Hemagglutinating activity of vibrio cholerae enterotoxin

Abstract The hemagglutinating activity and carbohydrate specificity of cholera toxin (cholera enterotoxin) was studied using hemagglutination and hemagglutination inhibition. Hemagglutination was obtained with cholera toxin at >108 μg/ml for human types A, B, and O erythrocytes, >216 μg/ml for chicken erythrocytes, and >865 μg/ml for sheep erythrocytes. When the erythrocytes were treated with either neuraminidase or pronase, the hemagglutinating activity of cholera toxin was enhanced about 8- to 32-fold. Hemagglutination of pronase-treated human type B erythrocytes induced by cholera toxin was inhibited by lactose, galactose, melibiose and l -arabinose. Lactose was the most effective of the mono-, di-, and polysaccharides used as inhibitors, being a slightly better inhibitor than galactose, and much more potent than melibiose. These results suggest that cholera toxin is a bacterial lectin specific for galactose and/or lactose.     

Specific binding of cholera toxin to rat erythrocytes revealed by analysis with a fluorescence-activated cell sorter

The direct binding of cholera toxin to the receptor on the native cell surface was analyzed with a fluorescence-activated cell sorter (FACS) by the direct membrane immunofluorescence technique using FITC-conjugated cholera toxin B subunit as a ligand and erythrocytes, but the binding was significantly affected by a change in pH, showing optimum pH of 7.2. The optimum conditions for analysis of the cholera toxin-binding with a FACS were reaction of the target cells with 0.2 M phosphate-buffer (pH 7.2) containing 0.025% of BSA and 0.175 M of NaCl at 4 degrees C for 40 min. The binding of cholera toxin B subunit to rat erythrocytes was linear in the range of 1.2 ng to 80 ng, which corresponded to 2,469 to 163,500 molecules of toxin per cell, and the latter was almost the saturated level of binding. although erythrocytes from different strains of rats possessed equal binding ability for the cholera toxin, no binding was observed with erythrocytes from mouse, guinea pig, cow, pig, man, or rabbit, indicating that the cholera-toxin binding occurs specifically on rat erythrocytes. This is in accord with our previous analytical deta on the absence of GM1 in erythrocytes of these animals except rat, of which erythrocytes contain GM1. Also, the structural specificity of the receptor for cholera toxin was assessed by a binding inhibition experiment using glycolipid-containing liposomes as inhibitors and GM1 was found to be the most potent inhibitor, showing complete inhibition of toxin (40 ng) binding to 5 x 10(6) erythrocytes at 505.6 pmol of GM1.     

The mechanism of action of cholera toxin in pigeon erythrocyte lysates.

The adenylate cyclase activity of intact pigeon erythrocytes begins to rise after about 20 min of exposure to cholera toxin. The maximum rate at which the cyclase activity increases appears to be limited by the number of toxin molecules which can reach an intracellular target. If the erythrocytes are made permeable to the toxin by a bacterial hemolysin, no such limit exists, and adenylate cyclase activity starts to rise immediately upon the addition of toxin, and continues to rise to a maximum at an initially constant rate which is dependent upon the concentration of toxin. On lysed erythrocytes, the addition of cholera antitoxin immediately prevents any further rise in adenylate cyclase activity, but does not reverse any activation already achieved. Erythrocyte lysates may also be activated by isolated peptide A1 of cholera toxin, although activation of adenylate cyclase of intact erythrocytes requires the complete toxin molecule. In the intact cells, toxin first attaches by its Component B to surface receptors of which there are about 30 per erythrocyte. Subsequently, peptide A1 but not Component B is inserted into the erythrocyte. It takes only about 1 min at 37 degrees for peptide A1 to be sufficiently deep within the cell membrane to be inaccessible to extracellular antitoxin, but its complete transit through the membrane appears to take longer. The surface receptors are used only once, for they remain blocked by Component B. The number of receptors available on the surface may be increased by soaking cells in ganglioside GM1. Cholera toxin also decreases the rate of apparently spontaneous loss of adenylate cyclase activity and increases the response to epinephrine. Theophylline inhibits the action of cholera toxin.     

Titration of Cholera Antitoxin in Human Sera by Microhemagglutination with Formalinized Erythrocytes

Microtiter hemagglutination tests employing formalinized sheep erythrocytes sensitized with either crude or purified cholera toxin were used to assay the cholera antitoxin content of human sera. Comparable results were obtained with either crude or purified toxin-sensitized cells with the exception of two sera that gave unusually high hemagglutination titers with the crude toxin. Sera from 13 convalescent cholera patients showed a high degree of correlation between antitoxin levels as determined in vitro by the hemagglutination test and in vivo by the skin permeability factor neutralization test. Fourfold or greater rises in antitoxin levels between acute and convalescent sera were detected in 9 of 15 patients with bacteriologically proven cholera. No significant increases in titer were observed in 14 cases of noncholera diarrhea. Cholera antitoxin was detected by hemagglutination in only 1 of 33 sera, obtained from eight countries, containing vibriocidal antibodies. Formalinized sheep erythrocytes sensitized with toxin and stored at 4 C in the presence of 1:10,000 thimerosal were stable and sensitive for at least 6 months (the longest time tested).     

Potential use of serological methods in detecting cholera toxin

In the study of 50 Vibrio cholerae museum strains, 45 of them producing cholerigenic effect in suckling rabbits, cholera toxin, determined by means of the passive immune hemolysis (PIH) test, has been detected in the supernatant of the culture fluid of only two strains: V. cholerae 569 B, a well-known producer of cholera toxin, and V. cholerae (eltor) 1310, from whose population a toxigenic variant has been obtained by selection. To study the capacity of V. cholerae for producing toxin in vitro, in six cholerigenic strains, besides the supernatant of their culture fluids, also protein fractions, cell lysates and membrane fractions have been studied in the PIH test. In all these strains cholera toxin has been detected only in membrane fractions, which should be taken into consideration in the serological evaluation of the toxigenicity of V. cholerae.     

Human platelets are defective in processing of cholera toxin.

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.     

Increased Intracellular Cyclic AMP Differentially Modulates Nerve Growth Factor Induction of Three Neuronal Recognition Molecules Involved in Neurite Outgrowth

The relative expression of the immunoglobulin superfamily members Thy-1 and L1 and the neural cell adhesion molecule (N-CAM) in PC12 cells grown in the presence of nerve growth factor (NGF), cholera toxin, or both has been quantified. Whereas NGF treatment induced increases in the cell surface expression of all three glycoproteins, treatment with cholera toxin resulted in the specific induction of L1. During the first few days of culture, cholera toxin acted synergistically with NGF to promote increases in neuritic outgrowth and the synthesis and cell surface accumulation of the 140- and 180-kilodalton subunits of N-CAM. In contrast, over the same period of culture, cholera toxin inhibited the NGF induction of Thy-1 and L1. Over longer periods of culture (3-5 days), cholera toxin inhibited the NGF induction of N-CAM and neurite outgrowth. A similar pattern of synergistic and inhibitory responses was observed when differentiation was induced by fibroblast growth factor (FGF) rather than NGF or when cholera toxin was replaced with forskolin. These data suggest that intracellular cyclic AMP can differentially modulate cell surface glycoprotein expression induced by either NGF or FGF. Of the three cell surface glycoproteins we have studied, temporal changes in N-CAM expression correlate best with the morphological differentiation status of PC12 cells.     

Effects of modulators of adenylyl cyclase on interleukin-2 production, cytosolic Ca2+ elevation, and K+ channel activity in Jurkat T cells

We have studied the effects of prostaglandin E2 (PGE2) and cholera toxin, two modulators of adenylyl cyclase, and 8-bromo cAMP (8-BrcAMP) on various parameters of lymphocyte activation using the human T cell line Jurkat. Our results show that PGE2 and cholera toxin inhibit, in a dose-related manner, the phytohemagglutinin (PHA)-dependent production of interleukin 2 by these cells. The data are consistent with the interpretation that the inhibition is due to an intracellular increase in cAMP, since the metabolically stable 8-BrcAMP analog produced the same inhibitory effect. However, PGE2 or 8-BrcAMP did not interfere with the PHA-induced elevation in the cytosolic concentration of Ca2+, suggesting that changes in the intracellular concentration of cAMP does not affect the internal release or the influx of Ca2+. In contrast, cholera toxin prevented the Ca2+ response of Jurkat cells to PHA. We studied the effects of PGE2, cholera toxin, and 8-BrcAMP on the amplitude of the K+ outward current using the patch clamp technique in the whole cell configuration. Results showed that PGE2, 8-BrcAMP, and cholera toxin inhibited K+ channel activity. For instance, the amplitude of the outward K+ current was reduced to 43 +/- 19%, 50 +/- 26%, and 46 +/- 16% of control values in the case of cells perfused in the presence of PGE2, 8-BrcAMP, and cholera toxin, respectively. Blocking K+ channels with tetraethylammonium ions did not prevent the characteristic Jurkat Ca2+ response to PHA. Our observations that cAMP inhibits K+ channel activity in a T cell line provide an additional explanation for its reported inhibition of lymphocyte activation. Increasing the intracellular concentration of cAMP may result in reduction of K+ movements and in negative modulation of signal transduction via G-proteins as previously suggested. These two effects could act in synergy to impair signal transduction.     

霍乱毒素B亚单位基因分泌型表达载体的构建及转化烟草的研究

为了探索利用植物分泌特性来表达重组蛋白的可行性,先构建了含钙网蛋白信号肽的植物双元载体pBIcal,再向该载体中插入霍乱毒素B亚单位编码基因,最后得到表达载体pBIcal—ctb。通过根癌农杆菌介导,该表达载体转化烟草,在卡那霉素抗性培养基上筛选,得到30棵抗性植株。经PCR鉴定,霍乱毒素B亚单位基因已经整合到烟草基因组中。初步表达分析表明,转基因烟草中含有具生物活性的霍乱毒素B亚单位蛋白。     

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

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 response to cholera toxin. Thus, in hepatic cells, a unique endocytic pathway was revealed following cholera toxin administration, with regulation specificity most probably occurring at the locus of the endosome and implicating endosomal proteases, such as cathepsin D, as well as organelle acidification.     

Construction of recombinant plasmids encoding the biosynthesis of the beta-subunit of cholera toxin

The structure of the cholera toxin operon and the location of A and B toxin subunits have been studied by the Southern blot hybridization on filters. The gene coding for the synthesis of the cholera toxin B-subunit has been cloned in the vector plasmid pBR322. The structural gene of A-subunit has been partially deleted by the restriction endonuclease Bal31 digestion. The size of the 250 b. p. deletion has been defined by electron microscopy. The production of the cholera toxin B-subunit in Escherichia coli K12 cells has been studied.     

Molecular Insights Into the Evolutionary Pathway of Vibrio cholerae O1 Atypical El Tor Variants

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.     

Calcium transport affinity,ion competition and cholera toxin effects on cytosolic Ca concentration

Summary The physiological relevance of an apparent ionophore activity of cholera toxin towards Ca²⁺ has been examined in several different systems designed to measure affinity, specificity, rates of ion transfer, and effects on intracellular ion concentrations. Half-maximal transfer rates across porcine jejunal brush-border vesicles were obtained at a concentration of 0.20 M Ca²⁺. When examined in the presence of competing ions the transfer process was blocked by very low concentrations of La³⁺ or Cd²⁺. Sr²⁺, Ba²⁺ and Mg²⁺ were relatively inefficient competitors for Ca²⁺ transport mediated by cholera toxin. The relative affinities observed would be compatible with a selectivity for Ca²⁺ transfer at physiological ion concentrations, as well as an inhibition of this ionophore activity by recognized antagonists of cholera toxin such as lanthanum ions. Entry rates of Ca²⁺ into brush-border vesicles exposed to cholera toxin were large enough to accelerate the collapse of a Ca²⁺ gradient generated by endogenous Ca, Mg-ATPase activity. The treatment of isolated jejunal enterocytes with cholera toxin caused a significant elevation in cytosolic Ca²⁺ concentrations as measured by Quin-2 fluorescence. This effect was specifically prevented by prior exposure of the cholera toxin to excess ganglioside G_M1. We conclude that cholera toxin has many of the properties required for promoting transmembranes Ca²⁺ movement in membrane vesicles and appears to be an effective Ca²⁺ ionophore in isolated mammalian cells.     

Vibrio cholerae infection of Drosophila melanogaster mimics the human disease cholera

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.     

Activation of cell membrane potassium conductance by mercury in cultured renal epithelioid (MDCK) cells

To elucidate mechanisms of mercury toxicity, the cell membrane potential has been determined continuously in cultured kidney (MDCK)-cells during reversible application of mercury ions to extracellular perfusate. Exposure of the cells to 1 microM mercury ions is followed by rapid, sustained, and slowly reversible hyperpolarization of the cell membrane, increase of cell membrane potassium selectivity, and decrease of cell membrane resistance. Thus, mercury ions enhance the potassium conductance of the cell membrane. Half maximal hyperpolarizing effect is elicited by approximately 0.2 microM. Higher concentrations of mercury ions (greater than 10 microM) eventually depolarize the cell membrane. At extracellular calcium activity reduced to less than 0.1 microM, 1 microM mercury ions still leads to a sustained hyperpolarization and increase of potassium selectivity of the cell membrane. As evident from fluorescence measurements, 10 microM, but not 1 microM mercury ions leads to a rapid increase of intracellular calcium activity. Pretreatment of the cells with either pertussis toxin or cholera toxin does not blunt the hyperpolarizing effect of mercury ions. In conclusion, mercury ions activate the potassium conductance by a mechanism independent of increase of intracellular calcium activity and of cholera toxin- or pertussis toxin-sensitive G-proteins. This activation of potassium conductance may account for early effects of mercury intoxication, such as kaliuresis.     

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxin's actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.     

Cholera toxin facilitates calcium transport in jejunal brush border vesicles

Cholera toxin is very well characterized in terms of the activation of adenylate cyclase. In some systems, however, this cyclase activation does not seem to account for all of the physiological responses to the toxin. On the premise that cholera toxin may also exert effects through other second messenger compounds we have studied the effect of cholera toxin on the rate of Ca2+ movement across the membrane of intestinal brush border vesicles. Increasing concentrations of cholera toxin progressively accelerated the passive uptake of Ca2+ into, and the efflux of Ca2+ from, an osmotically active space in brush border membrane vesicles. This effect of cholera toxin was saturable by excess Ca2+ and was relatively specific, as the toxin did not affect vesicle permeability to an uncharged polar solute. The toxin had two high affinity Ca2+ binding sites on the A subunit as measured by equilibrium dialysis. Ca2+ transport facilitated by cholera toxin was temperature dependent, required the holotoxin, and could be inhibited by preincubation of the toxin with excess free ganglioside GM1. This increased rate of Ca2+ influx caused by the in vitro addition of cholera toxin to brush border membrane vesicles may have physiological significance as it was comparable to rates observed with the Ca ionophore A23187. Similar effects occurring in vivo could permit cholera toxin to increase cytoplasmic Ca2+ concentrations and to produce accompanying second messenger effects.     

Structure-function studies of cholera toxin and its A and B protomers. Modification of tryptophan residues

The tryptophan residues on cholera toxin and its A and B protomers have been modified by reaction with 2-nitrophenylsulfenyl chloride and 2,4-dinitrophenylsulfenyl chloride. Modification of the tryptophan residues of cholera toxin results in complete loss of toxicity measured in a skin permeability assay. Modification of cholera toxin and its B protomer results in the complete loss of binding activity toward membrane receptors, the ganglioside galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylceramide (GM1), and the oligosaccharide moiety of the ganglioside GM1. Modification of cholera toxin and its A protomer results in a complete loss of the ADP-ribosylation activity exhibited by their native counterparts. Modification of the A protomer results in no apparent change in its physical properties by sedimentation velocity in the ultracentrifuge or by gel filtration chromatography. Modification of the B protomer, either directly or when it remains a component part of the holo toxin structure, results in a change in its sedimentation value and its elution from gel filtration columns. The changes are compatible with a conversion of the B protomer from a pentameric moiety in aqueous solvents to its existence as a monomer unit, i.e. to the individual polypeptide chains comprising the native B pentamer. Thiolysis of the 2,4-dinitrophenylsulfenyl chloride derivative of the B protomer reaggregates the individual-polypeptide chains but does not return its ability to interact with GM1.