g(m1)-ganglioside and Cholera

Topics: Adenylyl Cyclases; Adsorption; Animals; Chemical Phenomena; Chemistry; Cholera; Cholera Toxin; Cholera Vaccines; Cricetinae; Cyclic AMP; G(M1) Ganglioside; Humans; Mice; Rabbits; Rats; Receptors, Cell Surface; Receptors, Immunologic; Vibrio cholerae

The pathogenetic significance of toxin receptor interaction in disease is examplified by infectious diarrhoea. The world-wide problem of infectious diarrhoea is presented with regard to epidemiology, etiology, and pathophysiology. Enterotoxigenic diarrhoea is examined in the light of recent knowledge on toxin receptor interaction, which has greatly contributed to new vaccine as well as drug development in this disease condition.

Topics: Adenylyl Cyclases; Animals; Chlorpromazine; Cholera; Cholera Toxin; Cholera Vaccines; Combined Modality Therapy; Diarrhea; Enterotoxins; Enzyme Activation; Escherichia coli; Fluid Therapy; G(M1) Ganglioside; Guanylate Cyclase; Humans; Intestinal Mucosa; Intestinal Secretions; Rabbits; Receptors, Cell Surface; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Immunologic; Receptors, Peptide; Vibrio cholerae

Enteric infections cause more than a billion episodes of diarrhoeal disease in humans each year killing many millions of people, especially young children, in developing countries. Recent progress, reviewed in this article, has enabled that a specific pathogen now can be isolated in the majority of patients with acute diarrhoea, and has also elucidated fundamental pathogenic mechanisms and their pathophysiological effects for several of these agents. Based on this understanding it now seems possible to devise new techniques for the treatment and prevention of diarrhoeal disease to complement those based on fluid replacement therapy and sanitation; prospects for the development of new or improved vaccines, receptor-prophylactic binding agents, and antisecretory drugs are discussed.. Enteric infections cause more than 1 billion episodes of diarrheal disease in humans each year killing many millions of people, especially young children, in developing countries. Recent progress, reviewed here, has enabled that a specific pathogen can now be isolated in the majority of patients with acute diarrhea, and has also elucidated fundamental pathogenic mechanisms and their pathophysiological effects for several of these agents. Based on this understanding, it now seems possible to devise new techniques for the treatment and prevention of diarrheal disease to complement those based on fluid replacement therapy and sanitation. Prospects for the development of new or improved vaccines, receptor-prophylactic binding agents, and antisecretory drugs are discussed.

Topics: Antidiarrheals; Bacterial Vaccines; Calcium; Cholera; Cyclic AMP; Cyclic GMP; Diarrhea; Dysentery, Amebic; Escherichia coli Infections; G(M1) Ganglioside; Humans; Reoviridae Infections; Rotavirus; Viral Vaccines

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.

Topics: Adenylyl Cyclases; Cholera; Cholera Toxin; Cholera Vaccines; Enzyme Activation; G(M1) Ganglioside; Humans; Protein Conformation

Data from the literature on cholera pathogenesis in man and experimental animals are presented. The essence of secretion and filtration theories of rapid dehydration of the intestinal tract as well as that of the secretion-filtration theory proposed by the author are discussed. The data of both national and foreign authors are used in the discussion of the mechanisms of cholera toxin action. New observations on changes in the enzyme content (adenylate- and guanylate-cyclase systems) as well as the data on changes of the element composition of epithelial cells of different parts of the small intestine villi, on the role of local immunity in prevention of cholera toxicity are presented from the author's own observations and those of the laboratory staff members.

Topics: Adenylyl Cyclases; Animals; Cholera; Cholera Toxin; Cyclic AMP; Epithelium; G(M1) Ganglioside; Guanylate Cyclase; Humans; Intestinal Absorption; Intestine, Small; Microscopy, Electron; Rabbits; Receptors, Cyclic AMP

A study was undertaken to investigate whether toxin produced in the gut lumen contributes significantly to clinical illness and whether binding such toxin by GM1 ganglioside adsorbed onto charcoal could alter the clinical course of disease. 46 patients with severe cholera receiving standard intravenous therapy were randomly assigned to one of three groups: GM1 ganglioside-charcoal (16), charcoal alone (16), or water (14). The results demonstrated that patients were given sufficient GM1 ganglioside-charcoal to bind all luminal toxin produced by Vibrio cholerae in their intestines. Patients treated with GM1 ganglioside tended to have a greater reduction in purging than patients treated with either charcoal alone or water. This difference was statistically significant soon after beginning medication (8-15 h) and the reduction in fluid-loss was especially pronounced in patients with very severe initial purging who had been ill only for a short time before admission. These results suggest that toxin produced in the gut lumen increases fluid-loss early in cholera, but that later in the course of disease, toxin which is inaccessible to luminal binding agents in the major stimulus of purging.

Topics: Adolescent; Adsorption; Adult; Charcoal; Child; Cholera; Cholera Toxin; Clinical Trials as Topic; Dehydration; Feces; Female; Fluid Therapy; G(M1) Ganglioside; Gangliosides; Humans; Intestinal Mucosa; Intestines; Male

Cholera is a highly contagious and lethal waterborne disease induced by an infection with Vibrio cholerae (V. cholerae) secreting cholera toxin (CTx). Cholera toxin subunit B (CTxB) from the CTx specifically binds with monosialo-tetra-hexosyl-ganglioside (GM1) found on the exterior cell membrane of an enterocyte. Bioinspired by the pathological process of CTx, we developed an electrochemical biosensor with GM1-expressing Caco-2 cell membrane (CCM) on the electrode surface. Briefly, the electrode surface was functionalized with CCM using the vesicle fusion method. We determined the CTxB detection performances of Caco-2 cell membrane-coated biosensor (CCB) using electrochemical impedance spectroscopy (EIS). the CCB had an excellent limit of detection of ∼11.46 nM and a detection range spanning 100 ng/mL - 1 mg/mL. In addition, the CCB showed high selectivity against various interfering molecules, including abundant constituents of intestinal fluid and various bacterial toxins. The long-term stability of the CCBs was also verified for 3 weeks using EIS. Overall, the CCB has excellent potential for practical use such as point-of-care and cost-effective testing for CTxB detection in developing countries.

Topics: Biomimetics; Biosensing Techniques; Caco-2 Cells; Cholera; Cholera Toxin; G(M1) Ganglioside; Humans

Molecular modelling approach was used to investigate the intermolecular interactions of selected 20 polyphenolic compounds from three plants with CT using DOCK6. Based on intermolecular interactions, two phenolic acids, Ellagic acid (EA) and Chlorogenic acid (CHL); two flavonoids, Rutin (RTN) and Phloridzin (PHD) were selected along with their respective standards, Gallic acid (GA) and Quercetrin (QRTN). The stability of docked complexes was corroborated using molecular dynamics simulation. Furthermore, in vitro inhibitory activity of six compounds against CT was assessed using GM1 ELISA and cAMP assay. EA and CHL that showed prominent activity against CT in. The molecular modelling study revealed significant structural stability of the CT-EA, CT-CHL, and CT-PHD complexes compared to their respective controls. All the selected six compounds significantly reduced CT-induced cAMP levels, whereas EA, CHL, and PHD exhibited > 50% binding inhibition of CT to GM1. The EA and CHL that showed prominent neutralization activity against CT from

Topics: Animals; Cholera; Cholera Toxin; Diarrhea; G(M1) Ganglioside; Mice; Polyphenols; Pomegranate; Psidium

Careya arborea, Punica granatum, Psidium guajava, Holarrhena antidysenterica, Aegle marmelos, and Piper longum are commonly used traditional medicines against diarrhoeal diseases in India. This study investigated the inhibitory activity of these plants against cytotoxicity and enterotoxicity induced by toxins secreted by Vibrio cholerae. Cholera toxin (CT) and non-membrane damaging cytotoxin (NMDCY) in cell free culture filtrate (CFCF) of V. cholerae were quantified using GM1 ELISA and cell-based assays, respectively. Hydro-alcoholic extracts of these plants and lyophilized juice of P. granatum were tested against CT-induced elevation of cAMP levels in CHO cell line, binding of CT to ganglioside GM1 receptor and NMDCY-induced cytotoxicity. Significant reduction of cAMP levels in CFCF treated CHO cell line was observed for all extracts except P. longum. C. arborea, P. granatum, H. antidysenterica and A. marmelos showed >50% binding inhibition of CT to GM1 receptor. C. arborea, P. granatum, and P. guajava effectively decreased cytotoxicity and morphological alterations caused by NMDCY in CHO cell line. Further, the efficacy of these three plants against CFCF-induced enterotoxicity was seen in adult mice ligated-ileal loop model as evidenced by decrease in volume of fluid accumulation, cAMP levels in ligated-ileal tissues, and histopathological changes in intestinal mucosa. Therefore, these plants can be further validated for their clinical use against cholera.

Topics: Animals; CHO Cells; Cholera; Cholera Toxin; Cricetinae; Cytotoxins; G(M1) Ganglioside; Mice; Plants, Medicinal; Toxins, Biological; Vibrio cholerae

Cholera is a life-threatening diarrhoeal disease caused by the human pathogen Vibrio cholerae. Infection occurs after ingestion of the bacteria, which colonize the human small intestine and secrete their major virulence factor - the cholera toxin (CT). The GM1 ganglioside is considered the primary receptor of the CT, but recent studies suggest that also fucosylated receptors such as histo-blood group antigens are important for cellular uptake and toxicity. Recently, a special focus has been on the histo-blood group antigen Lewis

Topics: Binding Sites; Blood Group Antigens; Cholera; Cholera Toxin; G(M1) Ganglioside; Glycosylation; Humans; Protein Binding; Vibrio cholerae

Cholera is one of the most deadly diarrheal diseases that require new treatments. We investigated the neutralization of cholera toxin by five plant extracts obtained from the Rosaceae family that have been traditionally used in Poland to treat diarrhea (of unknown origin).. Hot water extracts were prepared from the dried plant materials and lyophilized before phytochemical analysis and assessment of antimicrobial activity using microdilution assays. The ability of the plant extracts to neutralize cholera toxin was analyzed by measurement of cAMP levels in cell cultures, enzyme-linked immunosorbent assay and electrophoresis, as well as flow cytometry and fluorescence microscopy studies of fluorescent-labeled cholera toxins with cultured human fibroblasts.. The antimicrobial assays displayed modest bacteriostatic potentials. We found that the plant extracts modulate the effects of cholera toxin on intracellular cAMP levels. Three plant extracts (Agrimonia eupatoria L., Rubus fruticosus L., Fragaria vesca L.) suppressed the binding of subunit B of cholera toxin to the cell surface and immobilized ganglioside GM. The traditional application of the Rosaceae plant infusions for diarrhea appears relevant to cholera, slowing the growth of pathogenic bacteria and either inhibiting the binding of cholera toxin to receptors or blocking toxin internalization. The analyzed plant extracts are potential complements to standard antibiotic treatment and Oral Rehydration Therapy for the treatment of cholera.

Topics: Agrimonia; Anti-Bacterial Agents; Cell Line; Cholera; Cholera Toxin; Fragaria; G(M1) Ganglioside; Humans; Plant Extracts; Rosaceae; Rubus; Vibrio cholerae

Stimuli-responsive receptors for the recognition unit of the cholera toxin (CTB) have been prepared by attaching multiple copies of its natural carbohydrate ligand, the GM1 oligosaccharide, to a thermoresponsive polymer scaffold. Below their lower critical solution temperature (LCST), polymers complex CTB with nanomolar affinity. When heated above their LCST, polymers undergo a reversible coil to globule transition which renders a proportion of the carbohydrate recognition motifs inaccessible to CTB. This thermally-modulated decrease in the avidity of the material for the protein has been used to reversibly capture CTB from solution, enabling its convenient isolation from a complex mixture.

Topics: Cholera; Cholera Toxin; G(M1) Ganglioside; Humans; Organoids; Phase Transition; Polymers; Protein Binding; Temperature; Vibrio cholerae

Diarrheal diseases are a major cause of morbidity and mortality worldwide. In many cases, antibiotic therapy is either ineffective or not recommended due to concerns about emergence of resistance. The pathogenesis of several of the most prevalent infections, including cholera and enteroxigenic Escherichia coli, is dominated by enterotoxins produced by lumen-dwelling pathogens before clearance by intestinal defenses. Toxins gain access to the host through critical host receptors, making these receptors attractive targets for alternative antimicrobial strategies that do not rely on conventional antibiotics. Here, we developed a new nanotechnology strategy as a countermeasure against cholera, one of the most important and prevalent toxin-mediated enteric infections. The key host receptor for cholera toxin, monosialotetrahexosylganglioside (GM1), was coated onto the surface of polymeric nanoparticles. The resulting GM1-polymer hybrid nanoparticles were shown to function as toxin decoys by selectively and stably binding cholera toxin, and neutralizing its actions on epithelial cells in vitro and in vivo. Furthermore, the GM1-coated nanoparticle decoys attenuated epithelial 3',5'-cyclic adenosine monophosphate production and fluid responses to infection with live Vibrio cholera in cell culture and a murine infection model. Together, these studies illustrate that the new nanotechnology-based platform can be employed as a non-traditional antimicrobial strategy for the management of enteric infections with enterotoxin-producing pathogens.

Topics: Animals; Binding Sites; Cell Line, Tumor; Cholera; Cholera Toxin; Cyclic AMP; Female; G(M1) Ganglioside; Intestinal Mucosa; Intestine, Small; Male; Mice; Mice, Inbred C57BL; Nanoparticles; Nanotechnology; Vibrio cholerae

Binding of cholera toxin subunit B (CTB) to its receptor and toxin transport into the intestinal epithelial cells are the causative events for the potentially lethal disease cholera. The five sugar mono-sialo ganglioside GM1 is the cell surface receptor for cholera toxin B-subunit. CTB binding was determined by use of immobilized GM1 to microtiter plates and by immunohistochemistry. Sections from the human colon and the human soft palate were incubated with FITC-conjugated CTB and with anti-MUC2. Both the luminal surface of the intestine and the secretory goblet cells exhibited strong binding. Addition of simple carbohydrates and milk to the incubation medium showed that a combination of lactose and non-fat dry milk was potent inhibitors of toxin- and mucin binding. Both CTB and ant-MUC2 stained to the cytoplasm (mucin granules) in the goblet cells from the human soft palate. In the colon CTB stained the entire cytoplasm of the goblet cells while anti-MUC2 detected only the supranuclear region of some cells, suggesting carbohydrate heterogeneity between goblet cell mucin granules in different regions of the human body. Both CTB- and MUC2 binding were inhibited when GM1 was added to the incubation medium. It is proposed that the human colonic goblet cells play a role in the secretory diarrhea in patients with cholera and that milk might have a prophylactic or therapeutic application in the management of cholera.

Topics: Cholera; Cholera Toxin; Epithelial Cells; G(M1) Ganglioside; Humans; Intestine, Large; Kinetics; Protein Binding; Vibrio cholerae

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 relatively easy synthesis and their resistance to glycolytic enzymes. One of the compounds links two toxin B-pentamers in the crystal structure, which may yield improved inhibition through the formation of toxin aggregates. These structures can spark the improved design of GM1 mimics, either alone or as multivalent inhibitors connecting multiple GM1-binding sites. Future developments may further include compounds that link the primary and secondary binding sites. Serving as decoys, receptor mimics may lessen symptoms while avoiding the use of antibiotics.

Topics: Bacterial Toxins; Binding Sites; Cholera; Cholera Toxin; Crystallography, X-Ray; Enterotoxins; G(M1) Ganglioside; Glycosides; Humans; Models, Molecular; Monosaccharides; Protein Binding; Protein Conformation

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.

Topics: ABO Blood-Group System; Acetylgalactosamine; Cell Engineering; Cholera; Cholera Toxin; Colforsin; CRISPR-Cas Systems; Cyclic AMP; G(M1) Ganglioside; Galactose; HT29 Cells; Humans; Protein Binding; Severity of Illness Index; Vibrio cholerae

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.

Topics: Cholera; Cholera Toxin; G(M1) Ganglioside; Humans; Membrane Microdomains; Vibrio cholerae

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.

Topics: Antitoxins; Calixarenes; Cholera; Cholera Toxin; G(M1) Ganglioside; Humans; Models, Molecular; Vibrio cholerae

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.

Topics: Animals; Binding Sites; Binding, Competitive; Carbohydrate Sequence; Chitin; Cholera; Cholera Toxin; Chromatography, Liquid; Epitopes; G(M1) Ganglioside; Glycosphingolipids; Host-Pathogen Interactions; Humans; Intestinal Mucosa; Intestines; Molecular Sequence Data; Rabbits; Spectrometry, Mass, Electrospray Ionization; Thymus Gland; Vibrio cholerae

When covalently bound to an appropriate ligand, iron oxide nanoparticles can bind to a specific target of interest. This interaction can be detected through changes in the solution's spin-spin relaxation times (T2) via magnetic relaxation measurements. In this report, a strategy of molecular mimicry was used in order to identify targeting ligands that bind to the cholera toxin B subunit (CTB). The cellular CTB-receptor, ganglioside GM1, contains a pentasaccharide moiety consisting in part of galactose and glucose units. We therefore predicted that CTB would recognize carbohydrate-conjugated iron oxide nanoparticles as GM1 mimics, thus producing a detectable change in the T2 relaxation times. Magnetic relaxation experiments demonstrated that CTB interacted with the galactose-conjugated nanoparticles. This interaction was confirmed via surface plasmon resonance studies using either the free or nanoparticle-conjugated galactose molecule. The galactose-conjugated nanoparticles were then used as CTB sensors achieving a detection limit of 40 pM. Via magnetic relaxation studies, we found that CTB also interacted with dextran-coated nanoparticles, and surface plasmon resonance studies also confirmed this interaction. Additional experiments demonstrated that the dextran-coated nanoparticle can also be used as CTB sensors and that dextran can prevent the internalization of CTB into GM1-expressing cells. Our work indicates that magnetic nanoparticle conjugates and magnetic relaxation detection can be used as a simple and fast method to identify targeting ligands via molecular mimicry. Furthermore, our results show that the dextran-coated nanoparticles represent a low-cost approach for CTB detection.

Topics: Animals; Cell Membrane; Chlorocebus aethiops; Cholera; Cholera Toxin; Dextrans; Ferric Compounds; G(M1) Ganglioside; Galactose; Ligands; Magnetics; Molecular Conformation; Molecular Mimicry; Nanoparticles; Surface Plasmon Resonance; Vero Cells

A new variant of enzyme immunoassay (EIA) has been developed on the basis of GM1 gangliosides to detect the toxin-producing Vibrio cholerae strains--GM1-dot-EIA. Experiments were run using a nitrocellulose membrane to bind GM1 gangliosides and polyclonal antitoxic serum to detect cholerogen. GM1-dot-EIA testing identified cholera toxin in 11 of 13 supernatants of V. cholerae eltor ctx(+) strains isolated from man and in 3 of 7 supernatants of V. cholerae eltor ctx(+) strains isolated from water. These data agree with those obtained in CM1-EIA. There was no reaction with the supernatants of other microorganisms. The sensitivity of the technique was 10 ng/ml. Thus, the simple and specific GM1-dot-EIA may be recommended to detect toxin-producing V cholerae strains isolated from man and water.

Topics: Cholera; Cholera Toxin; Collodion; Enzyme-Linked Immunosorbent Assay; G(M1) Ganglioside; Humans; Reproducibility of Results; Sensitivity and Specificity; Vibrio cholerae; Water

Vibrio cholerae, the causative agent for cholera, infects its host by expressing a protein consisting of two subunits: the pentameric cholera toxin B (CTB) and cholera toxin A (CTA). CTB frequently is used as an indicator of the presence of pathogenic V. cholerae and typically is detected using enzyme-linked immunosorbent assays (ELISAs). In lieu of an enzyme-linked detection method, we have developed GM(1) ganglioside-functionalized fluorescent dye-encapsulating liposomes for the detection of CTB produced by V. cholerae in a simple microtiter plate assay. Liposomes were compared with fluorescein-labeled antibodies and enzyme-linked secondary antibodies for quantification of purified CTB. A limit of detection for CTB using the liposomes was 340pg/ml, which was comparable to that using the ELISA but 18 times lower than that using the fluorescein-labeled anti-CTB antibodies for the same purpose. The sensitivity of the assay provided by the liposomes was substantial, and the working range improved when compared with that of the fluorescein-labeled antibodies and the ELISA. In addition, the liposomes required shorter assay times, exhibited greater precision, and were less expensive compared with the ELISA. The liposomes were optimized with respect to phospholipid and ganglioside concentrations. The optimized liposomes were then used to probe culture supernatants from V. cholerae El Tor C6706 grown in Dulbecco's modified Eagle's medium and AKI medium for the presence of CTB.

Topics: Antibodies; Cholera; Cholera Toxin; Cost-Benefit Analysis; Enzyme-Linked Immunosorbent Assay; Fluorescein; Fluorescent Dyes; G(M1) Ganglioside; Immunoassay; Liposomes; Reproducibility of Results; Sensitivity and Specificity; Vibrio cholerae

To elucidate mechanisms involved in M cell uptake and transcytosis of Vibrio cholerae, we used an in vitro model of human M-like cells in a Caco-2 monolayer. Interspersed among the epithelial monolayer of Caco-2 cells we detect cells that display M-like features with or without prior lymphocyte treatment and we have established key parameters for V. cholerae transcytosis in this model. Cholera toxin (CT) mutants lacking the A subunit alone or both the A and B subunits were deficient for transcytosis. We explored this finding further and showed that expression of both subunits is required for binding by whole V. cholerae to immobilized CT receptor, the glycosphingolipid GM1. Confocal microscopy showed CT associated with transcytosing bacteria, and transcytosis was inhibited by pre-incubation with GM1 before infection. Finally, heat treatment of the bacterial cells caused a loss of binding to GM1 that was correlated with a significant decrease in uptake and transcytosis by the monolayer. Our data support a model in which the ability of bacteria to interact with GM1 in a CT-dependent fashion plays a critical role in transcytosis of V. cholerae by M cells.

Topics: Antibodies, Bacterial; Biological Transport; Caco-2 Cells; Cholera; Cholera Toxin; DNA, Bacterial; G(M1) Ganglioside; Gene Expression Regulation, Bacterial; Humans; Intestinal Mucosa; Microbial Viability; Microscopy, Confocal; Mutation; Protein Binding; Transport Vesicles; Vibrio cholerae

In vitro and in vivo studies were conducted to assess the response of cholera toxin (CT) production to increasing iron concentrations in an aquatic environment. Production of CT by seven of eight Vibrio cholerae strains tested, including the Bengal strain (O139), was significantly enhanced in the presence of iron concentrations of 1.0 and 10 g/L. The exception (El Tor Ogawa) had a significant CT response only in the presence of 10 g of iron/L. Enhancement of CT production also occurred at iron concentrations less than 1.0 g/L, but not to a statistically significant degree. The high iron concentrations, which in this study were found to stimulate CT production, have been described by others in association with sediments, water plants, and chitinous fauna. Other investigators have shown a predilection by V. cholerae to attach to these sites in the aquatic environment. The importance of excess in vivo iron with respect to the pathogenicity of several gram-negative bacilli is well recognized. However, the possible impact of environmental iron on the in vitro toxigenicity of a microorganism, in this case V. cholerae in its aquatic environment, is to the best of our knowledge a new finding with important epidemiologic implications. These findings, coupled with the fact that iron concentration is considerably enhanced in industrially polluted waters and sediments, may reflect a causal link between the concurrent global upsurge of industrialization and pandemic occurrence of cholera during the latter half of the 20th century. Enhanced toxigenicity may also cause clinical disease following ingestion of lower than usual infective doses of cholera vibrios, thereby increasing the incidence of symptomatic cases and, possibly, of severe cases.

Topics: Animals; Cells, Cultured; Cholera; Cholera Toxin; Enzyme-Linked Immunosorbent Assay; G(M1) Ganglioside; Iron Compounds; Mice; Regression Analysis; Vibrio cholerae; Water Microbiology; Water Pollutants, Chemical

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.

Topics: Animals; Antibodies; Chlorocebus aethiops; Cholera; Cholera Toxin; Diarrhea; Female; G(M1) Ganglioside; Immune Sera; Mice; Mice, Inbred Strains; Mucous Membrane; Plants, Genetically Modified; Solanum tuberosum; Vaccines; Vero Cells

A ganglioside enzyme-linked immunosorbent assay (ELISA) was used to study and attempt to differentiate between antitoxin responses in persons infected with either Vibrio cholerae or Escherichia coli producing heat-labile enterotoxin. In most cases (69%-94%), experimentally infected North Americans and naturally infected Bangladeshis responded to either infection with significant (greater than twofold) increases in serum antibody titer to both heat-labile enterotoxin and cholera toxin. In all but one instance, the response was higher to the homologous than to the heterologous toxin, and for the Americans the homologous antitoxin titers remained significantly higher for at least one year. Determination of levels of antibodies to purified subunits A and B of cholera toxin by an ELISA showed that V. cholerae infection in most instances induced a significant response to subunit B but rarely to subunit A. E. coli infection, on the other hand, induced only slight increases in antibody titer to either subunit.

Topics: Adolescent; Adult; Aged; Antibodies, Bacterial; Antitoxins; Bacterial Toxins; Bangladesh; Child; Child, Preschool; Cholera; Cholera Toxin; Enterotoxins; Enzyme-Linked Immunosorbent Assay; Escherichia coli Infections; Escherichia coli Proteins; Female; G(M1) Ganglioside; Humans; Infant; Male; Middle Aged; North America

The immunogenicity and safety of procholeragenoid, a minimally toxic, heat-induced aggregate of cholera toxin (CT), were studied in enterically immunized rats and dogs. Although 99% less toxic than CT, procholeragenoid was only slightly less efficient in causing jejunal anti-CT responses in rats; in contrast, choleragenoid, the nontoxic B subunit pentamer of CT, was much less effective. The immunogenicity of procholeragenoid was due almost entirely to its large-molecular-weight components (MW = 10(6) to 10(7)) and was markedly reduced by preincubation with GM1 ganglioside or treatment with Formalin to eliminate residual toxicity. These findings suggest that molecular aggregation, binding to GM1 receptors on cell membranes, and stimulation of cellular adenylate cyclase each contributed to the effectiveness of procholeragenoid as a mucosal immunogen. In dogs, oral immunization with five 500-micrograms doses of procholeragenoid evoked vigorous anti-CT responses in jejunal mucosa without causing significant diarrhea. When subsequently challenged with virulent Vibrio cholerae, immunized dogs showed 83% protection against the development of severe or lethal diarrhea compared with non-immunized controls. These results confirm a protective role for mucosal antitoxin in experimental cholera and show that procholeragenoid is both safe and effective as an oral immunogen. Procholeragenoid, combined with other antigens of V. cholerae, may constitute a simple, safe, and effective oral vaccine for cholera.

Topics: Administration, Oral; Animals; Antitoxins; Cholera; Cholera Toxin; Dogs; Female; G(M1) Ganglioside; Immunization; Rats; Rats, Inbred Lew

Topics: Animals; Chlorpromazine; Cholera; Cholera Toxin; Cholera Vaccines; G(M1) Ganglioside; Humans; Rats; Vibrio cholerae