Monoclonal antibodies to Escherichia coli heat-labile enterotoxins: neutralising activity and differentiation of human and porcine LTs and cholera toxin

Forty-four monoclonal antibodies (MAbs) prepared against heat-labile enterotoxins (LTs) from human (LTh) or porcine (LTp) E. coli isolates were characterised, especially with regard to their reactivity with epitopes shared with the heterologous LT and/or cholera toxin (CT), and their toxin neutralising activity. Of 24 MAbs against LTh (all directed against the B subunit portion) 12 cross-reacted with LTp and CT, 4 with LTp but not CT, and 1 with CT but not LTp; 7 MAbs reacted with LTh epitope(s) not shared by either LTp or CT. Among 20 MAbs against LTp (9 directed against the B subunits and 11 against the A subunit) 2 cross-reacted with LTh as well as CT, 13 with LTh but not CT, and 5 MAbs were specific for LTp. Irrespective of whether the anti-LT MAbs were directed against shared or unshared epitopes, or against the A or B subunits, they neutralised their homologous toxin in direct proportion to their toxin-binding titre. The results show how minute differences in enterotoxin primary structures e.g., the LTh and LTp B chains differ in only 4 of 103 amino acid residues, are associated with antigenic epitopes against which toxin-differentiating MAbs with neutralising activity can be produced. Such MAbs are promising tools for species-specific diagnostic detection of enterotoxins in clinical specimens.     

Common antigenic determinant in the receptor binding sites of Escherichia coli enterotoxin and cholera toxin

Abstract A mutant (TUH No. 9) of a porcine strain of enterotoxigenic Escherichia coli (ETEC) produces as abnormal B subunit (B') of heat-labile enterotoxin (LT), which has aspartate instead of glycine at residue 33 from the N-terminus and does not bind to the receptor, GM1 ganglioside. The antigenicities of the receptor-binding site of LT were analyzed.
The antibody, which could not bind to the B' subunit in the anti-B subunit of porcine LT(LTp)-serum, could bind to cholera toxin (CT), LTp and LT produced by a human ETEC strain (LTh), suggesting that it recognizes a common epitope of LTp, LTh and CT. Thus glycine at residue 33 from the N-terminus in the B subunit of CT, LTh and LTp may be related to the common epitope of these three toxins. The bindings of CT, LTh and LTp to the antibody were inhibited by the GM1 ganglioside.
These data indicate that the antibody recognizes a common epitope in the receptor (GM1 ganglioside)-binding site of CT, LTh and LTp.     

Monoclonal antibodies against pertussis toxin subunits

Abstract Twenty monoclonal antibodies (mAbs) reacting with cholera toxin (CT) of Vibrio cholerae strain 569B were characterized in cross-section and G_M1 ganglioside inhibition assays. MAbs were characterized by reaction with CT and Escherichia coli heat-labile porcine strain (LT_p) and human strain (LTh) enterotoxins, and by G_M1 ganglioside inhibition of mAb binding. Eight of 10 CT-A specific and 3 of 10 CT-B-specific mAbs cross-reacted with LTh and LTp. G_M1 ganglioside inhibited reactions of the CT-B cross-reacting antibodies. Results showed that these epitodes common to the B subunit of CT and LT are located in or near the G_M1 ganglioside binding region, and that the G_M1 ganglioside-binding region of LT differs from that of CT.     

Interaction of cholera toxin and Escherichia coli heat-labile enterotoxin with glycoconjugates from rabbit intestinal brush border membranes: Relationship with ABH blood group determinants

The capacity of cholera toxin (CT) and type I heat-labile enterotoxin produced by Escherichia coli isolated from human intestine (LTh) to interact with glycoconjugates bearing ABH blood group determinants from rabbit intestinal brush border membranes (BBM) was studied. On the basis of the type of intestinal compounds related to the human ABH blood group antigens, rabbits were classified as AB or H. Toxin binding to the intestinal glycolipids and glycoproteins depends on the blood group determinant borne by the glycoconjugate and on the analyzed toxin. LTh was capable of interacting preferentially with several blood group A- and B-active BBM glycolipids compared to those isolated from animals lacking these antigens (H rabbits). Also, LTh preferably bound to several BBM glycoproteins from AB rabbit intestines compared to those from H ones. One of these glycoproteins, the sucrase-isomaltase complex (EC 3.2.1.48-10) isolated from AB and H rabbits showed the same differential LTh binding. Conversely, CT practically did not recognize either blood group A-, B-, or H-active glycolipids and glycoproteins. These results may be relevant for carrying out in vivo experiments in rabbits in order to disclose the role of ABH active-glycoconjugates in the secretory response induced by LTh in rabbit intestine.     

Primary structure of heat-labile enterotoxin produced by Escherichia coli pathogenic for humans

Heat-labile enterotoxin of Escherichia coli pathogenic for humans (LTh) or for piglets (LTp) and Vibrio cholerae enterotoxin (CT) are structurally and functionally similar toxins. We have determined the complete nucleotide sequence of the toxA gene which encodes the subunit A of LTh (LTh A). The deduced amino acid sequence consists of 258 residues including a signal peptide of 18 residues. According to the previously completed LTh B sequence (103 residues), the predicted holotoxin (1A5B) of LTh comprises 755 residues and has Mr = 87,866. With respect to LTh A and LTh B, secondary structures, local hydrophilicity, and sites for antigenic determinants were predicted. Both codon usage and G + C content of the toxA gene and the LTh B gene (toxB) were markedly different from those observed with several E. coli chromosomal genes. Its relatively low G + C content was rather close to that of the V. cholerae chromosome. Although the toxA gene shares a common ancestor with the LTp A gene (eltA), the two genes are apparently distinguishable from each other in their sequences. Like LTh B reported previously, the predicted sequence of the catalytic fragment LTh A1 also showed more homology to that of CT A1 than did that of LTp A1. In contrast, unique sequences were found in LTh A2.     

Activation of rat liver adenylate cyclase by cholera toxin requires toxin internalization and processing in endosomes

Involvement of acidic cell compartments in processing and action of cholera toxin (CT) in rat liver has been examined using subcellular fractionation. Liver cell fractions prepared various times after CT injection display, after a lag phase, a progressive increase in adenylate cyclase activity, detectable earlier in Golgi-endosomal fractions (20 min) than in plasma membrane fractions (30 min), with a maximum (3-fold basal activity) achieved by 60-90 min. Endosomes containing in vivo internalized CT display a time-dependent increase in their ability to bind anti-A-subunit antibodies and to stimulate exogenous adenylate cyclase, which kinetically parallels the generation of A1 peptide, suggesting a translocation of A-subunit (or A1 peptide) across the endosomal membrane. In vivo chloroquine treatment inhibits endocytosis of CT taken up into the liver, lengthens the lag phase for adenylate cyclase activation by CT, and reduces by 3- to 10-fold the apparent affinity of the toxin for the enzyme. Incubation of endosomes containing internalized toxin at 37 degrees C under isotonic conditions results in a pH-dependent increase in generation of A1 peptide, membrane translocation of A-subunit (or A1 peptide), and degradation of toxin, with a maximum at pH 5. Addition of ATP, by decreasing the internal endosomal pH, stimulates both generation of the A1 peptide and degradation of toxin at pH 6-8. It is concluded that activation of adenylate cyclase by CT in intact liver requires association and subsequent processing of toxin in an acidic cell compartment, presumably endosomal.     

Major outer membrane proteins in the phytopathogenic bacteria Erwinia carotovora subsp. carotovora and subsp. atroseptica

Abstract Immunological similarities of heat-labile Escherichia coli enterotoxins pathogenic for man (LTh) and piglets (LTp) and cholera enterotoxin (CT) were examined quantitatively by the reversed Mancini test. The following results were obtained by analysis of rabbit antisera against these toxins. (1) 86% and 61% of the immunoglobulins in anti-CT antisera were antibodies cross-reacting with LTh and LTp, respectively; (2) 77% and 66% of the immunoglobulins in anti-LTh antisera were antibodies cross-reacting with LTp and CT, respectively; (3) 75% and 59% of the immunoglobulins in anti-LTp antisera were antibodies cross-reacting with LTh and CT, respectively.     

Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.     

Purification of El Tor cholera enterotoxins and comparisons with classical toxin.

In 55 clinical isolates of Vibrio cholerae biotype El Tor, cholera toxin (CT) production was higher after growth in liquid medium first under relatively anaerobic conditions followed by excessive aeration (AKI conditions) as compared with growth under the optimal conditions for CT production from V. cholerae of classical biotype (median toxin level being 400 ng ml-1 and 1 ng ml-1 respectively, for the two different growth conditions). Large growth volumes further enhanced El Tor toxin production to levels at or above 3-5 micrograms ml-1 from several strains, which allowed for easy purification of toxin by salt precipitation, aluminium hydroxide adsorption and/or GM1 ganglioside affinity chromatography. However, such purified El Tor CT completely lacked the A subunit when examined by SDS-PAGE or by monoclonal anti-A subunit antibody GM1-ELISA. In contrast, when El Tor CT was prepared from bacteria grown in the presence of specific antiserum against soluble haemagglutinin/protease it contained the A subunit (unnicked) in the same proportion to the B subunit (1A:5B) as classical CT. Immunodiffusion-in-gel tests revealed that the B subunits of El Tor and classical CTs share major epitopes but also have one or more weaker biotype-specific epitopes. The two types of toxin were practically indistinguishable in various GM1-ELISA tests, and antisera raised against El Tor and classical CT, respectively, could also completely neutralize the heterologous as well as the homologous toxin activity in vivo. The results indicate that CTs from El Tor and classical V. cholerae, despite demonstrable epitope differences, are predominantly cross-reactive and give rise to antisera with strong cross-neutralizing activity.     

Modulation of toxin stability by 4-phenylbutyric acid and negatively charged phospholipids

AB toxins such as ricin and cholera toxin (CT) consist of an enzymatic A domain and a receptor-binding B domain. After endocytosis of the surface-bound toxin, both ricin and CT are transported by vesicle carriers to the endoplasmic reticulum (ER). The A subunit then dissociates from its holotoxin, unfolds, and crosses the ER membrane to reach its cytosolic target. Since protein unfolding at physiological temperature and neutral pH allows the dissociated A chain to attain a translocation-competent state for export to the cytosol, the underlying regulatory mechanisms of toxin unfolding are of paramount biological interest. Here we report a biophysical analysis of the effects of anionic phospholipid membranes and two chemical chaperones, 4-phenylbutyric acid (PBA) and glycerol, on the thermal stabilities and the toxic potencies of ricin toxin A chain (RTA) and CT A1 chain (CTA1). Phospholipid vesicles that mimic the ER membrane dramatically decreased the thermal stability of RTA but not CTA1. PBA and glycerol both inhibited the thermal disordering of RTA, but only glycerol could reverse the destabilizing effect of anionic phospholipids. In contrast, PBA was able to increase the thermal stability of CTA1 in the presence of anionic phospholipids. PBA inhibits cellular intoxication by CT but not ricin, which is explained by its ability to stabilize CTA1 and its inability to reverse the destabilizing effect of membranes on RTA. Our data highlight the toxin-specific intracellular events underlying ER-to-cytosol translocation of the toxin A chain and identify a potential means to supplement the long-term stabilization of toxin vaccines.     

Cholera toxin up-regulates endoplasmic reticulum proteins that correlate with sensitivity to the toxin

Cholera toxin (CT) contains one A chain and five B chains. The A chain is an enzyme that covalently modifies a trimeric G protein in the cytoplasm, resulting in the overproduction of cAMP. The B chain binds the glycosphingolipid G(M1), the cell surface receptor for CT, which initiates receptor-mediated endocytosis of the toxin. After endocytosis, CT enters the endoplasmic reticulum (ER) via retrograde vesicular traffic where the A chain retro-translocates through the ER membrane to reach the cytoplasm. The retro-translocation mechanism is poorly understood, but may involve proteins of the ER stress response, including the ER associated degradation (ERAD) pathway. We report here that treating cells with CT or CTB quickly up-regulates the levels of BiP, Derlin-1, and Derlin-2, known participants in the ER stress response and ERAD. CT did not induce calnexin, another known responder to ER stress, indicating that the CT-mediated induction of ER proteins is selective in this time frame. These data suggest that CT may promote retro-translocation of the A chain to the cytoplasm by rapidly up-regulating a set of ER proteins involved in the retro-translocation process. In support of this idea, a variety of conditions that induced BiP, Derlin-1, and Derlin-2 sensitized cells to CT and conditions that inhibited their induction de-sensitized cells to CT. Moreover, specifically suppressing Derlin-1 with siRNA protected cells from CT. In addition, Derlin-1 co-immunoprecipitated with CTA or CTB from CT-treated cells using anti-CTA or anti-CTB antibodies. Altogether, the results are consistent with the hypothesis that the B chain of CT up-regulates ER proteins that may assist in the retro-translocation of the A chain across the ER membrane.     

pH-induced transitions in cholera toxin conformation: a fluorescence study

Determination of the ratio of intrinsic fluorescence with dibrominated Bry 96 (F) relative to that with unbrominated Bry 96 (F0), at neutral pH and in the presence of 0.2 M NaCl, reveals that the A subunit of cholera toxin (CT A) has a somewhat higher affinity for this mild detergent than intact cholera toxin (CT) and its B subunit (CT B). Receptor (GM1 or oligo-GM1) binding has no influence on the very low detergent binding of CT and CT B. Activation of CT A by treatment with dithiothreitol (20 mM) also does not affect detergent binding. The weak hydrophobic nature of CT A is also reflected by the negative modulatory action of anionic phospholipids and deoxycholate on its mono-ADP-ribosyltransferase activity and the ability of the former to decrease its intrinsic fluorescence intensity in a salt-resistant way. Detergent binding of CT A is only slightly pH dependent whereas, upon lowering the pH, detergent binding to CT or CT B becomes significant. In the pH range 6.5-4.2 a gradual increase in detergent binding to CT and CT B occurs. In the narrow pH range 4.2-4.0 a sharp and time-dependent enhancement of brominated Bry 96 quenching is observed. The increase in detergent binding upon lowering the pH is fully reversible, salt dependent, and complete within 10 min (t1/2 = 2 min at 25 degrees C). Solute quenching experiments with the neutral polar quencher acrylamide reveal that upon lowering the pH to 5.0 a marked increase in the exposure of the lone Trp-88 residue in each beta-polypeptide chain of CT B occurs.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cholera toxin B-subunit variant that binds ganglioside G(M1) but fails to induce toxicity

Entry of cholera toxin (CT) into target epithelial cells and the induction of toxicity depend on CT binding to the lipid-based receptor ganglioside G(M1) and association with detergent-insoluble membrane microdomains, a function of the toxin's B-subunit. The B-subunits of CT and related Escherichia coli toxins exhibit a highly conserved exposed peptide loop (Glu(51)-Ile(58)) that faces the cell membrane upon B-subunit binding to G(M1). Mutation of His(57) to Ala in this loop resulted in a toxin (CT-H57A) that bound G(M1) with high apparent affinity, but failed to induce toxicity. CT-H57A bound to only a fraction of the cell-surface receptors available to wild-type CT. The bulk of cell-surface receptors inaccessible to CT-H57A localized to detergent-insoluble apical membrane microdomains (lipid rafts). Compared with wild-type toxin, CT-H57A exhibited slightly lower apparent binding affinity for and less stable binding to G(M1) in vitro. Rather than being transported into the Golgi apparatus, a process required for toxicity, most of CT-H57A was rapidly released from intact cells at physiologic temperatures or degraded following its internalization. These data indicate that CT action depends on the stable formation of the CT B-subunit.G(M1) complex and provide evidence that G(M1) functions as a necessary sorting motif for the retrograde trafficking of toxin into the secretory pathway of target epithelial cells.     

Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin

Cholera toxin (CT) enters host cells by binding to ganglioside GM1 in the apical plasma membrane (PM). GM1 carries CT retrograde from the PM to the endoplasmic reticulum (ER), where a portion of the toxin, the A1-chain, retro-translocates to the cytosol, causing disease. Trafficking in this pathway appears to depend on the association of CT–GM1 complexes with sphingomyelin (SM)- and cholesterol-rich membrane microdomains termed lipid rafts. Here, we find that in polarized intestinal epithelia, the conversion of apical membrane SM to ceramide by bacterial sphingomyelinase attenuates CT toxicity, consistent with the lipid raft hypothesis. The effect is reversible, specific to toxin entry via the apical membrane, and recapitulated by the addition of exogenous long-chain ceramides. Conversion of apical membrane SM to ceramide inhibits the efficiency of toxin endocytosis, but retrograde trafficking from the apical PM to the Golgi and ER is not affected. This result suggests that the cause for toxin resistance occurs at steps required for retro-translocation of the CT A1-chain to the cytosol.     

Evolutionary origin of pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae O1.

Three families of the evolutionarily related pathogenic determinants in enterotoxigenic Escherichia coli and Vibrio cholerae O1, a family of cholera enterotoxin (CT) and heat-labile enterotoxin (LT) including CT, LTh, and LTp, a family of heat-stable enterotoxin I (STI) including STIa and STIb, and a family of K88 enteroadhesion fimbriae including K88ab, K88ac, and K88ad were analyzed for synonymous (silent) nucleotide substitutions by using the gene nucleotide sequences of earlier reports and the LTp gene nucleotide sequence presented in this paper. The data suggested that the divergences between LT and CT and between STIa and STIb occurred in the remote past, whereas those between LTh and LTp and between members of the K88 family occurred very recently. We concluded that the LT gene is a foreign gene that has been acquired by E. coli to form an enteropathogen. This provides evolutionary evidence of species-to-species transfer of pathogenic determinants in procaryotes.     

Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism

Cholera toxin (CT) is a heterohexameric bacterial protein toxin belonging to a larger family of A/B ADP-ribosylating toxins. Each of these toxins undergoes limited proteolysis and/or disulfide bond reduction to form the enzymatically active toxic fragment. Nicking and reduction render both CT and the closely related heat-labile enterotoxin from Escherichia coli (LT) unstable in solution, thus far preventing a full structural understanding of the conformational changes resulting from toxin activation. We present the first structural glimpse of an active CT in structures from three crystal forms of a single-site A-subunit CT variant, Y30S, which requires no activational modifications for full activity. We also redetermined the structure of the wild-type, proenzyme CT from two crystal forms, both of which exhibit (i) better geometry and (ii) a different A2 "tail" conformation than the previously determined structure [Zhang et al. (1995) J. Mol. Biol. 251, 563-573]. Differences between wild-type CT and active CTY30S are observed in A-subunit loop regions that had been previously implicated in activation by analysis of the structure of an LT A-subunit R7K variant [van den Akker et al. (1995) Biochemistry 34, 10996-11004]. The 25-36 activation loop is disordered in CTY30S, while the 47-56 active site loop displays varying degrees of order in the three CTY30S structures, suggesting that disorder in the activation loop predisposes the active site loop to a greater degree of flexibility than that found in unactivated wild-type CT. On the basis of these six new views of the CT holotoxin, we propose a model for how the activational modifications experienced by wild-type CT are communicated to the active site.     

Rafting with cholera toxin: endocytosis and trafficking from plasma membrane to ER

Cholera toxin (CT), and members of the AB(5) family of toxins enter host cells and hijack the cell's endogenous pathways to induce toxicity. CT binds to a lipid receptor on the plasma membrane (PM), ganglioside GM1, which has the ability to associate with lipid rafts. The toxin can then enter the cell by various modes of receptor-mediated endocytosis and traffic in a retrograde manner from the PM to the Golgi and the endoplasmic reticulum (ER). Once in the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol so as to induce disease. GM1 is the vehicle that carries CT from PM to ER. Thus, the toxin pathway from PM to ER is a lipid-based sorting pathway, which is potentially meditated by the determinants of the GM1 ganglioside structure itself.     

The primary structure of the operons coding for Shigella dysenteriae toxin and temperature phage H30 shiga-like toxin

Nucleotide(nt) sequences were determined for the toxin (SHT) operon present in the chromosome of Shigella dysenteriae 1 and for the shiga-like toxin (SLT) operon found in the lambdoid phage H30 genome. The coding sequences of the sht and slt genes differ in 4 nt with 1 nt change responsible for an amino acid replacement. The deduced amino acid sequence in the A chain of the toxins is highly homologous to that of the A chain of ricin, a plant toxin. SHT-coding mRNAs were detected by mapping the 5' termini and using blot-hybridisation; one of them was more abundant and coded only for the B subunit of SHT while the other (bi-cistronic mRNA) encoded both subunits. An IS element related to the IS3 element of Escherichia coli was found in the chromosome of S. dysenteriae near the sht operon.