to be greatest in children less than 5 years of age. Every year about 91 000 people (uncertainty range: 28 000 to 142 000) die of cholera in endemic countries and 2500 people die of the disease in non-endemic countries. Conclusion The global burden of cholera, as determined through a systematic review with clearly stated assumptions, is high. The findings of this study provide a contemporary basis for planning public health interventions to control cholera. PMID:22461716

  16. The global burden of cholera.

    Science.gov (United States)

    Ali, Mohammad; Lopez, Anna Lena; You, Young Ae; Kim, Young Eun; Sah, Binod; Maskery, Brian; Clemens, John

    2012-03-01

    To estimate the global burden of cholera using population-based incidence data and reports. Countries with a recent history of cholera were classified as endemic or non-endemic, depending on whether they had reported cholera cases in at least three of the five most recent years. The percentages of the population in each country that lacked access to improved sanitation were used to compute the populations at risk for cholera, and incidence rates from published studies were applied to groups of countries to estimate the annual number of cholera cases in endemic countries. The estimates of cholera cases in non-endemic countries were based on the average numbers of cases reported from 2000 to 2008. Literature-based estimates of cholera case-fatality rates (CFRs) were used to compute the variance-weighted average cholera CFRs for estimating the number of cholera deaths. About 1.4 billion people are at risk for cholera in endemic countries. An estimated 2.8 million cholera cases occur annually in such countries (uncertainty range: 1.4-4.3) and an estimated 87,000 cholera cases occur in non-endemic countries. The incidence is estimated to be greatest in children less than 5 years of age. Every year about 91,000 people (uncertainty range: 28,000 to 142,000) die of cholera in endemic countries and 2500 people die of the disease in non-endemic countries. The global burden of cholera, as determined through a systematic review with clearly stated assumptions, is high. The findings of this study provide a contemporary basis for planning public health interventions to control cholera.

  17. 口服Ⅱ型胶原肽-霍乱毒素B亚单位-脂质体复合物诱导免疫耐受的研究%The research of tolerance induced by oral collagen type Ⅱ peptide-cholera toxin B subunit-liposome complex

    Institute of Scientific and Technical Information of China (English)

    毕莹; 赵欣欣; 张宁; 王宗谦

    2012-01-01

    Objective To investigate oral collagen type Ⅱ peptide-cholera toxin B subunit-liposome complex induce tolerance to collagen-induced arthritis (CIA).Methods DBA/1 mice were divided into four groups,group Ⅰ:normal control,group Ⅱ:CIA control,group Ⅲ:oral collagen type Ⅱ peptidecholera toxin B subunit-liposome complex,and group Ⅳ:for testing IgG2a (on day 14 after primary immunization).Arthritis scores and histopathologic assessment were analyzed.The levels of serum IgG2a were examined by ELISA.Results There was no arthritis development in group Ⅰ.The incidence of arthritis in group Ⅱ was higher than that in group Ⅲ ( 100% vs 28.6%,P<0.05).The arthritis score in group Ⅱ was higher than that in group Ⅲ (5.40 vs 0.4,3,P<0.01).Histopathologic score was higher in group Ⅱ than that in group Ⅲ (16.00 vs 2.85,p<0.05).Level of serum IgG2a of group Ⅰ was very 1ow(38 ng/ml).Mice of group Ⅱ produced significantly higher level of IgG2a than mice of group Ⅲ (3922 ng/ml vs 3219ng/ml,P<0.05).IgG2a of group Ⅳ was 98 ng/ml which was significantly higher than that of group Ⅰ (P<0.01 ).Conclusion Oral collagen type Ⅱ peptide-cholera toxin B subunit-liposome complex could inhibit CIA progression through immune tolerance.%目的 观察口服Ⅱ型胶原肽-霍乱毒素B亚单位-脂质体复合物对免疫耐受的诱导作用.方法 DBA/1小鼠分组,Ⅰ组:正常对照组,Ⅱ组:胶原诱导关节炎对照组,Ⅲ组:口服Ⅱ型胶原肽-霍乱毒素B亚单位-脂质体复合物组,Ⅳ组:免疫后14 d测定IgG2a组.记录分析关节炎评分及组织病理学评分,采用ELISA方法测定血清IgG2a水平.结果 正常对照组小鼠无关节炎发生.Ⅱ组关节炎发生率显著高于Ⅲ组(100% vs 28.6%,P<0.05);Ⅱ组关节炎评分显著高于Ⅲ组(5.40 vs0.43,P<0.01);Ⅱ组关节炎组织病理累计评分显著高于Ⅲ组(16.00 vs 2.85,P<0.05).Ⅰ组IgG2a水平极低,为38 ng/ml,Ⅱ组IgG2a

  18. The increased severity in patients presenting to hospital with diarrhea in Dhaka, Bangladesh since the emergence of the hybrid strain of Vibrio cholerae O1 is not unique to cholera patients.

    Science.gov (United States)

    Chowdhury, Fahima; Kuchta, Alison; Khan, Ashraful Islam; Faruque, A S G; Calderwood, Stephen B; Ryan, Edward T; Qadri, Firdausi

    2015-11-01

    A hybrid strain of Vibrio cholerae O1 El Tor that expresses a classical cholera toxin (CT) emerged in 2001. This hybrid variant rapidly replaced the previous El Tor strain around the world. The global emergence of this variant coincided with anecdotal reports that cholera patients were presenting with more severe dehydration and disease in many locations. A comparison was made of the severity of disease before and after the emergence of the hybrid strain in cholera patients attending an icddr,b hospital in Dhaka, Bangladesh. It was found that cholera patients presented with more severe dehydration and severe disease in the later period. However, this was also true for all non-cholera patients as well. In addition, in sub-analyses of patients who presented with rotavirus and enterotoxigenic Escherichia coli (ETEC), similar results were found. Comparing the two periods for differences in patient characteristics, nutritional status, vaccination status, and income, no plausible cause for patients presenting with more severe disease was identified in the later period. As a shift in severity for both cholera and non-cholera was observed, these results indicate that the altered El Tor strain cannot fully explain the difference in cholera severity before and after 2001. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Increased severity in patients presenting to hospital with diarrhea in Dhaka, Bangladesh Since emergence of the hybrid strain of Vibrio cholerae O1 is not unique to cholera patients

    Science.gov (United States)

    Chowdhury, Fahima; Kuchta, Alison; Khan, Ashraful Islam; Faruque, ASG; Calderwood, Stephen B.; Ryan, Edward T.; Qadri, Firdausi

    2015-01-01

    In 2001, a hybrid strain of Vibrio cholerae O1 El Tor that expresses a classical cholera toxin (CT) emerged and this hybrid variant rapidly replaced the previous El Tor strain around the world. The global emergence of this variant coincided with anecdotal reports that cholera patients were presenting with more severe dehydration and disease in many locations. We compared severity of disease in cholera patients from before and after emergence of the hybrid strain at a diarrheal hospital in Dhaka, Bangladesh. We did indeed find that cholera patients presented with more severe dehydration and severe disease in the latter period; however, this was also true for “all non-cholera patients” as well. In addition, in sub-analyses of patients who presented with rotavirus and enterotoxigenic E. coli (ETEC), we found similar results. Comparing the two periods for differences in patient characteristics, nutritional status, vaccination status and income, we were unable to detect a plausible cause for patients presenting with more severe disease in the latter period. Because we observed a shift in severity for both cholera and non-cholera, our results indicate that the altered El Tor strain cannot fully explain the differences in cholera severity before and after 2001 PMID:26409202

  20. Host intestinal signal-promoted biofilm dispersal induces Vibrio cholerae colonization.

    Science.gov (United States)

    Hay, Amanda J; Zhu, Jun

    2015-01-01

    Vibrio cholerae causes human infection through ingestion of contaminated food and water, leading to the devastating diarrheal disease cholera. V. cholerae forms matrix-encased aggregates, known as biofilms, in the native aquatic environment. While the formation of V. cholerae biofilms has been well studied, little is known about the dispersal from biofilms, particularly upon entry into the host. In this study, we found that the exposure of mature biofilms to physiologic levels of the bile salt taurocholate, a host signal for the virulence gene induction of V. cholerae, induces an increase in the number of detached cells with a concomitant decrease in biofilm mass. Scanning electron microscopy micrographs of biofilms exposed to taurocholate revealed an altered, perhaps degraded, appearance of the biofilm matrix. The inhibition of protein synthesis did not alter rates of detachment, suggesting that V. cholerae undergoes a passive dispersal. Cell-free media from taurocholate-exposed biofilms contains a larger amount of free polysaccharide, suggesting an abiotic degradation of biofilm matrix by taurocholate. Furthermore, we found that V. cholerae is only able to induce virulence in response to taurocholate after exit from the biofilm. Thus, we propose a model in which V. cholerae ingested as a biofilm has coopted the host-derived bile salt signal to detach from the biofilm and go on to activate virulence.

  1. The Influence of Ligand Valency on Aggregation Mechanisms for Inhibiting Bacterial Toxins

    NARCIS (Netherlands)

    Sisu, C.; Baron, A.J.; Branderhorst, H.M.; Connell, S.D.; Weijers, C.A.G.M.; Vries, de R.; Hayes, E.D.; Pukin, A.V.; Gilbert, M.; Pieters, R.J.; Zuilhof, H.; Visser, G.M.; Turnbull, W.B.

    2009-01-01

    Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat-labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic

  2. Physicochemical constraints of elevated pH affect efficient membrane interaction and arrest an abortive membrane-bound oligomeric intermediate of the beta-barrel pore-forming toxin Vibrio cholerae cytolysin.

    Science.gov (United States)

    Rai, Anand Kumar; Kundu, Nidhi; Chattopadhyay, Kausik

    2015-10-01

    Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging cytotoxic protein. VCC causes permeabilization of the target cell membranes by forming transmembrane oligomeric beta-barrel pores. Membrane pore formation by VCC involves following key steps: (i) membrane binding, (ii) formation of a pre-pore oligomeric intermediate, (iii) membrane insertion of the pore-forming motifs, and (iv) formation of the functional transmembrane pore. Membrane binding, oligomerization, and subsequent pore-formation process of VCC appear to be facilitated by multiple regulatory mechanisms that are only partly understood. Here, we have explored the role(s) of the physicochemical constraints, specifically imposed by the elevated pH conditions, on the membrane pore-formation mechanism of VCC. Elevated pH abrogates efficient interaction of VCC with the target membranes, and blocks its pore-forming activity. Under the elevated pH conditions, membrane-bound fractions of VCC remain trapped in the form of abortive oligomeric species that fail to generate the functional transmembrane pores. Such an abortive oligomeric assembly appears to represent a distinct, more advanced intermediate state than the pre-pore state. The present study offers critical insights regarding the implications of the physicochemical constraints for regulating the efficient membrane interaction and pore formation by VCC.

  3. Genomic science in understanding cholera outbreaks and evolution of Vibrio cholerae as a human pathogen.

    Science.gov (United States)

    Robins, William P; Mekalanos, John J

    2014-01-01

    Modern genomic and bioinformatic approaches have been applied to interrogate the V. cholerae genome, the role of genomic elements in cholera disease, and the origin, relatedness, and dissemination of epidemic strains. A universal attribute of choleragenic strains includes a repertoire of pathogenicity islands and virulence genes, namely the CTXϕ prophage and Toxin Co-regulated Pilus (TCP) in addition to other virulent genetic elements including those referred to as Seventh Pandemic Islands. During the last decade, the advent of Next Generation Sequencing (NGS) has provided highly resolved and often complete genomic sequences of epidemic isolates in addition to both clinical and environmental strains isolated from geographically unconnected regions. Genomic comparisons of these strains, as was completed during and following the Haitian outbreak in 2010, reveals that most epidemic strains appear closely related, regardless of region of origin. Non-O1 clinical or environmental strains may also possess some virulence islands, but phylogenic analysis of the core genome suggests they are more diverse and distantly related than those isolated during epidemics. Like Haiti, genomic studies that examine both the Vibrio core and pan-genome in addition to Single Nucleotide Polymorphisms (SNPs) conclude that a number of epidemics are caused by strains that closely resemble those in Asia, and often appear to originate there and then spread globally. The accumulation of SNPs in the epidemic strains over time can then be applied to better understand the evolution of the V. cholerae genome as an etiological agent.

  4. [Production and characteristics of monoclonal antibodies to the diphtheria toxin].

    Science.gov (United States)

    Valiakina, T I; Lakhtina, O E; Komaleva, R L; Simonova, M A; Samokhvalova, L V; Shoshina, N S; Kalinina, N A; Rubina, A Iu; Filippova, M A; Vertiev, Iu V; Grishin, E V

    2009-01-01

    Monoclonal antibodies to the diphtheria toxin were produced without cross reactivity with the thermolabile toxin (LT) from Escherichia coli; ricin; choleraic toxin; the SeA, SeB, SeE, SeI, and SeG toxins of staphylococcus; the lethal factor of the anthrax toxin; and the protective antigen of the anthrax toxin. A pair of antibodies for the quantitative determination of the diphtheria toxin in the sandwich variation of enzyme-linked immunosorbent assay (ELISA) was chosen. The determination limit of the toxin was 0.7 ng/ml in plate and 1.6 ng/ml in microchip ELISA. The presence of a secretion from the nasopharynx lavage did not decrease the sensitivity of the toxin determination by sandwich ELISA. The immunization of mice with the diphtheria toxin and with a conjugate of the diphtheria toxin with polystyrene microspheres demonstrated that the conjugate immunization resulted in the formation of hybridoma clones which produced antibodies only to the epitopes of the A fragment of the diphtheria toxin. The immunization with the native toxin caused the production of hybridoma clones which predominantly produced antibodies to the epitopes of the B fragment.

  5. Biological Activity of Recombinant Accessory Cholerae Enterotoxin (Ace on Rabbit Ileal Loops and Antibacterial Assay

    Directory of Open Access Journals (Sweden)

    Shaghayegh Anvari

    2012-01-01

    Full Text Available Objective: Vibrio cholerae (V. cholerae causes a potentially lethal disease named cholera. The cholera enterotoxin (CT is a major virulence factor of V. cholerae. In addition to CT, V. cholerae produces other putative toxins, such as the zonula occludens toxin (Zot and accessory cholera enterotoxin (Ace. The ace gene is the third gene of the V. cholerae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli (E. coli and determination of some characteristics of the recombinant Ace protein.Materials and Methods: In this experimental study, the ace gene was amplified from V. cholerae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli (DH5 α host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-β-D-galctoside (IPTG at a different concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus (S. aureus, and Pseudomonas aeruginosa (P. aeruginosa.Results: The recombinant Ace protein with a molecular weight of 18 kDa (dimeric form was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. cholerae polyclonal antibody. The Ace protein had antibacterial activity at a concentration of ≥200 μg/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test.Conclusion: This study described an E. coli cloning and expression system (E. coli BL21- pET-28a-ace for the Ace protein of V. cholerae. We confirmed the antibacterial properties and enterotoxin

  6. A case of non-O1/non-O139 Vibrio cholerae septicemia and meningitis in a neonate

    Directory of Open Access Journals (Sweden)

    Yingying Hao

    2015-06-01

    Full Text Available A case of septicemia with meningitis due to non-O1/non-O139 Vibrio cholerae in a neonate is reported. The genotype and phenotype of the isolate were examined in relation to the major virulence genes. The isolate was shown to be non-toxin but cytotoxin-producing, distinguished from the dominant clone of non-O1/non-O139 V. cholerae by multilocus sequence typing.

  7. Towards new cholera prophylactics and treatment: Crystal structures of bacterial enterotoxins in complex with GM1 mimics

    OpenAIRE

    Julie Elisabeth Heggelund; Alasdair Mackenzie; Tobias Martinsen; Joel Benjamin Heim; Pavel Cheshev; Anna Bernardi; Ute Krengel

    2017-01-01

    Cholera is a life-threatening disease in many countries, and new drugs are clearly needed. C-glycosidic antagonists may serve such a purpose. Here we report atomic-resolution crystal structures of three such compounds in complexes with the cholera toxin. The structures give unprecedented atomic details of the molecular interactions and show how the inhibitors efficiently block the GM1 binding site. These molecules are well suited for development into low-cost prophylactic drugs, due to their ...

  8. Genotypic and PFGE/MLVA analyses of Vibrio cholerae O1: geographical spread and temporal changes during the 2007-2010 cholera outbreaks in Thailand.

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    Kazuhisa Okada

    Full Text Available BACKGROUND: Vibrio cholerae O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of V. cholerae O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize V. cholerae O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time. METHODS/FINDINGS: A total of 343 isolates of V. cholerae O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (ctxB and El Tor rstR genes. Pulsed-field gel electrophoresis (PFGE differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA, and PCR to detect Vibrio seventh pandemic island II (VSP-II related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009-2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1-2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area. CONCLUSIONS: MLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of V. cholerae O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.

  9. RpoS controls the Vibrio cholerae mucosal escape response.

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    Alex Toftgaard Nielsen

    2006-10-01

    Full Text Available Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the "mucosal escape response," this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.

  10. RpoS controls the Vibrio cholerae mucosal escape response.

    Directory of Open Access Journals (Sweden)

    Alex Toftgaard Nielsen

    2006-10-01

    Full Text Available Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the "mucosal escape response," this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.

  11. From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen

    KAUST Repository

    Weynberg, Karen D.

    2015-12-08

    Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements.

  12. From cholera to corals: Viruses as drivers of virulence in a major coral bacterial pathogen

    Science.gov (United States)

    Weynberg, Karen D.; Voolstra, Christian R.; Neave, Matthew J.; Buerger, Patrick; van Oppen, Madeleine J. H.

    2015-01-01

    Disease is an increasing threat to reef-building corals. One of the few identified pathogens of coral disease is the bacterium Vibrio coralliilyticus. In Vibrio cholerae, infection by a bacterial virus (bacteriophage) results in the conversion of non-pathogenic strains to pathogenic strains and this can lead to cholera pandemics. Pathogenicity islands encoded in the V. cholerae genome play an important role in pathogenesis. Here we analyse five whole genome sequences of V. coralliilyticus to examine whether virulence is similarly driven by horizontally acquired elements. We demonstrate that bacteriophage genomes encoding toxin genes with homology to those found in pathogenic V. cholerae are integrated in V. coralliilyticus genomes. Virulence factors located on chromosomal pathogenicity islands also exist in some strains of V. coralliilyticus. The presence of these genetic signatures indicates virulence in V. coralliilyticus is driven by prophages and other horizontally acquired elements. Screening for pathogens of coral disease should target conserved regions in these elements. PMID:26644037

  13. Phenotypic and genetic characterization of Vibrio cholerae O1 isolated from various regions of Kenya between 2007 and 2010.

    Science.gov (United States)

    Mercy, Njeru; Mohamed, Ahmed Abade; Zipporah, Ng'ang'a; Chowdhury, Goutam; Pazhani, Gururaja Perumal; Ramamurthy, Thandavarayan; Boga, Hamadi I; Kariuki, Samuel M; Joseph, Oundo

    2014-01-01

    Cholera, a disease caused by Vibrio cholerae O1 and O139 remains an important public health problem globally. In the last decade, Kenya has experienced a steady increase of cholera cases. In 2009 alone, 11,769 cases were reported to the Ministry of Public Health and Sanitation. This study sought to describe the phenotypic characteristics of the isolated V. cholerae isolates. This was a laboratory based cross-sectional study that involved isolates from different cholera outbreaks. Seventy six Vibrio cholerae O1 strains from different geographical areas were used to represent 2007 to 2010 cholera epidemics in Kenya, and were characterized by serotyping, biotyping, polymerase chain r(PCR), pulsed-field gel electrophoresis (PFGE) and ribotyping along with antimicrobial susceptibility testing. Seventy six Vibrio cholerae O1 strains from different geographical areas were used to represent 2007 to 2010 cholera epidemics in Kenya. Serotype Inaba was dominant (88.2%) compared to Ogawa. The isolates showed varying levels of antibiotic resistance ranging from 100% susceptible to tetracycline, doxycycline, ofloxacin, azithromycin, norfloxacin and ceftriaxone to 100% resistant to furazolidone, trimethoprim-sulfamethoxazole, polymyxin-B and streptomycin. The isolates were positive for ctxA, tcpA (El Tor), rtxC genes and were biotype El Tor variant harboring classical ctxB gene. All the isolates were classified as cholera toxin (CT) genotype 1 as they had mutation in the ctxB at positions 39 and 68. All the isolates had genetically similar NotI PFGE and BglI ribotype patterns. The absence of any observed variation is consistent with a clonal origin for all of the isolates. Kenya experienced cholera numerous outbreak from 2007-2010. The clinical Vibrio cholerae O1 isolates from the recent cholera epidemic were serotypes Inaba and Ogawa, Inaba being the predominant serotype. The Vibrio cholerae O1 strains were biotype El Tor variants that produce cholera toxin B (ctx B) of the

  14. Single-Cell Analysis of the Plasmablast Response to Vibrio cholerae Demonstrates Expansion of Cross-Reactive Memory B Cells.

    Science.gov (United States)

    Kauffman, Robert C; Bhuiyan, Taufiqur R; Nakajima, Rie; Mayo-Smith, Leslie M; Rashu, Rasheduzzaman; Hoq, Mohammad Rubel; Chowdhury, Fahima; Khan, Ashraful Islam; Rahman, Atiqur; Bhaumik, Siddhartha K; Harris, Levelle; O'Neal, Justin T; Trost, Jessica F; Alam, Nur Haq; Jasinskas, Algis; Dotsey, Emmanuel; Kelly, Meagan; Charles, Richelle C; Xu, Peng; Kováč, Pavol; Calderwood, Stephen B; Ryan, Edward T; Felgner, Phillip L; Qadri, Firdausi; Wrammert, Jens; Harris, Jason B

    2016-12-20

    We characterized the acute B cell response in adults with cholera by analyzing the repertoire, specificity, and functional characteristics of 138 monoclonal antibodies (MAbs) generated from single-cell-sorted plasmablasts. We found that the cholera-induced responses were characterized by high levels of somatic hypermutation and large clonal expansions. A majority of the expansions targeted cholera toxin (CT) or lipopolysaccharide (LPS). Using a novel proteomics approach, we were able to identify sialidase as another major antigen targeted by the antibody response to Vibrio cholerae infection. Antitoxin MAbs targeted both the A and B subunits, and most were also potent neutralizers of enterotoxigenic Escherichia coli heat-labile toxin. LPS-specific MAbs uniformly targeted the O-specific polysaccharide, with no detectable responses to either the core or the lipid moiety of LPS. Interestingly, the LPS-specific antibodies varied widely in serotype specificity and functional characteristics. One participant infected with the Ogawa serotype produced highly mutated LPS-specific antibodies that preferentially bound the previously circulating Inaba serotype. This demonstrates durable memory against a polysaccharide antigen presented at the mucosal surface and provides a mechanism for the long-term, partial heterotypic immunity seen following cholera. Cholera is a diarrheal disease that results in significant mortality. While oral cholera vaccines are beneficial, they do not achieve equivalent protection compared to infection with Vibrio cholerae Although antibodies likely mediate protection, the mechanisms of immunity following cholera are poorly understood, and a detailed understanding of antibody responses to cholera is of significance for human health. In this study, we characterized the human response to cholera at the single-plasmablast, monoclonal antibody level. Although this approach has not been widely applied to the study of human bacterial infection, we were

  15. Improving immunization approaches to cholera.

    Science.gov (United States)

    Saha, Amit; Rosewell, Alexander; Hayen, Andrew; MacIntyre, C Raina; Qadri, Firdausi

    2017-03-01

    Cholera's impact is greatest in resource-limited countries. In the last decade several large epidemics have led to a global push to improve and implement the tools for cholera prevention and control. Areas covered: PubMed, Google Scholar and the WHO website were searched to review the literature and summarize the current status of cholera vaccines to make recommendations on improving immunization approaches to cholera. Oral cholera vaccines (OCVs) have demonstrated their effectiveness in endemic, outbreak response and emergency settings, highlighting their potential for wider adoption. While two doses of the currently available OCVs are recommended by manufacturers, a single dose would be easier to implement. Encouragingly, recent studies have shown that cold chain requirements may no longer be essential. The establishment of the global OCV stockpile in 2013 has been a major advance in cholera preparedness. New killed and live-attenuated vaccines are being actively explored as candidate vaccines for endemic settings and/or as a traveller's vaccine. The recent advances in cholera vaccination approaches should be considered in the global cholera control strategy. Expert commentary: The development of affordable cholera vaccines is a major success to improve cholera control. New vaccines and country specific interventions will further reduce the burden of this disease globally.

  16. Cholera Illness and Symptoms

    Science.gov (United States)

    ... are typically no long term consequences. Persons with cholera do not become carriers of the disease after they recover, but can be reinfected if ... Diseases (NCEZID) Division of Foodborne, Waterborne, and Environmental Diseases (DFWED) ... of Health & Human Services HHS/Open USA.gov Top

  17. Cross-protection against Vibrio cholerae infection by monoclonal antibodies against Vibrio vulnificus RtxA1/MARTXVv.

    Science.gov (United States)

    Lee, Tae Hee; Cha, Sun-Shin; Lee, Chang-Seop; Rhee, Joon Haeng; Woo, Hye Ryun; Chung, Kyung Min

    2016-11-01

    Gram-negative Vibrio species secrete multifunctional autoprocessing repeats-in-toxin (MARTX) toxins associated with bacterial pathogenesis. Here, the cross-reactivity and cross-protectivity of mAbs against V. vulnificus RtxA1/MARTXVv was evaluated. Passive administration of any of these mAbs (21RA, 24RA, 46RA, 47RA and 50RA) provided strong protection against lethal V. cholerae infection. Interestingly, 24RA and 46RA, which map to the cysteine protease domain of V. cholerae MARTXVc , inhibited CPD autocleavage in vitro; this process is involved in V. cholerae pathogenesis. These results generate new insight into the development of broadly protective mAbs and/or vaccines against Vibrio species with MARTX toxins. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

  18. Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model.

    Science.gov (United States)

    Duan, Faping; March, John C

    2010-06-22

    To investigate the possibility of using commensal bacteria as signal mediators for inhibiting the disease cholera, we stably transformed Escherichia coli Nissle 1917 (Nissle) to express the autoinducer molecule cholera autoinducer 1 (CAI-1) (shown previously to prevent virulence when present with another signaling molecule, autoinducer 2, at high concentrations) and determined the effect on Vibrio cholerae virulence gene expression and colonization in an infant mouse model. We found that pretreatment of mice for 8 h with Nissle engineered to express CAI-1 (Nissle-cqsA) greatly increased the mice's survival (92%) from ingestion of V. cholerae. Pretreatment with Nissle-cqsA for only 4 h increased survival by 77%, whereas ingesting Nissle-cqsA at the same time as V. cholerae increased survival rates by 27%. Immunostaining revealed an 80% reduction in cholera toxin binding to the intestines of mice pretreated for 8 h with Nissle-cqsA. Further, the numbers of V. cholerae in treated mouse intestines was reduced by 69% after 40 h. This finding points to an easily administered and inexpensive approach where commensal bacteria are engineered to communicate with invasive species and potentially prevent human disease.

  19. Multi-drug resistant toxigenic Vibrio cholerae O1 is persistent in water sources in New Bell-Douala, Cameroon

    Science.gov (United States)

    2013-01-01

    Background Cholera has been endemic in Douala, since 1971 when it was first recorded in Cameroon. Outbreaks have often started in slum areas of the city including New Bell. Despite the devastating nature of outbreaks, always resulting in high mortality and morbidity, a paucity of information exists on the reservoirs of the causative agent, V. cholerae, and factors maintaining its persistence. This has complicated disease prevention, resulting in frequent outbreaks of cholera. We investigated water sources in New Bell for contamination with V. cholerae O1 with pathogenic potential, to highlight their role in disease transmission. Antibiotic susceptibility pattern of isolates and the environmental factors maintaining its persistence were investigated. Method Water samples from various sources (taps, dug wells, streams) were analyzed for contamination with V. cholerae O1 using standard methods. Antibiotic susceptibility was determined by disc diffusion method. Pathogenic potential of isolates was determined by analyzing for genes for cholera toxin (ctx), toxin co-regulated pilus (tcpA), and zonula occludens toxin (zot) by PCR. Physico-chemical characteristics of water (pH, temperature and salinity) were investigated using standard methods. The Spearman’s Rank correlation was used to analyze the relationship between physico-chemical factors and the occurrence of V. cholerae O1. Differences were considered significant at P≤0.05. Results Twenty-five V. cholerae O1 strains were isolated from stream and well samples in both dry and rainy seasons. Twenty-three (92%) isolates were multidrug resistant. All isolates had genes for at least one virulence factor. Cholera toxin gene was detected in 7 isolates. Of the 15 isolates positive for tcpA gene, two had Classical type tcpA while 13 had tcpA El Tor. All tcpA Classical positive isolates were positive for ctx gene. Isolates were grouped into nine genotypes based on the genes analyzed. pH and salinity significantly

  20. RNA thermometer controls temperature-dependent virulence factor expression in Vibrio cholerae.

    Science.gov (United States)

    Weber, Gregor G; Kortmann, Jens; Narberhaus, Franz; Klose, Karl E

    2014-09-30

    Vibrio cholerae is the bacterium that causes the diarrheal disease cholera. The bacteria experience a temperature shift as V. cholerae transition from contaminated water at lower temperatures into the 37 °C human intestine. Within the intestine, V. cholerae express cholera toxin (CT) and toxin-coregulated pilus (TCP), two main virulence factors required for disease. CT and TCP expression is controlled by the transcriptional activator protein ToxT. We identified an RNA thermometer motif in the 5' UTR of toxT, with a fourU anti-Shine-Dalgarno (SD) element that base pairs with the SD sequence to regulate ribosome access to the mRNA. RNA probing experiments demonstrated that the fourU element allowed access to the SD sequence at 37 °C but not at 20 °C. Moreover, mutations within the fourU element (U5C, U7C) that strengthened base-pairing between the anti-SD and SD sequences prevented access to the SD sequence even at 37 °C. Translation of ToxT-FLAG from the native toxT UTR was enhanced at 37 °C, compared with 25 °C in both Escherichia coli and V. cholerae. In contrast, the U5C, U7C UTR prevented translation of ToxT-FLAG even at 37 °C. V. cholerae mutants containing the U5C, U7C UTR variant were unable to colonize the infant mouse small intestine. Our results reveal a previously unknown regulatory mechanism consisting of an RNA thermometer that controls temperature-dependent translation of toxT, facilitating V. cholerae virulence at a relevant environmental condition found in the human intestine.