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PubMed1983

Strains of CHO-K1 cells resistant to Pseudomonas exotoxin A and cross-resistant to diphtheria toxin and viruses.

We have investigated two phenotypically distinct types of mutants of CHO-K1 cells that are resistant to Pseudomonas exotoxin A due to a defect in the delivery of active toxin to the target site in the cell, elongation factor 2. Both types contain normal levels of toxin-sensitive elongation factor-2. Hybridization studies have shown that these cells fall into two distinct complementation groups. One group, designated DPVr, is resistant to Pseudomonas toxin, diphtheria toxin, and four enveloped RNA viruses. This group is also hypersensitive to ricin. The resistance of this group is apparently related to a defect in a mechanism for the acidification of endocytic vesicles. The other group, designated PVr, is resistant to Pseudomonas toxin and to three enveloped RNA viruses. The resistance of this group appears to be related to a defect in a cellular mechanism required for the maturation of Sindbis virus that is likewise required for the entry of active Pseudomonas toxin.

ADP Ribose Transferases↗
PubMed1980

Enhancement of cellular protein synthesis sensitivity to diphtheria toxin by interferon.

In attempts to determine whether, by analogy to cholera and tetanus toxins, diphtheria toxin (DT) can also relieve the antiviral effect of interferon (IF), we found that it rather enhanced the inhibitory effect of IF on the replication of murine leukemia virus in chronically infected NIH/3T3 cells. This enhancement was found to be a consequence of an increased sensitivity to DT of cellular protein synthesis in IF-treated cells. IF stimulated the anti-protein synthesis activity of DT in both mouse cells that are known to be highly resistant to this toxin and in human HeLa cells that are highly sensitive to this toxin. This stimulation was dependent on IF dose. The effect of IF on DT action was strictly species specific, indicating that it was not a consequence of the mere binding of IF to the cell membrane, but rather reflected the cellular changes that followed this initial binding. IF was found to be capable of potentiating intact DT, but could not potentiate its fragments in any combination. IF did not have any effect on the in vitro nicotinamide adenine dinucleotide glycohydrolase activity of DT, suggesting that the effect of IF is not due to molecular modification of the toxin.

Animals↗
PubMed1987

[Cloning of the structural gene for diphtheria toxin from Corynebacterium diphtheriae PW8 and expression of its deletion derivative in E. coli].

The coding region of diphtheria toxin gene from Corynebacterium diphtheriae PW8 was cloned in Escherichia coli on pUC19 plasmid. 5' end of the gene was deleted. The deleted gene codes for a toxin like protein truncated for 36 N-terminal aminoacids. The modified gene was fused with 5' terminal part of the lacZ gene on pUC19 plasmid having kept the translational frame intact. The hybrid gene transcription depends on lacP promoter, Mr of the resulting toxoid is 54,000. The toxoid is devoid of ADP-ribosyltransferase activity peculiar for diphtheria toxin and thus is not toxic. The constructed version of the toxin gene can be used for production of diphtheria anatoxin which does not require to be inactivated by formalin.

Chromosome Deletion↗
PubMed1993

Hypersensitivity to diphtheria toxin by mouse cells expressing both diphtheria toxin receptor and CD9 antigen.

DTS-II is a highly diphtheria toxin (DT)-sensitive cell line previously isolated by transfection of wild-type DT-resistant mouse L-M(TK-) cells with the cDNA encoding a monkey Vero cell DT receptor. DTS-II cells are as toxin-sensitive as Vero cells, have approximately 3-fold more receptors than Vero cells, and have approximately 10-fold lower affinity for DT than Vero cells. We now cotransfected DTS-II cells with a plasmid containing the Vero cell cDNA coding for CD9 antigen (pCD9) and with a plasmid containing the gene for hygromycin resistance (pHyg). The stably transfected hygromycin-resistant colonies were screened for DT hypersensitivity employing a replica plate system. A DT-hypersensitive colony was isolated and purified. The purified DT-hypersensitive cells, DTS-III, (i) are approximately 10-fold more toxin-sensitive than DTS-II and Vero cells and (ii) bear approximately 10(6) DT receptors per cell (i.e., approximately 20-fold and approximately 60-fold more receptors than DTS-II and Vero cells, respectively), but their receptor affinity is still approximately 10-fold lower than that of Vero cells. Cross-linking experiments employing 125I-labeled DT demonstrated that DTS-II and DTS-III cells have essentially the same profile of DT-binding cell-surface protein(s), suggesting that CD9 antigen, although expressed on the cell surface of DTS-III cells, may not be in close proximity to the DT-binding domain of the receptor. CD9 may affect DT receptor expression by increasing receptor density at the cell surface. By employing DTS-III cells it should be possible to purify and characterize the DT cell-surface receptor protein(s).

Animals↗
PubMed1991

An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor.

A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.

Animals↗
PubMed1972

Response of cultured mammalian cells to diphtheria toxin. IV. Isolation of KB cells resistant to diphtheria toxin.

KB cell strains which are resistant to the lethal effects of diphtheria toxin were isolated from the toxin-sensitive KB cell line. For one such strain, KB-R2, the concentration of diphtheria toxin required to cause a 50% reduction of incorporation of amino acids into protein was more than 2,000 times that required in the sensitive KB-S cell. The KB-R2 strain was cloned by a single-cell isolation method to yield uniform strains with an even greater resistance to toxin. Resistance is a stable, inheritable property, providing a good genetic marker, and appears to be related to some alteration in the cell membrane. Preliminary studies showed that these cell strains are resistant to poliovirus and vesicular stomatitis virus as well as to toxin.

Amino Acids↗
PubMed1987

Diphtheria toxin receptor. Identification of specific diphtheria toxin-binding proteins on the surface of Vero and BS-C-1 cells.

The biochemical characteristics of specific receptor molecules for diphtheria toxin on the surface of two toxin-sensitive cell lines (Vero and BS-C-1) were examined. Diphtheria toxin was found to bind to a number of different proteins in Nonidet P-40 solubilized extracts of 125I-labeled cells. In contrast, permitting diphtheria toxin to bind first to labeled intact cells, which were subsequently solubilized and subjected to immunoprecipitation with anti-diphtheria toxin, resulted in a far more restricted profile of diphtheria toxin-binding proteins that possessed Mrs in the range of 10,000-20,000. Direct chemical cross-linking of radioiodinated diphtheria toxin to cell surface proteins resulted in the appearance of several predominant bands possessing Mrs of approximately 80,000. The Mr approximately 80,000 complexes were shown to be composed of radiolabeled diphtheria toxin (Mr 60,000) and unlabeled Mr approximately 20,000 cellular proteins. These complexes were judged to be a result of specific binding in that their appearance could be preferentially inhibited by the addition of a 100-fold excess of unlabeled diphtheria toxin. The formation of the Mr approximately 80,000 complexes was sensitive to prior trypsin treatment of the cells and to known inhibitors of diphtheria toxin binding. Furthermore, prior incubation of the cells with diphtheria toxin at 37 degrees C ("down regulation") markedly and specifically reduced the subsequent formation of the Mr approximately 80,000 cross-linked complexes, and these down-regulated cells were less sensitive to diphtheria toxin in cytotoxicity assays. Further incubation of down-regulated cells at 37 degrees C restored their ability to form Mr approximately 80,000 complexes; this regeneration requires protein synthesis and restores the cells' sensitivity to diphtheria toxin-mediated cytotoxicity. These results strongly suggest that a Mr 10,000-20,000 cell surface protein is, or constitutes a portion of, the functional diphtheria toxin receptor.

Animals↗
PubMed1984

Entry of diphtheria toxin into cells: possible existence of cellular factor(s) for entry of diphtheria toxin into cells was studied in somatic cell hybrids and hybrid toxins.

Ehrlich ascites tumor cells were found to be very insensitive to diphtheria toxin. We formed 37 hybrids from Ehrlich tumor cells and diphtheria toxin-sensitive human fibroblasts. The effects of diphtheria toxin on protein synthesis in those hybrids were examined. The hybrids were divided into three groups on the basis of toxin sensitivity. Group A hybrids were as sensitive to diphtheria toxin as human fibroblasts, Group C were as resistant as Ehrlich tumor cells, and Group B had intermediate sensitivity. Group A hybrids had diphtheria toxin-binding sites but Group B and C had no detectable binding sites. Elongation factor-2 of all the hybrids was susceptible to ADP-ribosylation by fragment A of diphtheria toxin. Cells of Group A and B became more sensitive to CRM 45 (cross-reacting material 45 of diphtheria toxin) after they were exposed to low pH (pH = 4.5). The resistance of Group C to CRM 45 was not affected by the same treatment. Group A and B hybrids and human fibroblasts had similar sensitivities to a hybrid toxin composed of wheat germ agglutinin and fragment A of diphtheria toxin, but Group C and Ehrlich tumor cells were resistant to this hybrid toxin. All the hybrids and Ehrlich tumor cells were more sensitive to a hybrid toxin composed of wheat germ agglutinin and subunit A of ricin than were human fibroblasts. On subcloning of Group B hybrids, one Group C hybrid was obtained, but no Group A hybrid. These facts suggest that Ehrlich ascites tumor cells differ from human fibroblasts in the expression of a factor(s) that is involved in entry of fragment A of diphtheria toxin into the cytoplasm after the toxin binds to its surface receptors.

Adenosine Diphosphate Ribose↗
PubMed1998

Cytotoxicity and specificity of directed toxins composed of diphtheria toxin and the EGF-like domain of heregulin beta1.

As a step in the design of directed toxins, aimed at cells that overexpress HER receptors, particularly breast carcinoma cells, we studied the properties of a chimera of diphtheria toxin (DT) and heregulin beta1. The EGF-like growth hormone heregulin is a ligand for the HER3 and HER4 receptors and their heterodimers with HER2. The 60-residue EGF-like domain (hrg) of heregulin elicits a biological response and binds to these receptors primarily through its N terminus. We tested a fusion protein in which hrg replaces the C-terminal receptor-binding domain of DT (DT(389)hrg) and an alternative design in which this domain is fused to the N terminus of DT(389). Of those two constructs, the N-terminal fusion was not active as a directed toxin but elicited a growth response. The C-terminal fusion of hrg to DT(389) yielded a functional toxin and showed cell line specific cytotoxicity that is consistent with heregulin specificity. The binding of hrg to its cognate receptor is not impaired as shown by receptor activation, direct binding, and competition with free hrg. Cytotoxicity is dependent on high-affinity binding of DT(389)hrg to HER3 and HER4 receptors and is not mediated by HER2 overexpression alone. For those cell lines exhibiting high-affinity binding sites, the level of cytotoxicity correlates with the rate of internalization. Thus DT(389)hrg chimeras offer a possible avenue toward directed toxins against cells that overexpress HER receptors.

Binding Sites↗
PubMed2003

Detection of differences in the nucleotide and amino acid sequences of diphtheria toxin from Corynebacterium diphtheriae and Corynebacterium ulcerans causing extrapharyngeal infections.

While Corynebacterium ulcerans can mimic classical diphtheria, extrapharyngeal infections are extremely rare. Sequencing of the diphtheria toxin (DT)-encoding tox gene of two C. ulcerans isolates from extrapharyngeal infections revealed differences from C. diphtheriae DT sequences, mainly in the translocation and receptor-binding domains. C. ulcerans supernatants were much less potent than supernatant from C. diphtheriae. A C. ulcerans DT-specific PCR is described below.

Adult↗
PubMed1996

Targeted delivery of diphtheria toxin via immunoliposomes: efficient antitumor activity in the presence of inactivating anti-diphtheria toxin antibodies.

Diphtheria toxin (DT) has attracted considerable attention for anti-cancer therapy. However, its extensive use is prohibited by (i) its non-specific action which can result in substantial toxicity, (ii) most patients have low serum levels of anti-DT antibodies (AT antibodies) which can inactivate DT and (iii) its immunogenicity will boost the circulating AT antibody level, thereby further compromising the antitumor activity. To overcome these limitations, we have developed a new approach for targeted delivery of DT utilizing immunoliposomes. In this approach, protection against the non-specific action of DT is combined with efficient antitumor activity even in the presence of inactivating AT antibodies.

Animals↗
PubMed2003

Transgenic mice expressing the diphtheria toxin receptor are sensitive to the toxin.

Whereas diphtheria and the mechanism of action of diphtheria toxin, the bacterial molecule that induces the disease, have been studied and understood for some time, the receptor that allows animal cells to bind the toxin escaped identification until recently. The receptor was identified by its ability to confer toxin-sensitivity to mouse cells, which are normally toxin-resistant. Although mice are also naturally resistant, we now demonstrate that transgenic mice expressing the diphtheria toxin receptor are as sensitive to the toxin as are humans and other toxin-sensitive animals. These transgenic mice provide a suitable model for studying modern antidotes for diphtheria.

Animals↗
PubMed1996

Reactivity of murine cytokine fusion toxin, diphtheria toxin390-murine interleukin-3 (DT390-mIL-3), with bone marrow progenitor cells.

Myeloid leukemias can express interleukin-3 receptors (IL-3R). Therefore, as an antileukemia drug, a fusion immunotoxin was synthesized consisting of the murine IL-3 (mIL-3) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mIL-3 hybrid gene was cloned into a vector under the control of an inducible promote. The fusion protein was expressed in Escherichia coli and then purified from inclusion bodies. The fusion toxin was potent because it inhibited FDC-P1, an IL-3R-expressing murine myelomonocytic tumor line (IC50 = 0.025 nmol/L or 1.5 ng/mL). Kinetics were rapid and cell-free studies showed that DT390-mIL-3 was as toxic as native DT. DT390-mIL-3 was selective because anti-mIL-3 monoclonal antibody, but not irrelevant antibody, inhibited its ability to kill. Cell lines not expressing IL-3R were not inhibited by the fusion protein. Because the use of DT390-mIL-3 as an antileukemia agent could be restricted by its reactivity with committed and/or primitive progenitor cells, bone marrow (BM) progenitor assays were performed. DT390-mIL-3 selectively inhibited committed BM progenitor cells as measured by in vitro colony-forming unit-granulocyte-macrophage and in vivo colony-forming unit-spleen colony assays. To determine if this fusion protein was reactive against BM progenitor cells required to rescue lethally irradiated recipients, adoptive transfer experiments were performed. Eight million DT390-mIL-3-treated C57BL/6 Ly5.2 BM cells, but not 4 million, were able to rescue lethally irradiated congenic C57BL/6 Ly5.1 recipients, suggesting that progenitor cells might be heterogenous in their expression of IL-3R. This idea was supported in competitive repopulation experiments in which DT390-mIL-3 treated C57BL/6 Ly5.2 BM cells were mixed with nontreated C57BL/6 Ly5.1 BM cells and used to reconstitute C57BL/6 Ly5.1 mice. A significant reduction, but not elimination, of Ly5.2-expressing cells 95 days post-BM transplantation and secondary transfer experiments indicated that IL-3R is not uniformly expressed on all primitive progenitor cells. The fact that some early progenitor cells survived DT390-mIL-3 treatment indicates that this fusion toxin may be useful in the treatment of myeloid leukemias that express the IL-3R.

Animals↗
PubMed2004

Prediction of DtxR regulon: identification of binding sites and operons controlled by Diphtheria toxin repressor in Corynebacterium diphtheriae.

BACKGROUND: The diphtheria toxin repressor, DtxR, of Corynebacterium diphtheriae has been shown to be an iron-activated transcription regulator that controls not only the expression of diphtheria toxin but also of iron uptake genes. This study aims to identify putative binding sites and operons controlled by DtxR to understand the role of DtxR in patho-physiology of Corynebacterium diphtheriae. RESULT: Positional Shannon relative entropy method was used to build the DtxR-binding site recognition profile and the later was used to identify putative regulatory sites of DtxR within C. diphtheriae genome. In addition, DtxR-regulated operons were also identified taking into account the predicted DtxR regulatory sites and genome annotation. Few of the predicted motifs were experimentally validated by electrophoretic mobility shift assay. The analysis identifies motifs upstream to the novel iron-regulated genes that code for Formamidopyrimidine-DNA glycosylase (FpG), an enzyme involved in DNA-repair and starvation inducible DNA-binding protein (Dps) which is involved in iron storage and oxidative stress defense. In addition, we have found the DtxR motifs upstream to the genes that code for sortase which catalyzes anchoring of host-interacting proteins to the cell wall of pathogenic bacteria and the proteins of secretory system which could be involved in translocation of various iron-regulated virulence factors including diphtheria toxin. CONCLUSIONS: We have used an in silico approach to identify the putative binding sites and genes controlled by DtxR in Corynebacterium diphtheriae. Our analysis shows that DtxR could provide a molecular link between Fe+2-induced Fenton's reaction and protection of DNA from oxidative damage. DtxR-regulated Dps prevents lethal combination of Fe+2 and H2O2 and also protects DNA by nonspecific DNA-binding. In addition DtxR could play an important role in host interaction and virulence by regulating the levels of sortase, a potential vaccine candidate and proteins of secretory system.

Bacterial Proteins↗
PubMed1986

Interactions between diphtheria toxin entry and anion transport in Vero cells. IV. Evidence that entry of diphtheria toxin is dependent on efficient anion transport.

Entry of prebound diphtheria toxin at low pH occurred rapidly in the presence of isotonic NaCl, NaBr, NaSCN, NaI, and NaNO3, but not in the presence of Na2SO4, 2-(N-morpholino)ethanesulfonic acid neutralized with Tris, or in buffer osmotically balanced with mannitol. SCN- was the most efficient anion to facilitate entry. Uptake studies with radioactively labeled anions showed that SCN- was transported into cells 3 times faster than Cl-, while the entry of SO2-4 occurred much more slowly. The anion transport inhibitors 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and piretanide inhibited entry at low pH even in the presence of permeant anions. When cells with bound toxin were exposed to low pH in the absence of permeant anions, then briefly exposed to neutral pH and subsequently exposed to pH 4.5 in the presence of isotonic NaCl, toxin entry was induced. The data indicate that efficient anion transport at the time of exposure to low pH is required for entry of surface-bound diphtheria toxin into the cytosol. Since insertion of diphtheria toxin into the membrane occurs even in the absence of permeant anions, the results indicate that low pH is required not only for insertion of fragment B into the membrane, but also for the subsequent entry of fragment A into the cytosol.

Animals↗