Heterologous Toxin Research Articles

Fluorescent analyses of Bacillus thuringiensis Cry1Fa and Cry1Ab toxin binding sites on brush border membrane vesicles of Ostrinia nubilalis (Hübner), Diatraea grandiosella (Dyar), and Helicoverpa zea (Boddie) larvae

Bacillus thuringiensis (Bt) Cry1Fa and Cry1Ab proteins are important Cry toxins due to their high, selective toxicity against a number of lepidopteran species, including important pests of corn and cotton. Competition binding assays are a classical tool for investigating Cry toxin interactions with target pest insects. We developed a fluorescence-based binding assay and assessed Cry1Fa and Cry1Ab toxin binding to brush border membrane preparations from lepidopteran corn pests including Ostrinia nubilalis (European corn borer, ECB), Diatraea grandiosella (south western corn borer, SWCB), and Helicoverpa zea (corn earworm, CEW). Homologous and heterologous competition binding assays with fluorophore-(Alexa488)-labeled Cry1Fa toxin showed that Cry1Fa shares binding site(s) with Cry1Ab toxin in ECB, and SWCB for which Cry1Ab has higher affinity than Cry1Fa. Apart from the shared binding sites, Cry1Ab and Cry1Fa bind an additional site(s) in ECB and SWCB. In CEW, Cry1Fa and Cry1Ab each, has a high affinity binding site(s), which binds the heterologous toxin with low affinity. The Cry1Ab-Cry1Fa toxin binding models for ECB, SWCB and CEW based on our results are considered in the context of what is known about acquired cross-resistance against Cry1Ab and Cry1Fa toxins.

Recombinant Botulinum Neurotoxin Hc Subunit (BoNT Hc) and Catalytically Inactive Clostridium botulinum Holoproteins (ciBoNT HPs) as Vaccine Candidates for the Prevention of Botulism.

There are few available medical countermeasures against botulism and the discontinuation of the pentavalent botulinum toxoid vaccine by the Centers for Disease Control and Prevention in 2011 has resulted in the need for a safe and effective prophylactic alternative. Advances in genetic engineering have resulted in subsequent vaccine efforts being primarily focused on the production of highly purified recombinant protein antigens representing one or more domains of the botulinum neurotoxin. Recombinant subunit vaccines based on the carboxy one-third of the toxin (Hc) developed in our lab against serotypes A-F have been shown to be safe and effective. However, in response to the identification of an ever increasing number of BoNT subtypes with significant amino acid heterogeneity, we have developed catalytically inactive BoNT holoproteins (ciBoNT HPs) in an attempt to elicit greater protective immunity to address these toxin variants. Here we report the production of ciBoNT/B1 HP, ciBoNT/C1 HP, ciBoNT/E1 HP and ciBoNT/F1 HP and compare the immunological and protective abilities of ciBoNT HPs and BoNT/A Hc, BoNT/B Hc, BoNT/C Hc, BoNT/E Hc and BoNT/F Hc vaccines when challenged with homologous and heterologous toxins. Our results suggest the ciBoNT HP vaccines exhibit superior potency after single vaccinations but multiple vaccinations with BoNT/Hc antigens resulted in increased survival rates at the toxin challenge levels used.

Self-replicating shuttle vectors based on pANS, a small endogenous plasmid of the unicellular cyanobacterium Synechococcus elongatus PCC 7942.

To facilitate development of synthetic biology tools for genetic engineering of cyanobacterial strains, we constructed pANS-derived self-replicating shuttle vectors that are based on the minimal replication element of the Synechococcus elongatus strain PCC 7942 plasmid pANS. To remove the possibility of homologous recombination events between the shuttle plasmids and the native pANS plasmid, the endogenous pANS was cured through plasmid incompatibility-mediated spontaneous loss. A heterologous toxin-antitoxin cassette was incorporated into the shuttle vectors for stable plasmid maintenance in the absence of antibiotic selection. The pANS-based shuttle vectors were shown to be able to carry a large 20 kb DNA fragment containing a gene cluster for biosynthesis of the omega-3 fatty acid eicosapentaenoic acid. Based on quantitative PCR analysis, there are about 10 copies of pANS and 3 copies of the large native plasmid pANL per chromosome in S. elongatus. Fluorescence levels of GFP reporter genes in a pANS-based vector were about 2.5-fold higher than when in pANL or integrated into the chromosome. In addition to its native host, pANS-based shuttle vectors were also found to replicate stably in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. There were about 27 copies of a pANS-based shuttle vector, 9 copies of a pDU1-based shuttle vector and 3 copies of an RSF1010-based shuttle vector per genome when these three plasmids co-existed in Anabaena cells. The endogenous pANS from our S. elongatus laboratory strain was cloned in Escherichia coli, re-sequenced and re-annotated to update previously published sequencing data.

K2 killer toxin-induced physiological changes in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells produce killer toxins, such as K1, K2 and K28, that can modulate the growth of other yeasts giving advantage for the killer strains. Here we focused on the physiological changes induced by K2 toxin on a non-toxin-producing yeast strain as well as K1, K2 and K28 killer strains. Potentiometric measurements were adjusted to observe that K2 toxin immediately acts on the sensitive cells leading to membrane permeability. This correlated with reduced respiration activity, lowered intracellular ATP content and decrease in cell viability. However, we did not detect any significant ATP leakage from the cells treated by killer toxin K2. Strains producing heterologous toxins K1 and K28 were less sensitive to K2 than the non-toxin producing one suggesting partial cross-protection between the different killer systems. This phenomenon may be connected to the observed differences in respiratory activities of the killer strains and the non-toxin-producing strain at low pH. This might also have practical consequences in wine industry; both as beneficial ones in controlling contaminating yeasts and non-beneficial ones causing sluggish fermentation.

Comparisons of Native Shiga Toxins (Stxs) Type 1 and 2 with Chimeric Toxins Indicate that the Source of the Binding Subunit Dictates Degree of Toxicity

Shiga toxin (Stx)-producing E. coli (STEC) cause food-borne outbreaks of hemorrhagic colitis. The main virulence factor expressed by STEC, Stx, is an AB5 toxin that has two antigenically distinct forms, Stx1a and Stx2a. Although Stx1a and Stx2a bind to the same receptor, globotriaosylceramide (Gb3), Stx2a is more potent than Stx1a in mice, whereas Stx1a is more cytotoxic than Stx2a in cell culture. In this study, we used chimeric toxins to ask what the relative contribution of individual Stx subunits is to the differential toxicity of Stx1a and Stx2a in vitro and in vivo. Chimeric stx1/stx2 operons were generated by PCR such that the coding regions for the A2 and B subunits of one toxin were combined with the coding region for the A1 subunit of the heterologous toxin. The toxicities of purified Stx1a, Stx2a, and the chimeric Stxs were determined on Vero and HCT-8 cell lines, while polarized HCT-8 cell monolayers grown on permeable supports were used to follow toxin translocation. In all in vitro assays, the activity of the chimeric toxin correlated with that of the parental toxin from which the B subunit originated. The origin of the native B subunit also dictated the 50% lethal dose of toxin after intraperitoneal intoxication of mice; however, the chimeric Stxs exhibited reduced oral toxicity and pH stability compared to Stx1a and Stx2a. Taken together, these data support the hypothesis that the differential toxicity of the chimeric toxins for cells and mice is determined by the origin of the B subunit.

Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme

Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins

Recombinant botulinum Hc (rBoNT Hc) vaccines for serotypes C1 and D were produced in the yeast Pichia pastoris and used to determine protection against four distinct BoNT C and D toxin subtypes. Mice were vaccinated with rBoNT/C1 Hc, rBoNT/D Hc, or with a combination of both vaccines and challenged with BoNT C1, D, C/D, or D/C toxin. Mice receiving monovalent vaccinations were partially or completely protected against homologous toxin and not protected against heterologous toxin. Bivalent vaccine candidates completely survived challenges from all toxins except D/C toxin. These results indicate the recombinant C1 and D Hc vaccines are not only effective in a monovalent formula but offer complete protection against both parental and C/D mosaic toxin and partial protection against D/C mosaic toxin when delivered as a bivalent vaccine.

Genetic toxoids of Shiga toxin types 1 and 2 protect mice against homologous but not heterologous toxin challenge

Shiga toxin type 1 (Stx1) and type 2 (Stx2) are produced by Escherichia coli O157:H7 and are responsible for the life-threatening sequela, the hemolytic uremic syndrome. Whether antisera to Stx1 or Stx2 are cross-neutralizing remains controversial, so we constructed genetic toxoids of Stx1 and Stx2 and evaluated them as vaccines. Antisera from mice immunized with a single toxoid type recognized and neutralized the homologous toxin but not the heterologous toxin. Furthermore, only mice immunized with Stx1 and Stx2 toxoids were protected against a lethal challenge of both toxins. We conclude that Stx1 and Stx2 are distinct antigens for mice.

Cross-protection against challenge by intravenous Escherichia coli verocytotoxin 1 (VT1) in rabbits immunized with VT2 toxoid.

Rabbits challenged intravenously with Escherichia coli verocytotoxin (VT1, Shiga toxin 1, Stx1) die after developing diarrhea and paralysis, and this outcome can be prevented by pre-immunization with VT1 toxoid. In nonimmune rabbits, intravenously administered 125I-VT1 binds to the central nervous system and gastrointestinal tract, whereas in immunized animals, these organs are spared and the toxin localizes in the liver and spleen. In rabbits immunized with either VT1 or VT2 toxoids, both the homologous or heterologous toxins are prevented from binding to target organs. This has lead to the advancement of a hypothesis that cross-protection in vivo can be induced to both toxins by immunization with a toxoid even though these toxins do not exhibit cross-neutralization in vitro. It was shown that rabbits immunized with VT2 were fully protected from the intravenous administration of 10 LD50 and 50 LD50 of VT1, and this correlated directly with the protection from binding of this toxin to target organs. These findings have important implications on the design of the vaccination strategies to prevent human VT-mediated diseases and also validate the concept of testing for immunity to VT by monitoring the inhibition of binding of the 125I-VT to target organs in preference to performing LD50 assays.

Binding analyses of Bacillus thuringiensis Cry delta-endotoxins using brush border membrane vesicles of Ostrinia nubilalis.

Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit (125)I-Cry1Ab binding to BBMV. Cry1F inhibited (125)I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.

Toxicity, binding, and permeability analyses of four Bacillus thuringiensis Cry1 delta-endotoxins using brush border membrane vesicles of Spodoptera exigua and Spodoptera frugiperda.

The binding and pore formation properties of four Bacillus thuringiensis Cry1 toxins were analyzed by using brush border membrane vesicles from Spodoptera exigua and Spodoptera frugiperda, and the results were compared to the results of toxicity bioassays. Cry1Fa was highly toxic and Cry1Ac was nontoxic to S. exigua and S. frugiperda larvae, while Cry1Ca was highly toxic to S. exigua and weakly toxic to S. frugiperda. In contrast, Cry1Bb was active against S. frugiperda but only marginally active against S. exigua. Bioassays performed with iodinated Cry1Bb, Cry1Fa, and Cry1Ca showed that the effects of iodination on toxin activity were different. The toxicities of I-labeled Cry1Bb and Cry1Fa against Spodoptera species were significantly less than the toxicities of the unlabeled toxins, while Cry1Ca retained its insecticidal activity when it was labeled with 125I. Binding assays showed that iodination prevented Cry1Fa from binding to Spodoptera brush border membrane vesicles. 125I-labeled Cry1Ac, Cry1Bb, and Cry1Ca bound with high-affinities to brush border membrane vesicles from S. exigua and S. frugiperda. Competition binding experiments performed with heterologous toxins revealed two major binding sites. Cry1Ac and Cry1Fa have a common binding site, and Cry1Bb, Cry1C, and Cry1Fa have a second common binding site. No obvious relationship between dissociation of bound toxins from brush border membrane vesicles and toxicity was detected. Cry1 toxins were also tested for the ability to alter the permeability of membrane vesicles, as measured by a light scattering assay. Cry1 proteins toxic to Spodoptera larvae permeabilized brush border membrane vesicles, but the extent of permeabilization did not necessarily correlate with in vivo toxicity.

Immunochemical cross-reactivity of two phospholipase A 2 neurotoxins, agkistrodotoxin and crotoxin

V. Choumet, M.-S. Jiang, I. Specker and C. Bon. Immunochemical cross-reactivity of two phospholipase A 2 neurotoxins, agkistrodotoxin and crotoxin. Toxicon 29, 441–451, 1991.—Polyclonal rabbit antisera were raised against the phospholipase A 2 neurotoxin agkistrodotoxin (AGTX) from Agkistrodon blomhoffii brevicaudus venom and against the phospholipase A 2 subunit (component-B, CB) of crotoxin from Crotalus durissus terrificus venom. Anti-AGTX antibodies cross-reacted strongly with crotoxin and crotoxin-like molecules and more weakly with other phospholipases A 2 from the venoms of Viperidae and Crotalidae. On the other hand, anti-CB antibodies cross-reacted with AGTX, and also recognized ammodytoxin A and the phospholipase A 2 from Vipera berus venom, but not other phospholipases A 2 from Crotalidae and Viperidae. Anti-AGTX and anti-CB antibodies were able to inhibit the phospholipase A 2 activity and to neutralize the lethal potency of the homologous and heterologous toxins (AGTX or crotoxin). Immunoaffinity chromatography columns were used to isolate anti-AGTX antibodies which recognized CB (91% of the total anti-AGTX antibodies), and anti-CB antibodies which recognized AGTX (52% of the total anti-CB antibodies). Immunochemical investigations performed with each type of antibody indicated that the majority of AGTX antigenic determinants are present on crotoxin component-B and on phospholipases A 2 from Viperidae venoms, and that some of these determinants are involved in the neutralization of lethal potency and in the inhibition of enzymatic activity of AGTX and crotoxin.

Production of a monoclonal antibody to Vibrio cholerae non-O1 heat-stable enterotoxin (ST) which is cross-reactive with Yersinia enterocolitica ST

A monoclonal antibody (MAb) against synthetic heat-stable enterotoxin of Vibrio cholerae non-O1 (NAG-ST) was produced. The MAb, namely, 2F, belonged to the immunoglobulin G1 class. Ascitic fluid drawn from pristane-primed BALB/c mice injected with a 2F-producing clone demonstrated anti-NAG-ST activity which could be detected in enzyme-linked immunosorbent assay even at a dilution of 1:128,000. Fifty-fold-diluted ascitic fluid could completely neutralize the activity of NAG-ST (synthetic and native) and Vibrio mimicus ST (identical to NAG-ST) in suckling mice. In the same assay, 2F could also neutralize Yersinia enterocolitica ST (Y-ST) but did not neutralize Escherichia coli STh and STp. A similar pattern of reactivity occurred in a competitive enzyme-linked immunosorbent assay with homologous and heterologous toxins. Competitive inhibition curves with synthetic peptides representing NAG-ST and its shorter analogs revealed that aspartic acid located at position 2 from the N terminus of NAG-ST was the essential residue of the recognized epitope. Significantly, in Y-ST, to which 2F cross-reacted, aspartic acid is in the corresponding position as that of NAG-ST, thereby confirming our conclusions that the epitope defining this MAb is aspartic acid.

Staphylococcal enterotoxin B and toxic shock syndrome toxin-1 bind to distinct sites on HLA-DR and HLA-DQ molecules.

Staphylococcal enterotoxins (SE) activate human T cells in vitro. This requires the presence of Ia+ accessory cells but does not require processing of the toxin by the accessory cell. We and others have recently demonstrated direct binding of SE to human MHC class II molecules. In this study, we have compared in detail the ability of class II molecules to bind two SE, toxic shock syndrome toxin-1 (TSST-1) and SEB. Scatchard analysis of equilibrium binding data indicate that SEB binds to Ia+ human cell lines with a 10-fold lower affinity than TSST-1. Likewise, SEB precipitates HLA-DR alpha- and beta-chains from detergent lysates of Ia+ cells less efficiently than TSST-1. The binding of TSST-1 and SEB to transfected L cells expressing HLA-DR and HLA-DQ gene products was differentially inhibited by anti-HLA-DR mAb. There was virtually no cross-inhibition of TSST-1 and SEB in competitive binding assays. We conclude that TSST-1 and SEB bind to two MHC class II sites which can be distinguished by their relative accessibility to blocking by anti-class II mAb and heterologous toxin.

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.

Protection of rats against cholera toxin and cholera-like enterotoxins by immunization with enteric-coated cholera toxin

Pure cholera toxin (CT) given as a booster in enteric-coated tablets to rats produced a humoral and intestinal immune response similar to the result of instilling the boosting dose of CT directly into the duodenum. This method protects the antigen against gastric acid and allows delivery of the immunogen to intestinal mucosa, an essential step in producing intestinal secretory IgA. Immunization gave protection against pure CT during intestinal perfusion but also significantly protected against the secretory effects of E. coli LT and CT-like toxin of A. sobria. The use of enteric-coated vaccines offers advantages for mass immunization programmes and our results suggest that immunization with preparations containing CT holotoxin may protect against heterologous toxins which cross-react with CT.

Inhibition of staphylococcal α-hemolysis by monoclonal antibodies against oligomer 12 S α-toxin

Oligomer 12 S α-toxin as well as 3 S α-toxoid of Staphylococcus aureus induced the formation of monoclonal antibodies (mabs). Mabs against the 12 S α-toxin could be demonstrated in 31 and those against 3 S α-toxoid in 18 of 120 hybrid cell colonies. Each of these mab-preparations reacted with 12 S, 3 S α-toxin and 3 S α-toxoid. The reactions were more pronounced with the homologous than the heterologous toxin preparations. Mabs against 12 S α-toxin inhibited the hemolytic effects of native 3 S α-toxin as well or better than the respective polyclonal antisera.

Differences in cross-protection in rats immunized with the B subunits of cholera toxin and Escherichia coli heat-labile toxin.

Although cholera toxin (CT), Escherichia coli heat-labile toxin (LT), and their B subunits are known to be immunologically related, the ability of each to raise an antitoxin response that provides equally strong cross-protection against active challenge with pure heterologous toxin has not been examined previously. We immunized rats with pure preparations of the B subunits of human LT, porcine LT, and CT. Immunization with either of the LT B subunits raised greater than or equal to fourfold increases in specific mucosal immunoglobulin A antitoxin titers to homologous and heterologous LT and CT B subunits, thereby providing strong protection against active challenge in ligated ileal loops with all three respective holotoxins and with a viable LT-producing E. coli strain. In contrast, immunization with the CT B subunit raised a greater than or equal to fourfold increase in antitoxin titers only to itself and provided strong protection only against challenge with the CT holotoxin. Conjugation of the CT B subunit with the E. coli heat-stable toxin by the carbodiimide reaction yielded a cross-linked immunogen with equal antigenicity for both components; immunization with this conjugate raised greater than or equal to fourfold increases in antitoxin titers to both components, but it provided significant protection only against challenge with a viable heat-stable toxin-producing E. coli strain and not to an LT-producing E. coli strain. These observations indicate that immunization with the LT B subunits raises a heterologous antitoxin response that extends to the CT B subunit, thereby providing equally strong protection against LT and CT; however, immunization with the CT B subunit raises principally a homologous antitoxin response, so that this immunogen provides strong protection only against CT.

Inhibition of bovine rod outer segment GTPase by Bordetella pertussis toxin.

Incubation of photolyzed bovine rod outer segment (ROS) membranes with pertussis toxin led to an inhibition of GTPase activity in an NAD-dependent reaction. In the presence of [32P]NAD, pertussis toxin catalyzed the [32P]ADP-ribosylation of a 39-kDa ROS protein and the 39-kDa alpha-subunit of transducin purified from ROS membranes. The decrease in GTPase activity was paralleled by an increase in the extent of [32P]ADP-ribosylation of the 39-kDa ROS protein. [32P]ADP-ribosylation of the 39-kDa protein occurred in both photolyzed and dark ROS membranes. Neither ATP nor guanylyl imidodiphosphate were required for either ADP-ribosylation or inhibition of GTPase. It was demonstrated previously that choleragen catalyzed the NAD-dependent inhibition of ROS GTPase (Abood, M. E., Hurley, J. B., Pappone, M-C., Bourne, H. R., and Stryer, L. (1982) J. Biol. Chem. 257, 10540-10543). Choleragen-catalyzed inhibition of GTPase and [32P]ADP-ribosylation of the 39-kDa protein required guanylyl imidodiphosphate and did not occur in dark ROS membranes. The effects of choleragen and pertussis toxin were not additive. Incubation of ROS with choleragen or pertussis toxin prevented the subsequent [32P]ADP-ribosylation by the homologous but not the heterologous toxin, consistent with the hypothesis that the two toxins act at different sites on the 39-kDa protein. Pertussis toxin is known to ADP-ribosylate the inhibitory guanine nucleotide-binding subunit of adenylate cyclase (Ni) but not the stimulatory subunit (Ns); choleragen is known to ADP-ribosylate Ns but not Ni. Since both toxins ADP-ribosylate the same subunit of transducin, it appears that this protein may possess characteristics of both Ns and Ni.

Serologic differentiation between antitoxin responses to infection with Vibrio cholerae and enterotoxin-producing Escherichia coli.

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