Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin

This study examined the binding of purified 125I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4 degrees C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4 degrees C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 4 degrees C dissociated from MVM when the temperature was raised to 37 degrees C, but reassociated when the temperature was again brought to 4 degrees C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.     

Immunological control mechanism against cholera toxin: interference with toxin binding to intestinal receptors.

The immunological control mechanism against cholera toxin (CT) in the small intestine of rats was studied in vivo. CT binding to intestinal receptors was determined by injected radiolabeled CT into the loops of rat small intestine and subsequently separating purified microvillus membranes from mucosal scrapings of those loops. substantial radioactivity (10(5) cpm/mg of microvillus protein) was present in microvillus fractions of small intestine exposed to 125I-labeled CT compared to radioactivity (10(2) cpm/mg) in fractions from intestine exposed to radiolabeled bovine serum albumin (BSA) used as a control. CT binding to intestinal receptors was significantly inhibited (P less than 0.02) in rats immunized with crude toxin by a combined intraperitoneal and oral method compared to CT binding in animals immunized with BSA or controls, suggesting a specific relationship between intestinal antitoxin and inhibition of binding. Furthermore, ligated ileal loops from CT-immunized animals showed a significant decrease in fluid accumulation when exposed to CT compared to loops from control or BSA-immunized animals, suggesting that antitoxins also interfered with the biological action of CT under conditions of immunization. These studies provide direct evidence that intestinal antitoxins protect against CT-induced diarrhea by interfering with the attachment of the toxin to the intestinal microvillus surface.     

Differential stimulation of intestinal mucin secretion by cholera toxin and carbachol

 Cholinergic stimulation triggers the secretion of apically stored, preformed mucin from goblet cells but the pathway of cAMP-stimulated mucin secretion is not known. In this study the effect of cholera toxin on mucin secretion in the human colonic goblet cell line HT-29/B6 was investigated and compared to the action of carbachol. PAS staining of mucin blotted onto nitrocellulose served to quantify the secretion of total mucin. Metabolic labelling was used to evaluate the secretion of newly synthesized mucin. The mucinous nature of the detected material was confirmed with an immunoblot employing a well-characterized polyclonal antibody reacting with MUC2-mucin. Cholera toxin caused a 116-fold increase of intracellular cAMP and strongly stimulated the secretion of both preformed and newly synthesized mucin for more than 20 h. Carbachol only triggered the release of preformed mucin immediately after addition. The secretory response to cholera toxin could be partly inhibited by the protein kinase A inhibitor H8 and the microtubule inhibitor colchicine. The action of carbachol was not affected by these agents. In conclusion, we demonstrate a direct cAMP-dependent effect of cholera toxin on mucin secretion by intestinal goblet cells. In contrast to carbachol, the action of cholera toxin involves de novo synthesis of mucin molecules and microtubule-mediated secretion. There seem to be distinct secretion pathways for muscarinic or cAMP-dependent stimulation of mucin secretion. Received: 10 June 1996 / Received after revision: 19 September 1996 / Accepted: 30 September 1996     

Changes in intestinal fluid and mucosal immune responses to cholera toxin in Giardia muris infection and binding of cholera toxin to Giardia muris trophozoites.

The effect of Giardia muris infection on the diarrheal response and gut mucosal antibody response to cholera toxin was examined in mice. The results obtained showed that the fluid accumulation in intestinal loops exposed to cholera toxin was increased in mice infected with a low number (5 X 10(4) ) of G. muris cysts compared with the response in noninfected mice. This effect was associated with a marked reduction in absorption of oral rehydration fluid from the intestine. In contrast, mice infected with a high dose (2 X 10(5) ) of cysts showed a marked decrease in fluid accumulation in response to the toxin. This decrease might be related to the finding that both G. muris and Giardia lamblia trophozoites can bind significant amounts of cholera toxin. Evidence is presented which suggests that the gut mucosal antibody response, mainly immunoglobulin A but also immunoglobulin G, to an immunization course with perorally administered cholera toxin was depressed in mice infected with G. muris. The reduction in antibody levels was particularly evident when the primary immunization was made very early after infection. The serum antitoxin antibodies to the oral immunization with cholera toxin were, however, not affected. Likewise, the delayed-type hypersensitivity response against sheep erythrocytes in animals primed subcutaneously with sheep erythrocytes was not modified during the course of G. muris infection.     

Monoclonal immunoglobulin A antibodies directed against cholera toxin prevent the toxin-induced chloride secretory response and block toxin binding to intestinal epithelial cells in vitro.

Secretory immunoglobulin A (IgA) antibodies directed against cholera toxin (CT) are thought to be important in resistance to oral challenge with virulent Vibrio cholerae, although alternative mechanisms for protection of intestinal epithelia against CT-induced fluid secretion have been proposed. The ability of anti-CT IgA to block the effects of CT on human enterocytes has not been directly tested because of the lack of a well-defined in vitro intestinal epithelial cell system to directly measure toxin action and the limited availability of purified anti-CT IgA antibodies. We have generated hybridomas that produce monoclonal IgA and IgG antibodies directed against CT by fusion of Peyer's patch cells with mouse myeloma cells after oral-systemic immunization of mice with CT and CT B-subunit protein. All of the anti-CT antibodies recognized the B subunit. Three clones (designated anti-CTB IgA-1, IgA-2, and IgA-3) which produced IgA antibodies in dimeric and polymeric forms were selected. Checkerboard immunoblotting demonstrated that IgA-1 recognized an epitope distinct from that recognized by IgA-2 and IgA-3 and that none of the antibodies were directed against the binding site of GM1, the intestinal cell membrane toxin receptor. The protective capacity of these IgAs was tested in vitro with human T84 colon carcinoma cells grown on permeable supports as confluent monolayers of polarized enterocytes. When each anti-CTB IgA was mixed with 10 nM CT and applied to the apical surfaces of T84 cell monolayers, all three IgAs blocked CT-induced Cl- secretion in a dose-dependent manner and completely inhibited binding of rhodamine-labelled CT to apical cell membranes. Thus, monoclonal anti-CTB IgA antibodies are sufficient to protect human enterocytes in vitro against CT binding and action.     

Suppression of the intestinal immune response to cholera toxin by specific serum antibody.

The possibility that preexisting specific serum antibody could suppress a defined mucosal immune response to a topically applied antigen was studied in rats. Hyperimmune serum antibody induced by parenteral immunization of rats with cholera toxoid markedly suppressed the mucosal immune response to enterically applied cholera toxin. Such antibody was far more suppressive than antibody induced by primary parenteral immunization, apparently due to its greater avidity. Transfusion of small amounts (25 to 100 microliter) of hyperimmune serum suppressed the primary mucosal antitoxin response, the development of specific memory in the mucosal immune system, and, somewhat less effectively, the secondary mucosal antitoxin response. Suppression was due largely to a direct effect of serum antibody upon the interaction of absorbed enteric antigen with lymphoid tissue in Peyer's patches and, possible, mesenteric lymph nodes; interference with antigen absorption played little or no role in the observed suppression. These results do not explain the previously reported suppressive effect of primary parenteral immunization on the mucosal immune response to cholera toxin. However, they support the notions that repeated parenteral immunization can evoke avid serum antibody without necessarily stimulating mucosa-associated lymphoid tissue and that such antibody can markedly suppress primary and secondary phases of the local immune response to mucosally applied antigen. Thus, a mechanism is demonstrated by which repeated parenteral immunization may adversely affect efforts to initiate or sustain protective mucosal immune responses.     

The involvement of intramural nerves in cholera toxin induced intestinal secretion

In previous reports we have suggested that nervous reflexes are involved in the pathophysiology of cholera secretion and that these nervous reflexes involve a cholinergic synapse and a neuron with vasoactive intestinal polypeptide (VIP) as neurotransmitter. These proposals were further analyzed in this study. Tetrodotoxin (TTX) and lidocaine applied on the serosal surface inhibited cholera secretion in segments of rat small intestine. Fluid absorption in control rats was not significantly changed. Hexamethonium given i. v. decreased cholera secretion in the cat. No additional inhibition of cholera secretion was observed after giving TTX close i. a. Furthermore, the intestinal secretion evoked by VIP was not influenced by hexamethonium given i. v. or TTX given close i. a. The present observations support the hypothesis of a role for nervous reflexes in cholera secretion. The results suggest that at least a major part of the proposed nervous reflex(es) in cholera have a cholinergic synapse. Furthermore, the VIP-ergic neuron is situated “distal” to the cholinergic neuron in the reflex(es) closer to the effector cells.     

Ganglioside localization on myelinated nerve fibres by cholera toxin binding

Summary G_M1 ganglioside has been localized on the surfaces of myelinated, peripheral nerve fibres by using immunofluorescence to detect cholera toxin receptors. Unfixed, mouse sciatic nerves were teased into individual, intact fibres in order to expose their extracellular surfaces. Cholera toxin binding sites were abundant at all nodes of Ranvier; they were scarce on the internodal fibre surfaces. The nodal receptors were resistant to various degradative enzymes, including trypsin and proteinase K. Proteases did not unmask receptors on the internodal surfaces. Exogenous G_M1 successfully competed for the toxin binding sites on the fibres. From this evidence and the specificity of cholera toxin binding, we conclude that G_M1 ganglioside is abundantly present on the membrane surfaces of peripheral nodes of Ranvier and is not present on the internodal Schwann cell surfaces in an appreciable amount. The patterns of fluorescence within the node suggest that the axon and Schwann cell structures are sites where G_M1 is localized.Treatment of the teased fibres withVibrio cholerae neuraminidase, which is known to reduce polysialogangliosides to the monosialoganglioside G_m1, induced cholera toxin binding on the internodal Schwann cell surfaces. The induced receptors, as well as their precursors, were resistant to trypsin and proteinase K. We conclude that the internodal Schwann cell surface is rich in an unidentified polysialoganglioside(s) that can be converted to G_M1 by neuraminidase.     

Fixation of Clostridium difficile toxin A and cholera toxin to intestinal brush border membranes from axenic and conventional mice.

We have tested the in vitro binding of Clostridium difficile toxin A (enterotoxin) and cholera toxin to intestinal brush border membranes prepared from either conventional or axenic mice. Membranes from axenic mice were shown to be saturated at a lower toxin A concentration (at least 2.5 times lower). Because there were no significant differences between membranes from axenic and conventional mice in binding at low toxin A concentrations, the presence of the normal microflora seems to increase the number but not the affinity of brush border membrane receptors on the enterocyte surface. Corroborating the in vitro results, we observed that conventional mice were more sensitive to the pathological effects of toxin A given intragastrically than were axenic mice. In contrast, there was no difference in the binding characteristics of cholera toxin between membranes from conventional and axenic mice. We conclude that the presence of the mouse intestinal bacteria increases the number of C. difficile toxin A intestinal receptors but does not influence cholera toxin receptors.     

Role of local IgA antitoxin-producing cells for intestinal protection against cholera toxin challenge

We examined in mice, perorally immunized with cholera toxin (CT) or cholera B subunit (CTB), the association between protection against intestinal toxin challenge and frequency and function of gut mucosal IgA antitoxin-forming cells. The in vitro production of IgA antitoxin by isolated cells and the toxin-neutralizing ability of culture supernatants were determined. Repeated oral immunizations with CT gave rise to high numbers of IgA antitoxin 'spot-forming' cells (SFC) in the lamina propria as well as to protection against challenge with CT in ligated intestinal loops. In contrast, mice immunized with purified CTB, gave poor IgA antitoxin SFC responses in the lamina propria and little or no protection. When a small amount of CT was used to adjuvant the response to CTB, many IgA antitoxin SFC were found; however, protection in intestinal loops remained poor. This discrepancy was explained by the predominant localization of antitoxin SFC in the proximal small intestine following oral CTB/CT-adjuvant immunization, whereas relatively few SFC were found further down in the intestine where the loop-protection test was performed. Thus, when lamina propria plasma cells were isolated from challenged loops and cultured in vitro, they released only low titers of IgA antitoxin and CT-neutralizing antibodies in culture supernatants; this was in contrast to cells from optimally immunized mice which gave supernatants with high IgA antitoxin and toxin-neutralizing antibody titers. Increasing the dose of CT, added as adjuvant to the CTB, resulted in better protection and higher numbers of IgA antitoxin SFC in more distal parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusions to the cholera toxin B subunit: influence on pentamerization and GM1 binding

The cholera toxin B (CTB) subunit has been used extensively in vaccine research as a carrier for peptide immunogens due to its immunopotentiating properties, where coupling has been obtained either by genetic fusion or chemical conjugation. For genetically fused immunogens both N- and C-terminal fusions have been used. Only shorter extensions have previously been evaluated and in some reports these fusions have impaired the biological functions of CTB, such as the ability to form pentamers and to adhere to its cell receptor, the GM1 ganglioside. Here we report the first systematic study where the same fusion partner has been used for either C-terminal, N-terminal or dual fusions to CTB. The serum albumin binding region (BB, approximately 25 kDa) from streptococcal protein G, which is known to fold independently of N- or C-terminal fusions, was selected as fusion partner. The three fusion proteins CTB-BB, BB-CTB and BB-CTB-BB were expressed in Escherichia coli, where they were efficiently secreted to the periplasmic space, and could be purified by affinity chromatography on human serum albumin (HSA) columns. The CTB fusion proteins were compared for their ability to form pentamers, by gel electrophoresis and size-exclusion chromatography, and it was concluded that all three fusion proteins were able to pentamerize. Interestingly, the C-terminal fusion to CTB showed most efficient pentamerization, while the dual fusion was much less efficient. Purified pentamer fractions from all three fusions where found to react to a monoclonal antibody described to react only to pentameric forms of CTB. In addition, the purified pentamer fractions were analyzed in an enzyme-linked immunosorbent assay (ELISA) for their ability to bind GM1, and it was found that the C-terminal fusion (CTB-BB) showed significant GM1-binding, but that also the N-terminal and dual CTB fusion proteins bound GM1, although less efficiently. The implications of the results for the design and use of CTB fusion proteins as subunit vaccines are discussed.     

Binding of cholera toxin to Giardia lamblia.

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.     

Specific antibodies to cholera toxin in rabbit milk are protective against Vibrio cholerae-induced intestinal secretion.

Breast feeding helps to protect the nursing infant against infectious diarrhoeas, but the relative importance of antibodies compared with other components present in milk is unsettled. In order to aid in resolving this issue we evaluated the ability of milk, collected from rabbits not immunized or immunized enterally during pregnancy with toxinogenic, live Vibrio cholerae, to inhibit water secretion induced by V. cholerae in rat ileal loops. Non-immune milk was not inhibitory, whereas immune milk was. The inhibitory component of the immune milk was immunoglobulin by virtue of its molecular weight and absorption by an anti-rat immunoglobulin immunosorbent. In addition, the inhibitory antibodies were principally antibodies to cholera toxin because they could be removed from the milk by a cholera toxin immunosorbent but were only partially removed by incubation with whole V. cholerae. Thus, in rabbit milk, we could implicate specific antibodies in protection against intestinal water secretion induced by V. cholerae.     

Differential interaction of Escherichia coli heat-labile toxin and cholera toxin with pig intestinal brush border glycoproteins depending on their ABH and related blood group antigenic determinants.

The ability of glycoproteins from pig intestinal brush border membranes (BBM) to bind cholera toxin (CT) or heat-labile toxins from strains of Escherichia coli isolated from human (LTh) or pig (LTp) intestines was studied. Glycoproteins capable of binding the toxins are also recognized by antibodies or lectins specific for ABO(H) blood group and related antigens. Pigs expressing A, H, or I antigenic determinants were used for comparison. The toxin-binding capacity of a glycoprotein depends on the toxin type and the blood group epitope borne by the glycoprotein. LTh and LTp preferably bound to several blood group A-active glycoproteins rather than H-active glycoproteins. By contrast, CT practically did not recognize either blood group A- or blood group H-active glycoproteins, while glycoproteins from pigs expressing I antigenic determinants were able to interact with LTh, LTp, and CT. LTh, LTp, or CT glycoprotein binding was selectively inhibited by specific lectins or monosaccharides. Affinity purification of the toxin binding brush border glycoproteins on the basis of their blood group reactivity suggests that such glycoproteins are hydrolytic enzymes. BBM from A+ pigs contain about 27 times more LTh binding sites, in addition to those recognized by CT, than an equivalent membrane preparation from H+ pigs. The present findings may help clarify some previous unclear results on LTh binding to intestinal BBM glycoproteins obtained by use of animals not typed by their ABO(H) blood group phenotype.     

Major outer membrane proteins from many Campylobacter species cross-react with cholera toxin

We have previously shown that Campylobacter jejuni strains do not produce a functional cholera toxin-like toxin (CTLT) detectable in a Chinese hamster ovary cell assay. Instead, the 53-kDa major outer membrane protein (OMP) of C. jejuni, PorA, reacts with cholera toxin (CT) antibody on immunoblots. Here, we have extended this observation to other species of Campylobacter, including C. coli, C. lari, C. fetus, C. hyointestinalis, and C. upsaliensis, the common 53-kDa OMP of which reacted with CT antibody in immunoblotting assays. There were additional reactive bands for C. fetus. As with C. jejuni, this finding may lead to the erroneous conclusion that these additional species produce a functional CTLT. However, this common cross-reactive OMP can be explored as a vaccine candidate to prevent campylobacteriosis.     

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

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.     

Role of Peyer''s patch in the intestinal immune response to cholera toxin in enterically immunized rats.

Cholera toxin has been widely used to obtain insight into the cellular dynamics of the antigen-specific mucosal immune response. The present study was undertaken to clarify the influence of the organized intestinal lymphoid tissue (Peyer's patches [PP]) on the distribution of anti-cholera-toxin-containing cells (ACC) after intraperitoneal immunization and intraduodenal challenge with purified cholera toxin. This was done in rats which were surgically deprived of all visible PP. In comparison with sham-operated animals, each PP-deprived rat had nearly the same amount of ACC in the spleen, the mesenteric lymph nodes, and, surprisingly, the thoracic duct lymph. In contrast, the ACC in the duodenum, the jejunum, and the ileum of each PP-deprived animal were drastically reduced. Therefore the PP are suggested as an important organizing structure for the buildup of a local antigen-specific immune response.     

Inhibition of cholera toxin binding to membrane receptors by pig gastric mucin-derived glycopeptides: differential effect depending on the ABO blood group antigenic determinants.

The capacity of pig gastric mucin-derived glycopeptides to interfere with the binding of cholera toxin (CT) to membrane receptors was studied. Two types of glycopeptide preparations with or without human blood group A antigenic activity were assayed for comparison in a system in which the target for the toxin was rat erythrocyte ghosts. Blood group A-active glycopeptides (A+ glycopeptides) were more potent inhibitors for the toxin binding than those lacking group A activity (A- glycopeptides). The mean values of the 50% inhibitory dose revealed that the A+ glycopeptide preparations were 6.6-fold-more potent inhibitors than the A- ones (P less than 0.001). The inhibitory capacity of the different A+ glycopeptide preparations was not directly proportional to the group A antigenic titer. The A+ glycopeptides showed a higher capacity than the A- glycopeptides to interact with the toxin as revealed by CT-glycopeptide complex formation, which could be detected by Sephacryl S-400 chromatography. This result suggests that glycopeptide inhibition of CT binding to the erythrocyte ghosts is mediated by a competition between the GM1 receptors and the glycopeptides for the toxin. The differential effect between both types of glycoconjugates was independent of the way of measuring the amount of glycopeptides used (dry weight, carbohydrate or protein content). The existence in the gastrointestinal tract of mucins not carrying or carrying different ABO blood group determinants, which could behave as more or less potent inhibitors of CT binding to membrane receptors, may help to explain the relationship between ABO blood groups and severity of cholera.     

Binding of cholera toxin to pig intestinal mucosa glycosphingolipids: relationship with the ABO blood group system.

A search for compounds from intestinal mucosa of pigs carrying and not carrying blood group A-active substances (A+ and A- pigs, respectively) capable of binding cholera toxin (CT) was performed. Glycolipid extracts from a pool of pig intestinal mucosa resolved in thin-layer chromatography (TLC) revealed the presence of six to eight compounds capable of binding 125I-CT, two of them running as the ganglioside standards GM1 and GD1b. When intestinal mucosa glycolipids from single pigs were assayed by TLC for CT-binding capacity, two different patterns of labeling were observed. The main difference was at the level of compounds running below GD1b. The A+ pigs but not the A- pigs showed CT binding at this level. The major CT-binding compound detected only in A+ pigs was purified and some properties were determined. After TLC developed with different solvent systems, the purified compound bound CT and also immunoreacted with anti-A and anti-AB antisera but not with anti-B antiserum. The compound was also able to inhibit the hemagglutination of human A erythrocytes caused by anti-A antiserum, but inhibition was not observed with the B-anti-B or O (H)-Ulex europaeus lectin systems. A partial chemical characterization indicated that the active compound is a neutral glycosphingolipid containing glucose, fucose, galactose, and hexosamine. The existence of a blood group-active substance(s) able to interact with CT may help to explain the relationship between ABO blood groups and the diarrheal disease caused by infection with Vibrio cholerae.