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PubMed1989

X-ray grade crystals of the enzymatic fragment of diphtheria toxin.

The enzymatic fragment of diphtheria toxin, fragment A (Mr = 21,167), complexed to the dinucleotide adenosine 3',5'-uridine (ApU), has been crystallized at two different values of pH by hanging drop vapor diffusion. Crystals grown at a pH value of 5.0 (from I) belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 71.2 A, b = 73.0 A, c = 139.8 A and four protomers in the asymmetric unit. Crystals grown at a pH value of 8.1 (form II) belong to the monoclinic space group C2, with unit cell parameters a = 65.2 A, b = 85.6 A, c = 34.6 A, beta = 103.0 degrees and one protomer in the asymmetric unit. Both crystal forms diffract to 2.5 A resolution. The molecular structures of fragment A obtained from these two crystal forms may illuminate the pH-dependent transition of diphtheria toxin during membrane translocation.

Crystallization↗
PubMed1973

Characteristics of human antibody to diphtheria toxin.

Human antibody to diphtheria toxin was analyzed with an ammonium sulfate radioimmunoassay. In 15 immune sera with titers of between 0.03 and 25 units/ml, the avidity was similar to that of U.S. standard antitoxin. The proportion of the post-immunization anti-diphtheria antibody specific for fragment A was variable, but in the 10 sera tested constituted a substantial fraction of the antibody. Approximately 2% of healthy adult males tested failed to make an antidiphtheria toxin response after two booster doses of 1.5 Lf adsorbed diphtheriatetanus toxoid, although these nonresponders made precipitating antibody to tetanus toxoid and had normal immunoglobulin levels.

Adult↗
PubMed1982

Receptor-mediated endocytosis of diphtheria toxin by cells in culture.

The binding and uptake of fluorescently labeled diphtheria toxin by cells in culture has been examined by using epifluorescence video intensification microscopy. Rhodamine-labeled diphtheria toxin retained significant toxicity on bioassay and in cell culture and was tested for uptake by human WI-38 and mouse 3T3 fibroblasts grown in culture. When added to cells at 37 degrees C, toxin was observed to become concentrated and internalized in discrete vesicles in both cell lines. The appearance of fluorescent clusters could be prevented by addition of excess unlabeled diphtheria toxin to the medium or by addition of ATP (which has been shown to block toxin binding to cells), indicating that the rhodamine-labeled toxin was binding to diphtheria toxin-specific cell surface binding sites. When the simultaneous uptake of rhodamine-labeled diphtheria toxin and fluorescein-labeled alpha 2-macroglobulin was monitored, the two proteins appeared in the same clusters indicating that the toxin undergoes receptor-mediated endocytosis. Despite the difference in susceptibility to diphtheria toxin of cells derived from sensitive (human) and resistant (mouse) tissues, the behavior of the rhodamine-labeled derivative in both cell lines was indistinguishable in terms of toxin required for formation of clusters or inhibition by unlabeled toxin or by ATP. These results demonstrate that diphtheria toxin-specific cell surface binding sites occur on both insensitive and sensitive cells and suggest that toxin is processed similarly by both cell types during its initial cell surface binding and internalization by this pathway. The possible involvement of this uptake system in the mechanism of action of diphtheria toxin in cells is discussed.

Animals↗
PubMedDate not supplied

[Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin].

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin is not detected, and free antibodies account for 68 to 98% of the original amount. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80-100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalency of serum antibodies, and monovalency of the toxin epitopes. A new model of the toxin-antibody interactions is proposed.

Antibodies↗
PubMed1997

Differential toxicity of ricin and diphtheria toxin for bloodstream forms of Trypanosoma brucei.

The cytotoxicity of ricin and diphtheria toxin was studied in culture-adapted bloodstream forms of Trypanosoma brucei. Although ricin is endocytosed at a rate comparable to that of other internalized macromolecules, it is nontoxic to bloodstream-form trypanosomes. The resistance lies partly in low susceptibility of the targeted ribosomes: T. brucei cell-free protein biosynthesis is only partially inhibited by ricin A chain. In addition, ricin is degraded before it reaches the ribosomes, as the toxin is delivered to lysosomes. In contrast, diphtheria toxin shows similar cytotoxicities for bloodstream-form trypanosomes and mouse myeloma cells. Both trypanosome and myeloma cells are more than 1000-fold less sensitive to the action of the toxin than most other mammalian cell lines, although nicked reduced diphtheria toxin inhibits cell-free protein synthesis of T. brucei and myeloma cells to the same extent as that of a rabbit reticulocyte lysate. The effect of diphtheria toxin on T. brucei in vitro translation is NAD+ dependent, suggesting that ADP-ribosylation of elongation factor 2 could be the cause of the inhibition as it is in mammalian cells. Thus, the toxic moiety of diphtheria toxin is suitable for preparation of cell-type-specific cytotoxic reagents directed towards trypanosomes.

Animals↗
PubMed2002

Construction and characterization of transposon insertion mutations in Corynebacterium diphtheriae that affect expression of the diphtheria toxin repressor (DtxR).

Transcription of the bacteriophage-borne diphtheria toxin gene tox is negatively regulated, in response to intracellular Fe(2+) concentration, by the chromosomally encoded diphtheria toxin repressor (DtxR). Due to a scarcity of tools, genetic analysis of Corynebacterium diphtheriae has primarily relied on analysis of chemically induced and spontaneously occurring mutants and on the results of experiments with C. diphtheriae genes cloned in Escherichia coli or analyzed in vitro. We modified a Tn5-based mutagenesis technique for use with C. diphtheriae, and we used it to construct the first transposon insertion libraries in the chromosome of this gram-positive pathogen. We isolated two insertions that affected expression of DtxR, one 121 bp upstream of dtxR and the other within an essential region of the dtxR coding sequence, indicating for the first time that dtxR is a dispensable gene in C. diphtheriae. Both mutant strains secrete diphtheria toxin when grown in medium containing sufficient iron to repress secretion of diphtheria toxin by wild-type C. diphtheriae. The upstream insertion mutant still produces DtxR in decreased amounts and regulates siderophore secretion in response to iron in a manner similar to its wild-type parent. The mutant containing the transposon insertion within dtxR does not produce DtxR and overproduces siderophore in the presence of iron. Differences in the ability of the two mutant strains to survive oxidative stress also indicated that the upstream insertion retained slight DtxR activity, whereas the insertion within dtxR abolished DtxR activity. This is the first evidence that DtxR plays a role in protecting the cell from oxidative stress.

Bacterial Proteins↗
PubMed1995

Structure of the isolated catalytic domain of diphtheria toxin.

The structure of the isolated catalytic domain of diphtheria toxin at pH 5.0 was determined by X-ray crystallography at 2.5 A resolution and refined to an R-factor of 19.7%. The domain is bound to its endogenous inhibitor adenylyl(3'-->5')uridine 3'-monophosphate (ApUp). The structure of this 190-residue domain, which was expressed in and isolated from Escherichia coli, is essentially identical to the structure of the catalytic domain within whole diphtheria toxin determined at pH 7.5. However, there are two adjacent surface loops (loop 66-78 and loop 169-176) that exhibit clear differences when compared to the structure of the catalytic domain in whole diphtheria toxin. Although both loops are at the surface of the protein and are relatively flexible, the chain trace is well-defined in the electron density. The main structural difference is the closer approach of loops 66-78 and 169-176. We ascribe this structural change mainly to the absence of the neighboring transmembrane domain in the isolated catalytic domain as compared to whole diphtheria toxin. We suggest that this change represents the first step of the structural transition from the catalytic domain in whole diphtheria toxin to the translocated form of the domain. The changes are described in detail, and their implications for membrane translocation are discussed.

Binding Sites↗
PubMed2000

[Diagnostic value of Diamed AG latex gel kit for detection of diphtheria toxin].

ID-PaGIA diphtheria toxin polymer particle diagnostic agent manufactured by DiaMed AG, Switzerland, was tried at bacteriological laboratory of Institute of Childhood Infections in St. Petersburg. The trials were carried out using two methods, direct and capture, which differ by the duration of incubation of the studied C. diphtheriae cultures. Ouchterloney's immunoprecipitation test in agar was the control method. Ninety-seven toxigenic strains were tested by direct test and 76 by capture test; in addition, 19 nontoxigenic strains were tested. The results coincided with control tests in 100% cases. The advantages of each method are defined and the possibility of their utilization at bacteriological laboratories of infectious hospitals and State Sanitary and Epidemiological Surveillance is evaluated.

Diphtheria Toxin↗
PubMed2005

Prolylpeptide binding by the prokaryotic SH3-like domain of the diphtheria toxin repressor: a regulatory switch.

Diphtheria toxin repressor (DtxR) regulates the expression of iron-sensitive genes in Corynebacterium diphtheriae, including the diphtheria toxin gene. DtxR contains an N-terminal metal- and DNA-binding domain that is connected by a proline-rich flexible peptide segment (Pr) to a C-terminal src homology 3 (SH3)-like domain. We determined the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by use of NMR spectroscopy. The structure of the intramolecularly bound Pr segment differs from that seen in eukaryotic prolylpeptide-SH3 domain complexes. The prolylpeptide ligand is bound by the SH3-like domain in a deep crevice lined by aliphatic amino acid residues and passes through the binding site twice but does not adopt a polyprolyl type-II helix. NMR studies indicate that this intramolecular complex is present in the apo-state of the repressor. Isothermal equilibrium denaturation studies show that intramolecular complex formation contributes to the stability of the apo-repressor. The binding affinity of synthetic peptides to the SH3-like domain was determined using isothermal titration calorimetry. From the structure and the binding energies, we calculated the enhancement in binding energy for the intramolecular reaction and compared it to the energetics of dimerization. Together, the structural and biophysical studies suggest that the proline-rich peptide segment of DtxR functions as a switch that modulates the activation of repressor activity.

Amino Acid Sequence↗
PubMed1984

Anion requirement and effect of anion transport inhibitors on the response of vero cells to diphtheria toxin and modeccin.

The anion requirement for toxic action of diphtheria toxin and modeccin was studied. In Cl- -free Hepes buffer made isotonic with mannitol, cells were insensitive to diphtheria toxin and modeccin. Just 2 mM NaCl was sufficient to obtain full toxic activity of modeccin, whereas 140 mM NaCl was required for maximal intoxication with diphtheria toxin. Br- could substitute for Cl-. NO3-,l-, and ClO3- were less efficient than Cl-, whereas SO42- and SCN- were unable to replace Cl- . Cl- deprivation both reduced the ability of cells to bind diphtheria toxin and prevented bound toxin from intoxicating the cells. The binding of modeccin was not reduced. SITS (4-acetamide-4'-isothiocyano-stilbene-2,2'-disulfonic acid), an inhibitor of Cl- entry, protected against diphtheria toxin and modeccin, indicating that Cl- transport is required for intoxication.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1997

Use of Trp mutations to evaluate the conformational behavior and membrane insertion of A and B chains in whole diphtheria toxin.

The structure of diphtheria toxin was examined using its Trp fluorescence. To examine the interactions of the A and B chains of the toxin independently, mutants were constructed in which Trp residues were restricted to either the A or the B chain. The conformation and stability of the mutants were very similar to those of the wild-type protein. In addition, they underwent the low-pH conformational transition and membrane insertion at about the same pH as wild-type toxin. This shows Trp do not play a critical role in these processes which are necessary for the translocation of toxin across endosomal membranes in vivo. There was a shift in fluorescence of the Trp mutants which showed the low-pH-induced transition increases exposure of both the A and B Trp to a more polar environment. This supports a model in which the interdomain interactions present at neutral pH break down at low pH. To evaluate the location of the A and B chains in the membrane, the fluorescence quenching of model membrane inserted toxin was measured. Comparison of the amount of quenching by lipid labeled with nitroxides localized at shallow, medium, or deep depths within the bilayer demonstrated that both the A and B chains insert deeply, but the A chain Trp are somewhat less deeply inserted. Trp on the A chain are also less exposed to lipid than on the B chain, as judged by their weaker quenching by the nitroxide-labeled lipid. This conclusion was supported by the observation that the Trp of membrane-inserted isolated A chain is more lipid-exposed than when the A chain is part of the whole toxin. Both the A and B chain Trp become less exposed to lipid after neutralizing pH. However, both chains remain inserted, with at least part of the B chain remaining deeply inserted. These results support the "partial wrapper" model in which both the A and B chains are inserted but contacts between the two chains significantly reduce the exposure of the A chain to lipid.

Diphtheria Toxin↗
PubMed1985

Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain.

CRM197, CRM176, and CRM228 are products of single or multiple missense mutations in the diphtheria toxin gene. CRM197 differs from wild-type toxin in 1 amino acid residue of the fragment A region, and also CRM176 and CRM228 have amino acid substitution(s) in fragment A. We compared the binding properties of CRM197 to toxin-sensitive Vero cells with those of diphtheria toxin and other CRMs. Nicked CRM197 is about 50 times more effective than intact CRM197 in inhibiting the action of diphtheria toxin on sensitive cells, as shown by inhibition of diphtheria toxin cytotoxicity or inhibition of binding of 125I-diphtheria toxin. The binding of native toxin or other CRMs was not significantly affected by nicking. Moreover, the binding of CRM197 to cells was unaffected by ATP, although ATP clearly inhibits binding of diphtheria toxin, CRM176, and CRM228. Two kinds of hybrid protein were formed using fragment B of CRM197: one with fragment A of diphtheria toxin and one with fragment A of CRM228. ATP inhibited the binding of these hybrid proteins. Furthermore, the affinities of these hybrid proteins for diphtheria toxin-sensitive cells were the same as that of native toxin. Thus, it was concluded that the altered binding properties of CRM197 were due to alteration of fragment A and what the interaction of diphtheria toxin with ATP involves both fragments. The results also suggest that fragment A plays a role in diphtheria toxin-receptor interaction.

Adenosine Triphosphate↗
PubMed1993

Subcloning and characterization of the binding domain of fragment B of diphtheria toxin.

The binding domain (R domain) of diphtheria toxin as defined from the recently published crystal structure [Choe, Bennett, Fujii, Curmi, Kantardjieff, Collier and Eisenberg (1992) Nature (London) 357, 216-222] was subcloned. The 17 kDa peptide containing amino acids 378-535 from fragment B of diphtheria toxin preceded by the tripeptide Met-His-Gly bound specifically and with high affinity to diphtheria-toxin receptors. It efficiently inhibited the toxicity of full-length toxin. The binding domain entered the detergent phase of Triton X-114 at pH values below 6, indicating that it exposed hydrophobic regions at acidic pH.

Animals↗
PubMedDate not supplied

[The estimation of the amount of diphtheria toxin and its activity by various tests in the dynamic cultivation of Corynebacterium diphtheriae PW8].

Direct correlation between the results of tests for the biological activity of diphtheria toxin, carried out in vivo guinea pigs and in vitro in the microcytotoxicity test in CHO cell culture, has been established, which makes it possible to use the latter as one of the methods for the rapid, reproducible and economic evaluation of diphtheria toxin. The qualitative and quantitative evaluation of diphtheria toxin in the enzyme immunoassay with the use of monoclonal antibodies and in the microcytotoxicity test demonstrates that these two tests, when used for controlling cultivation processes, have essential advantages over the flocculation test as regards their specificity and information content.

Animals↗
PubMed2002

Immunochromatographic strip test for rapid detection of diphtheria toxin: description and multicenter evaluation in areas of low and high prevalence of diphtheria.

An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of Corynebacterium spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively.

Antibodies, Bacterial↗