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.     

Association of protease activity in Vibrio cholerae vaccine strains with decreases in transcellular epithelial resistance of polarized T84 intestinal epithelial cells

Culture supernatants prepared from reactogenic strains of Vibrio cholerae cause a decrease in the transcellular epithelial resistance of T84 intestinal cells. This decrease correlates with the presence of hemagglutinin/protease but not with the presence of other potential accessory toxins or proteases. These data suggest a possible role for hemagglutinin/protease in reactogenicity, although other factors may also contribute.     

Bacteroides fragilis toxin exhibits polar activity on monolayers of human intestinal epithelial cells (T84 cells) in vitro.

Strains of Bacteroides fragilis associated with diarrhea in children (termed enterotoxigenic B. fragilis, or ETBF) produce a heat-labile ca. 20-kDa protein toxin (BFT). The purpose of this study was to examine the activity of BFT on polarized monolayers of human intestinal epithelial cells (T84 cells). In Ussing chambers, BFT had two effects. First, BFT applied to either the apical or basolateral surfaces of T84 monolayers diminished monolayer resistance. However, the time course, magnitude, and concentration dependency differed when BFT was applied to the apical versus basolateral membranes. Second, only basolateral BFT stimulated a concentration-dependent and short-lived increase in short circuit current (Isc; indicative of C1- secretion). Time course experiments indicated that Isc returned to baseline as resistance continued to decrease, indicating that these two electrophysiologic responses to BFT are distinct. Light microscopic studies of BFT-treated monolayers revealed only localized cellular changes after apical BFT, whereas basolateral BFT rapidly altered the morphology of nearly every cell in the monolayer. Transmission and scanning electron microscopy after basolateral BFT confirmed a striking loss of cellular microvilli and complete dissolution of some tight junctions (zonula occludens) and zonula adherens without loss of desmosomes. The F-actin structure of BFT-treated monolayers (stained with rhodamine-phalloidin) revealed diminished and flocculated staining at the apical tight junctional ring and thickening of F-actin microfilaments in focal contacts at the basolateral monolayer surface compared to those in similarly stained control monolayers. BFT did not injure T84 monolayers, as assessed by lactic dehydrogenase release and protein synthesis assays. These studies indicate that BFT is a nonlethal toxin which acts in a polar manner on T84 monolayers to stimulate C1- secretion and to diminish monolayer resistance by altering the apical F-actin structure of these cells. BFT may contribute to diarrheal disease associated with ETBF infection by altering epithelial barrier function and stimulating C1- secretion.     

Adhesion of Helicobacter pylori to polarized T84 human intestinal cell monolayers is pH dependent.

Epithelial cells, which form tight polarized monolayers on porous substrates, constitute ideal model systems to study bacterial adhesion and invasion. The binding of Helicobacter pylori to the apical membrane of T84 cells, an epithelial cell line derived from a human colon carcinoma, was assessed biochemically and morphologically. Attachment was rapid, and binding remained constant over time, with a significant (P < 0.01, Mann-Whitney U test) ca. fourfold increase at pH 5.4 (76% +/- 22%) compared with pH 7.4 (18% +/- 7%). In contrast, adhesion of enteropathogenic Escherichia coli was not enhanced at pH 5.4. The transepithelial electrical resistance of the T84 cell monolayers was not affected by pH or by H. pylori. Following binding, H. pylori induced a reorganization of the brush border as reflected by actin condensation, facilitating the intimate association of the bacteria with the apical plasma membrane. H.pylori was not internalized, as shown by confocal microscopy. Some bacteria, found in deep invaginations of the apical membrane, were probably inaccessible to gentamicin, thus accounting for the observed tolerance to the antibiotic. These data provide the first evidence that an acidic environment favors Helicobacter adhesion and that binding is followed by survival of the survival of the bacteria in pockets of the apical membrane.     

Inhibition of cholera toxin production by thiols in Vibrio cholerae.

We found that thiols reduced the amount of cholera toxin produced by Vibrio cholerae 569B in vitro. A sulfhydryl group at least was necessary for the reduction of cholera toxin production by thiols.     

Interaction of cholera toxin and toxin derivatives with lymphocytes. II. Modulating effects of cholera toxin on in vivo humoral and cellular immune responses.

The in vivo effects of cholera toxin on lymphoid organ structure and function in mice were investigated. It was found that within a day following intravenous injection of 1 mug of toxin, thymus as well as spleen weight decreased but the animals remained healthy. Histological studies suggested that the involution of lymphoid organs was due to cell death. Injection of cholera toxin into adrenalectomized mice was lethal within 36 h. In these animals no decrease in lymphoid organ weight was noted. Thymus cells from toxin-treated mice were found to be much inferior to thymocytes of untreated animals in their in vitro response to Concanavalin A, whereas the response of spleen cells from toxin-treated animals to mitogens was slightly increased. 1 mug of cholera toxin increased primary antibody formation when given to mice together with antigen (sheep erythrocytes) and decreased primary antibody formation when given before or after the antigen. The toxin also increased secondary antibody formation when injected simultaneously with or after the booster antigen dose, and decreased the antibody formation when given a few days before the booster injection. Treatment of mice with toxin was found to increase the capacity of spleen cells from these animals to induce the parental effect on antibody formation and to induce graft-versus-host reactions. The mechanisms behind the observed effects are discussed. It is suggested that cholera toxin affects different types of cells involved in immune responses primarily by a direct inhibitory action on cellular proliferation but also indirectly by causing release of adrenal gland hormones.     

Hemolysin and the multifunctional autoprocessing RTX toxin are virulence factors during intestinal infection of mice with Vibrio cholerae El Tor O1 strains

The seventh cholera pandemic that started in 1961 was caused by Vibrio cholerae O1 strains of the El Tor biotype. These strains produce the pore-forming toxin hemolysin, a characteristic used clinically to distinguish classical and El Tor biotypes. Even though extensive in vitro data on the cytolytic activities of hemolysin exist, the connection of hemolysin to virulence in vivo is not well characterized. To study the contribution of hemolysin and other accessory toxins to pathogenesis, we utilized the model of intestinal infection in adult mice sensitive to the actions of accessory toxins. In this study, we showed that 4- to 6-week-old streptomycin-fed C57BL/6 mice were susceptible to intestinal infection with El Tor strains, which caused rapid death at high doses. Hemolysin had the predominant role in lethality, with a secondary contribution by the multifunctional autoprocessing RTX (MARTX) toxin. Cholera toxin and hemagglutinin/protease did not contribute to lethality in this model. Rapid death was not caused by increased dissemination due to a damaged epithelium since the numbers of CFU recovered from spleens and livers 6 h after infection did not differ between mice inoculated with hemolysin-expressing strains and those infected with non-hemolysin-expressing strains. Although accessory toxins were linked to virulence, a strain defective in the production of accessory toxins was still immunogenic since mice immunized with a multitoxin-deficient strain were protected from a subsequent lethal challenge with the wild type. These data suggest that hemolysin and MARTX toxin contribute to vaccine reactogenicity but that the genes for these toxins can be deleted from vaccine strains without affecting vaccine efficacy.     

Sensitivity of polarized epithelial cells to the pore-forming toxin aerolysin

Aerolysin is one of the major virulence factors produced by Aeromonas hydrophila, a human pathogen that produces deep wound infection and gastroenteritis. The toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface. Surprisingly, we found that all three cell types were sensitive to the toxin from both the apical and the basolateral sides. Apical sensitivity was always higher, even for FRT cells. In contrast, FRT cells were more sensitive from the basolateral than from the apical side to the related toxin Clostridium septicum alpha-toxin, which also binds to GPI-anchored proteins but lacks the lectin binding domain found in aerolysin. These observations are consistent with the notion that a shuttling mechanism involving low-affinity interactions with surface sugars allows aerolysin to gradually move toward the membrane surface, where it can finally encounter the glycan cores of GPI-anchored proteins.     

Characterisation of volume-activated ion transport across epithelial monolayers of human intestinal T84 cells

The effects of hypo-osmolarity upon transepithelial ion transport in human intestinal cell layers have been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (I _sc). This transient stimulation of inward current by hypo-osmotic media was abolished by 100 mol/l 4,4-diisothiocyanostilbene 2,2-disulphonic acid (DIDS). After prestimulation of inward I _sc by vasoactive intestinal peptide (VIP) or by combinations of carbachol and prostaglandin E₁ hypoosmotic exposure of the basal-lateral surfaces resulted in a further transient stimulation of I _sc. The stimulation of I _sc in these conditions was largely insensitive to DIDS inhibition. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a stimulation of loop-diuretic-insensitive ⁸⁶Rb efflux across the basal-lateral surfaces. In addition, hypo-osmotic exposure of T₈₄ cells is also associated with an increase in cytosolic Ca²⁺. It is concluded that the effects of hypo-osmotic exposure of T₈₄ cells on secretory I _sc are consistent with the activation of a DIDS-sensitive apical Cl^– conductance and a basal-lateral K⁺ conductance. With prior activation of inward I _sc by VIP via a cAMP-activated DIDS-insensitive apical Cl^– conductance, augmentation of the secretory current by hypo-osmotic exposure is likely to result primarily from increased basal-lateral K⁺ current and loop-diuretic-sensitive Cl^– uptake.     

Weak serum and intestinal antibody responses to Vibrio cholerae soluble hemagglutinin in cholera patients.

A soluble hemagglutinin/protease from Vibrio cholerae has been suggested to be a putative virulence factor and protective antigen. However, clinical cholera infection gave rise to detectable serum antibody responses to soluble hemagglutinin in only 2 of 10 Bangladeshi patients or 1 of 17 cholera-infected North American volunteers. A gut mucosal immunoglobulin A antibody response to soluble hemagglutinin was seen in 4 of 8 Bangladeshi patients, but in 0 of 10 North American volunteers. These responses were much weaker than those to cholera toxin or lipopolysaccharide.     

Translocation of Shiga toxin across polarized intestinal cells in tissue culture.

Escherichia coli strains producing Shiga toxins (Stx) 1 and 2 colonize the lower gastrointestinal tract in humans and are associated with gastrointestinal and systemic diseases. Stx are detectable in the feces of infected patients, and it is likely that toxin passes from the intestinal tract lumen to underlying tissues. The objective of this study was to develop an in vitro model to study the passage of Stx across intact, polarized cell monolayers. Translocation of biologically active Stx was examined in four cell lines grown on polycarbonate filters. Stx1 translocated across intestinal cell monolayers (CaCo2A and T84 cells) in an energy-requiring and saturable manner, while the monolayers maintained a high level of electrical resistance. Stx1 had no effect on electrical resistance or inulin movement across these cell lines for at least 24 h. Induction of specific Stx receptors with sodium butyrate reduced the proportion of toxin translocated across CaCo2A monolayers but had no major effect on the movement of horseradish peroxidase or [3H]inulin. We have shown that biologically active Stx1 is capable of moving across intact polarized intestinal epithelial cells without apparent cellular disruption, probably via a transcellular pathway. The data also suggest that the presence of Stx receptors on the apical surface of intestinal epithelial cells may offer some protection against the absorption of luminal Stx1.     

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.     

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.     

New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor.

A new medium that stimulates in vitro production of cholera toxin by Vibrio cholerae O1 El Tor (El Tor vibrios) was developed. The medium contains 0.5% NaCl, 0.3% NaHCO3, 0.4% yeast extract, and 1.5% Bacto-Peptone. El Tor vibrios were cultured in a stationary test tube at 37 degrees C for 20 h. The culture supernatant was assayed for cholera toxin by a reversed passive latex agglutination method. Most vibrios grown in this medium produced 10 to 20 times more toxin than in traditional syncase medium. The number of live vibrios at the end of culture was about 10(8)/ml in the new medium (AKI medium) and about 10(10)/ml in the syncase medium. As a result, each individual organism in the new medium should have produced as much as 1,000 times more toxin than in syncase medium. Sodium bicarbonate was found to be the most important factor in toxin production by El Tor vibrios in the new medium. We recommend this new medium because of its high yield of cholera toxin and its technical simplicity.     

Large production of cholera toxin by Vibrio cholerae O1 in yeast extract peptone water.

A large amount of cholera toxin (CT) was produced by Vibrio cholerae O1 cultured in yeast extract-peptone water. The organisms were cultured initially in a stationary test tube (small surface-to-volume ratio) until the end of the exponential phase and subsequently cultured in a shaking flask for 15 to 20 h. By this method (previously reported as the AKI-SW method), most cholera vibrios produced an abundance of CT (up to 64 micrograms/ml), regardless of their biotype and serotype. A substantial amount of CT was produced even in basic peptone water (2% peptone, 0.5% NaCl). Use of sodium bicarbonate, which markedly stimulated CT production in the stationary test tube culture, was undesirable for CT production by the culture method used here. CT production was greatly influenced by culture conditions but was not significantly affected by the composition of the medium.     

Role of Vibrio cholerae neuraminidase in the function of cholera toxin.

Vibrio cholerae neuraminidase (NANase) is hypothesized to act synergistically with cholera toxin (CT) and increase the severity of a secretory response by increasing the binding and penetration of CT to enterocytes. To test this hypothesis, the NANase gene (nanH) from V. cholerae Ogawa 395 was first cloned and sequenced. Isogenic wild-type and NANase- V. cholerae 395 strains were then constructed by using suicide vector-mediated mutagenesis. The influence of NANase on CT binding and penetration was examined in vitro by using culture filtrates from these isogenic strains. Fluorescence due to binding of fluorescein-conjugated CT to C57BL/6 and C3H mouse fibroblasts exposed to NANase+ filtrates increased five- and eightfold, respectively, relative to that with NANase- filtrates. In addition, NANase+ filtrates increased the short-circuit current measured in Ussing chambers 65% relative to that with NANase- filtrates, although this difference decreased as production of CT increased. The role of NANase in V. cholerae pathogenesis was examined in vivo by intragastric inoculation of the isogenic strains into CD1 suckling mice. No difference in fluid accumulation ratios was seen at doses of 10(4) to 10(8) CFU, but NANase+ strains produced 18% higher fluid accumulation ratios at 10(9) CFU than NANase- strains when inoculated into nonfasted suckling mice. It is concluded that NANase plays a subtle but significant role in the binding and uptake of CT by susceptible cells under defined conditions.     

Effect of Helicobacter pylori on polymorphonuclear leukocyte migration across polarized T84 epithelial cell monolayers: role of vacuolating toxin VacA and cag pathogenicity island

Helicobacter pylori infection can induce polymorphonuclear leukocyte (PMNL) infiltration of the gastric mucosa, which characterizes acute chronic gastritis. The mechanisms underlying this process are poorly documented. The lack of an in vitro model has considerably impaired the study of transepithelial migration of PMNL induced by H. pylori. In the present work, we used confluent polarized monolayers of the human intestinal cell line T84 grown on permeable filters to analyze the epithelial PMNL response induced by broth culture filtrates (BCFs) and bacterial suspensions from different strains of H. pylori. We have evaluated the role of the vacuolating cytotoxin VacA and of the cag pathogenicity island (PAI) of H. pylori in PMNL migration via their effects on T84 epithelial cells. We noted no difference in the rates of PMNL transepithelial migration after epithelial preincubation with bacterial suspensions or with BCFs of VacA-negative or VacA-positive H. pylori strains. In contrast, PMNL transepithelial migration was induced after incubation of the T84 cells with cag PAI-positive and cagE-positive H. pylori strains. Finally, PMNL migration was correlated with a basolateral secretion of interleukin-8 by T84 cells, thus creating a subepithelial chemotactic gradient for PMNL. These data provide evidence that the vacuolating cytotoxin VacA is not involved in PMNL transepithelial migration and that the cag PAI, with a pivotal role for the cagE gene, provokes a transcellular signal across T84 monolayers, inducing a subepithelial PMNL response.     

Manipulation of intestinal immune responses against ovalbumin by cholera toxin and its B subunit in mice.

We studied the effect of mucosal presentation of ovalbumin (OVA) conjugated to cholera toxin (CT) or cholera toxin B subunit (CTB) on the intestinal immune responses against OVA. Mice were primed intraperitoneally (i.p.) with OVA in a water-in-oil emulsion and boosted intraduodenally (i.d.) with OVA conjugated to CT or CTB in various molar ratios. Responses were evaluated by testing intestinal secretions for OVA-specific antibodies and by quantifying the OVA-specific antibody secreting cells (ASC) in the lamina propria of the small intestine. OVA-CT conjugates were tested in a molar ratio ranging from 1.8:1 to 4500:1. OVA-CTB conjugates were tested in a molar ratio ranging from 0.25:1 to 500:1. The optimum intestinal immune response was reached at a molar ratio of 1.8:1 for OVA-CT and 5:1 for OVA-CTB. The binding capacity of OVA-CTB, but not of OVA-CT, to GM1 ganglioside corresponded with the capacity to enhance the intestinal immune response. The effect of conjugating CTB or CT to OVA on the immune response against OVA was more striking when mice were not only boosted i.d., but also primed i.d. Both OVA-CT and OVA-CTB induced detectable immune responses, whereas free OVA did not. Therefore, the carrier effect of CT or CTB is essential to trigger a mucosal immune response against OVA when presented mucosally only. We conclude that enhancing antigen uptake greatly facilitates mucosal immune responses.