Binding of ATP by pertussis toxin and isolated toxin subunits.

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [3H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP greater than ATP greater than GTP greater than CTP greater than TTP for pertussis toxin and ATP greater than GTP greater than TTP greater than CTP for the B oligomer. Phosphate ions inhibited the binding of [3H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [3H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.     

Crystallization of diphtheria toxin.

Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.     

Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli.

A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of CTP3 and beta-galactosidase, followed by a booster injection of a sub-immunizing amount (1 microgram) of cholera toxin, led to a substantial level of neutralizing antibodies against both cholera toxin and the heat-labile toxin of Escherichia coli.     

Stoichiometry of anthrax toxin complexes.

After being proteolytically activated, the protective antigen (PA) moiety of anthrax toxin self-associates to form symmetric, ring-shaped heptamers. Heptameric PA competitively binds the enzymatic moieties of the toxin, edema factor and lethal factor, and translocates them across the endosomal membrane by a pH-dependent process. We used two independent approaches to determine how many of the seven identical EF/LF binding sites of the PA heptamer can be occupied simultaneously. We measured isotope ratios in complexes assembled from differentially radiolabeled toxin subunits, and we determined the molecular masses of unlabeled complexes by multiangle laser light scattering. Both approaches yielded the same value: the PA heptamer in solution binds three molecules of protein ligand under saturating conditions. This suggests that each bound ligand sterically occludes the binding sites of two PA subunits. According to this model, a ligand-saturated heptamer is asymmetric, with the sites of six of the seven subunits occluded. These results contribute to the conceptual framework for understanding the mechanism of membrane translocation by anthrax toxin.     

Undescribed toxin in pseudomembranous colitis.

A girl aged 12 developed pseudomembranous colitis after a short course of oral penicillin. She had no history of adverse reaction to penicillin before or after the illness. No pathogenic bacteria, mycoplasmas, or viruses were found in her faeces, but they did contain a toxin. Toxin was also found in four of five other patients with pseudomembranous colitis but not in six specimens obtained from patients with diarrhoea caused by other disorders. Further studies may show that pseudomembranous colitis is caused by a bacterial toxin.     

Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments.

Tetanus toxin was digested with papain, yielding one major polypeptide (Fragment C) with a molecular weight corresponding to 47,000 +/- 5%, thus comprising about one-third of the toxin molecule. Fragment C was antigenically active, atoxic, and stimulated the formation of antibodies neutralizing the lethal action of tetanus toxin in vivo. Furthermore, a second split product (Fragment B) was isolated from the papain digest, containing two polypeptide chains linked together via a disulfide bond. Fragment B (Mr = 95,000 +/- 5%) was atoxic and showed a reaction of nonidentity with Fragment C on immunodiffusion analysis against tetanus antitoxin. The basic two-chain structure (heavy and light chain polypeptide, cf. Matsuda, M., and Yoneda, M. (1975) Infect. Immun. 12, 1147-1153) of tetanus toxin has been confirmed and the relationship between Fragments B and C within this framework has been established. Fragment C was distinguished from the light chain by electrophoresis in sodium dodecyl sulfate and by immunodiffusion analysis, indicating that this fragment constitutes a portion of the heavy chain polypeptide. Fragment B showed a reaction of partial identity with the light as well as the heavy chain from tetanus toxin. Reduction of Fragment B with dithiothreitol followed by gel chromatography yielded a fraction which was indistinguishable from the light chain portion of the toxin molecule. It is concluded that Fragment B comprises the complementary portion of the heavy chain (remaining after scission of the polypeptide bond(s) releasing Fragment C) linked to the light chain by a disulfide bond.     

Regions of toxin A involved in toxin A excretion in Pseudomonas aeruginosa.

Toxin A is excreted by Pseudomonas aeruginosa as a mature 66,583-dalton protein. In this study, we used molecular cloning and deletion analysis to define specific regions of the toxin molecule involved in its excretion. Subclones that express either the amino terminus, the carboxy terminus, or toxin A molecules with internal deletions were constructed. The hypotoxigenic mutant PAO-T1 was used as a host for the expression of the toxin constructs. When overexpressed (by the presence of extra copies of the toxin A-positive regulatory gene, regA, in trans), toxin A-cross-reactive materials produced by most of these constructs were detected in the supernatant of PAO-T1. The supernatant of P. aeruginosa PAO-T1 contained proteolytic activity that degraded toxin A-derived products but not the intact toxin molecule. A single SalI intragenic deletion (coding for the leader peptide, the first 30 amino acids, and the last 305 amino acids of the toxin) resulted in a relatively stable product in the supernatant of PAO-T1. The product of the carboxy terminus construct (which codes for the last 305 amino acids of the toxin) was detected in the lysate of PAO-T1 only. The data suggest that the amino terminus region of toxin A (the leader peptide plus the first 30 amino acid of the mature protein) is sufficient for its excretion, and that a second region, amino acids 309 through 413, protects an internally truncated toxin A molecule from the proteolytic activity in the supernatant of P. aeruginosa PAO-T1.     

The cytolethal distending toxin family.

Cytolethal distending toxins are produced by a small but diverse group of bacterial pathogens. This newly discovered toxin family can cause a variety of mammalian cells to become irreversibly blocked in the G2 phase of the cell cycle. How this novel effect is accomplished is unknown but the study of these fascinating toxins promises to reveal new methods of host-pathogen interaction.     

Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits.

The A-subunit polypeptides of Shiga toxin, the Shiga-like toxins (SLTs), and the plant lectin ricin inactivate eucaryotic ribosomes by enzymatically depurinating 28S rRNA. Comparison of the amino acid sequences of the members of the Shiga toxin family and ricin revealed two regions of significant homology that lie within a proposed active-site cleft of the ricin A chain. In previous studies, these conserved sequences of the SLT-I and ricin A subunits have been implicated as active sites. To establish the importance of these regions of homology, we used site-directed mutagenesis to alter the A-subunit sequences of two members of the Shiga toxin family. Substitution of an aspartic acid for glutamic acid 166 of the Slt-IIA subunit decreased the capacity of the polypeptides to inhibit protein synthesis at least 100-fold in a cell-free translation system. However, this mutation did not prevent the expression of immunoreactive, full-length Slt-IIA. In addition, SLT-II holotoxin containing the mutated A subunit was 1,000-fold less toxic to Vero cells. Finally, site-directed mutagenesis was used to delete sequences encoding amino acids 202 through 213 of the Shiga toxin A subunit. Although this deletion did not prevent holotoxin assembly, it abolished cytotoxic activity.     

Adenylate cyclase toxin from Bordetella pertussis. Conformational change associated with toxin activity

Adenylate cyclase (AC) toxin from Bordetella pertussis interacts with and enters eukaryotic cells to catalyze the production of supraphysiologic levels of cyclic AMP. Although the calmodulin-activated enzymatic activity (ability to convert ATP to cyclic AMP in a cell-free assay) of this molecule is calcium independent, its toxin activity (ability to increase cyclic AMP levels in intact target cells) requires extracellular calcium. Toxin activity as a function of calcium concentration is biphasic, with no intoxication occurring in the absence of calcium, low level intoxication (200-300 pmol of cyclic AMP/mg of Jurkat cell protein) occurring with free calcium concentrations between 100 nM and 100 microM and a 10-fold increase in AC toxin activity at free calcium concentrations above 300 microM. The molecule exhibits a conformational change when free calcium concentrations exceed 100 microM as demonstrated by shift in intrinsic tryptophan fluorescence, an alteration in binding of one anti-AC monoclonal antibody, protection of a fragment from trypsin-mediated proteolysis, and a structural modification as illustrated by electron microscopy. Thus, it appears that an increase in the ambient calcium concentration to a critical point and the ensuing interaction of the toxin with calcium induces a conformational change which is necessary for its insertion into the target cell and for delivery of its catalytic domain to the cell interior.