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PubMed1968

Site in cell-free protein synthesis sensitive to diphtheria toxin.

The effects of diphtheria toxin on cell-free protein synthesis in a bacterial system, and preparations obtained from animals that were sensitive and resistant to toxin were examined. In the presence of nicotinamide adenine dinucleotide (NAD), toxin inhibited the incorporation of amino acids by endogenous and synthetic polynucleotides in both rat liver and guinea pig liver cell-free systems that were exposed to 6 Lf units per ml of toxin. A cell-free system derived from Streptococcus faecalis was resistant to high concentrations of toxin. Dialyzed toxin-antitoxin floccules that are formed in the presence of NAD and the 105,000 x g supernatant fluid from rat liver contain NAD. Such floccules are also active in protein synthesis in the absence of added transferase I or II. An operational model presents the view that the intoxication complex is formed at the ribosomal level and occurs in two steps. First, the toxin molecule binds to transferase II and alters its stereospecific relationship to transferase I, but it does not result in an inactive complex. Second, the stereospecific alteration in transferase I, but it does not result in an inactive complex. Second, the stereospecific alteration in transferase II caused by the binding of diphtheria toxin allows NAD to bridge between transferase I and II, which then results in an inactivated complex. The sensitivity of the cell-free system derived from the normally resistant rat implies that in some cells the cell membrane serves as a permeability barrier to the toxin molecule. The resistance of bacterial cell-free protein synthesizing systems to diphtheria toxin may reflect basic differences between transferase enzymes from bacterial and mammalian sources.

Amino Acids↗

PubMed1999

Granulocyte-macrophage colony-stimulating factor induces expression of heparin-binding epidermal growth factor-like growth factor/diphtheria toxin receptor and sensitivity to diphtheria toxin in human neutrophils.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a widely expressed EGF superfamily member that induces mitogenic and/or chemotactic activities toward different cell types through binding to EGF receptors 1 or 4. Membrane-bound HB-EGF exerts growth activity and adhesion capabilities and possesses the unique property of being the receptor for diphtheria toxin (DT). Using molecular and functional techniques, we show that human polymorphonuclear granulocytes (PMN), which did not express HB-EGF in resting conditions, expressed it at mRNA and protein level, following incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Other classic agonists for PMN (including lipopolysaccharide, phagocytable particles, tumor necrosis factor-alpha, or G-CSF) failed to induce HB-EGF. The effects of GM-CSF on HB-EGF mRNA levels were concentration-dependent, reached a plateau after 1 to 2 hours of stimulation, and did not require protein synthesis. After GM-CSF treatment, membrane-bound HB-EGF was detected by flow cytometry. At the same time, PMN acquired sensitivity to the apoptosis-promoting effect of DT, which, moreover, specifically suppressed the GM-CSF-induced priming of formyl-methionyl-leucyl-phenylalanine-stimulated superoxide anion release. Finally, soluble HB-EGF was detected in the PMN culture medium by a specific enzyme-linked immunosorbent assay. Thus, we provide evidence that HB-EGF is specifically inducible by GM-CSF in PMN and represents a novel peptide to be included in the repertoire of PMN-derived cytokines.

Diphtheria Toxin↗

PubMed2006

Fresh frozen plasma and platelet concentrates may increase plasma anti-diphtheria toxin IgG concentrations: Implications for diphtheria fusion protein therapy.

We are developing two fusion proteins consisting of a diphtheria toxin (DT) linked to either granulocyte macrophage colony stimulating factor (DT388-GMCSF) or interleukin-3 (DT388-IL3). In trials, patients with anti-DT IgG concentrations >2.5 microg/ml had significantly lower concentrations of either fusion protein. DT389-IL2 is currently FDA approved for the treatment of cutaneous T-cell lymphomas. We noted increased concentrations of anti-DT IgG after administration of platelet concentrates (PC). Because many of these patients require transfusions, we measured the anti-DT IgG content of FFP and PC. We assayed 14 bags of FFP and 12 bags of single-donor PCs for anti-DT IgG by an enzymoimmunoassay against DT389-IL2, DT388-IL3, DT388-GMCSF. The median (percent of samples positive) of anti-DT IgG concentrations in PC against DT388-GMCSF, DT388-IL3, DT389-IL2 was 0.8 microg/ml (83%), 0.7 microg/ml (83%), and 0.3 microg/ml (58%), respectively. The median (percent of samples positive) anti-DT IgG concentration in FFP against DT388-GMCSF, DT388-IL3, and DT389-IL2 was 2.1 microg/ml (86%), 1.9 microg/ml (93%), and 1.4 microg/ml (86%), respectively. There was a strong association between anti-DT389IL2 IgG, anti-DT388IL3 IgG, and anti-DT388GMCSF IgG concentrations in both the FFP (95.6%) and PC (76.3%). Assuming a plasma volume of 3 l in a 70 kg patient, a single FFP unit would increase the plasma anti-DT389IL2 IgG, anti-DT388IL3 IgG, and anti-DT388GMCSF IgG by 0.13 microg/ml, 0.17 microg/ml, and 0.19 microg/ml, respectively. For PC, a single unit would increase plasma anti-DT389-IL2 IgG, anti-DT388-IL3 IgG, and anti-DT388-GMCSF IgG by 0.03 microg/ml, 0.06 microg/ml, and 0.07 microg/ml, respectively. In conclusion, a single FFP or PC appears to minimally increase anti-DT IgG concentrations, but multiple units may significantly do such.

Blood Platelets↗

PubMed1999

Endocytic mechanisms responsible for uptake of GPI-linked diphtheria toxin receptor.

We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.

Adolescent↗

PubMed1976

Interaction of diphtheria toxin and its active subunit, fragment A, with toxin-sensitive and toxin-resistant cells.

Diphtheria toxin and purified fragment A, the active subunit of the toxin, were tested on toxin-sensitive and permeability class toxin-resistant cultured mammalian cells. Protein synthesis was inhibited to the same degree in sensitive or resistant cells by active concentrations of purified fragment A. In contrast, resistant cells required a concentration of whole toxin 5 to 6 logs greater than that required by sensitive cells to achieve the same degree of inhibition. On a molar basis, the toxicity of fragment A was equivalent to that of whole toxin on resistant cells. These results are evidence for the existence of two independent mechanisms for the entry of toxin or its active moiety into cells. One mechanism is a highly efficient, toxin-specific entry mechanism, involving surface receptor and fragment B-mediated association, and is active in sensitive cells. The other is a less efficient, nonspecific mechanism, probably related to endocytosis, which is operative in sensitive and resistant cells but is inapparent in sensitive cells when they are exposed to whole toxin because of the action of the specific mechanism.

Ammonium Chloride↗

PubMed1989

Cellular regulation of diphtheria toxin cell surface receptors.

The cellular regulation of diphtheria toxin cell surface receptors was studied. Treatment of Vero cells with cycloheximide reduced their diphtheria toxin binding capacity, while cells treated with actinomycin D did not lose their ability to bind diphtheria toxin. A non-toxic analogue of diphtheria toxin, CRM 197, produced a dose-related depletion of cell surface diphtheria toxin binding capacity that was reversible upon washing the cells. Vero cells depleted of toxin receptors by CRM 197 did not restore their ability to bind diphtheria toxin in the presence of cycloheximide. Phospholipase C treatment of Vero cells reduced their diphtheria toxin binding capacity in a dose-dependent manner. The loss of diphtheria toxin binding capacity was recovered within 2 hr after removal of the enzyme. Protein synthesis inhibition blocked this recovery while actinomycin D partially inhibited it. Receptors prebound with toxin were resistant to phospholipase C treatment, suggesting that the action of the enzyme was directly on the receptor. Inhibition of glycosylation with tunicamycin did not prevent reappearance of toxin receptors after CRM 197 or phospholipase C treatment. These data establish the requirement of a continuous protein synthesis for the maintenance of diphtheria toxin cell surface receptors and also suggest that these receptors do not recycle after binding ligand. A hypothesis is put forward that the diphtheria toxin receptor might be a lipid-linked cell surface protein.

Animals↗

PubMed1980

Binding of diphtheria toxin to phospholipids in liposomes.

Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine/cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of diphtheria toxin to cells.

Cardiolipins↗

PubMed1992

Membrane translocation of diphtheria toxin carrying passenger protein domains.

For diphtheria toxin to be cytotoxic, the enzymatically active part (fragment A) must be translocated to the cytosol. We here demonstrate that additional proteins linked as N-terminal extensions can be translocated along with fragment A across the plasma membrane of toxin-sensitive cells. Thus, an extra fragment A of diphtheria toxin and some of apolipoprotein AI were translocated as passenger proteins along with mutant diphtheria toxin fragment A. Translocation was monitored by the cytotoxic effect of the additional fragment A as well as by the translocation of [35S]methionine-labelled protein to a compartment protected from externally added pronase. Cytotoxicity experiments indicated that double A fragments can also be translocated across the membrane of intracellular vesicles. The results demonstrate that the translocation apparatus used for toxin translocation is not limited to a single A fragment but can accommodate additional proteins as well. The fact that proteins as large as 20 kDa can be brought into cells by way of diphtheria toxin under both in vitro and in vivo conditions opens up the possibility of using diphtheria toxin mutants for introducing molecules with biological activity into cells.

Amino Acid Sequence↗

PubMed1988

Specific cleavage of diphtheria toxin by human urokinase.

Diphtheria toxin must undergo a specific cleavage reaction and subsequent reduction to express the enzymatic ADP-ribosyltransferase activity that is responsible for its toxicity. In an effort to identify potential cellular enzymes that might be involved in this process we have found that a human urinary plasminogen activator, urokinase, is capable of specifically cleaving diphtheria toxin to yield an enzymatically active A fragment (more homogeneous than that produced by trypsin cleavage) and a B fragment (with an identical amino-terminal sequence to that produced by trypsin cleavage). The results raise the possibility that urokinase or urokinase-like enzymes play a role in diphtheria toxin-mediated intoxication.

Amino Acid Sequence↗

PubMed1982

Inhibition of mitochondrial protein synthesis and energy coupling by fragment A of diphtheria toxin.

The effect of intact diphtheria toxin and its fragment A on energy-dependent functions in mouse liver mitochondria/mitoplasts has been studied. Fragment A was found to inhibit protein synthesis in mitoplasts to the same extent (approximately 80%) as the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone, but had no similar effect in lysed mitoplasts. Intact diphtheria toxin had no effect in either case. Fragment A was found to function as a potent uncoupler in isolated mitochondria and mitoplasts, inhibiting oxidative phosphorylation by approximately 80% at a concentration (2 micrograms fragment A/mg of protein) that did not inhibit protein synthesis. In contrast, intact toxin slightly increased the tightness of energy coupling in isolated mitoplasts. 125I-labelled intact diphtheria toxin was bound to mitoplasts to about the same extent as labelled fragment A. At concentrations which efficiently inhibited mitochondrial protein synthesis, fragment A had no effect on the intramitochondrial concentration of nicotinamide adenine dinucleotides, nor was it capable of ADP-ribosylating mitochondrial proteins, indicating that the well known enzymatic activity of fragment A is not involved in the observed effect in mitochondria. The results indicate that fragment A of diphtheria toxin inhibits protein synthesis in mitochondria and mitoplasts by inhibiting mitochondrial energy transduction. The detailed mechanism of the uncoupling effect and its possible significance in intact cells remain to be elucidated.

Animals↗

PubMed2004

Transition state structure for ADP-ribosylation of eukaryotic elongation factor 2 catalyzed by diphtheria toxin.

Bacterial protein toxins are the most powerful human poisons known, exhibiting an LD(50) of 0.1-1 ng kg(-)(1). A major subset of such toxins is the NAD(+)-dependent ADP-ribosylating exotoxins, which include pertussis, cholera, and diphtheria toxin. Diphtheria toxin catalyzes the ADP ribosylation of the diphthamide residue of eukaryotic elongation factor 2 (eEF-2). The transition state of ADP ribosylation catalyzed by diphtheria toxin has been characterized by measuring a family of kinetic isotope effects using (3)H-, (14)C-, and (15)N-labeled NAD(+) with purified yeast eEF-2. Isotope trapping experiments yield a commitment to catalysis of 0.24 at saturating eEF-2 concentrations, resulting in suppression of the intrinsic isotope effects. Following correction for the commitment factor, intrinsic primary kinetic isotope effects of 1.055 +/- 0.003 and 1.022 +/- 0.004 were observed for [1(N)'-(14)C]- and [1(N)-(15)N]NAD(+), respectively; the double primary isotope effect was 1.066 +/- 0.004 for [1(N)'-(14)C, 1(N)-(15)N]NAD(+). Secondary kinetic isotope effects of 1.194 +/- 0.002, 1.101 +/- 0.003, 1.013 +/- 0.005, and 0.988 +/- 0.002 were determined for [1(N)'-(3)H]-, [2(N)'-(3)H]-, [4(N)'-(3)H]-, and [5(N)'-(3)H]NAD(+), respectively. The transition state structure was modeled using density functional theory (B1LYP/6-31+G) as implemented in Gaussian 98, and theoretical kinetic isotope effects were subsequently calculated using Isoeff 98. Constraints were varied in a systematic manner until the calculated kinetic isotope effects matched the intrinsic isotope effects. The transition state model most consistent with the intrinsic isotope effects is characterized by the substantial loss in bond order of the nicotinamide leaving group (bond order = 0.18, 1.99 A) and weak participation of the attacking imidazole nucleophile (bond order = 0.03, 2.58 A). The transition state structure imparts strong oxacarbenium ion character to the ribose ring even though significant bond order remains to the nicotinamide leaving group. The transition state model presented here is asymmetric and consistent with a dissociative S(N)1 type mechanism in which attack of the diphthamide nucleophile lags behind departure of the nicotinamide.

ADP Ribose Transferases↗

PubMed1991

Purification of diphtheria toxin receptor from Vero cells.

Diphtheria toxin receptor has been solubilized from Vero cell membranes with octyl beta-D-glucoside. CRM197, the product of a mutated diphtheria toxin gene, was used for the identification of the receptor. The binding activity of the solubilized receptor was assayed by precipitating the receptor with acetone in the presence of phospholipids and carrier proteins. The solubilized receptor was purified by the combination of several chromatographic steps in the presence of the detergent, resulting in about a 10(6)-fold purification of the receptor. The purified receptor showed essentially a single band of 14.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When partially purified receptor fractions were subjected to ligand blotting analysis using 125I-CRM197 as the probe, the 14.5-kDa protein and a few minor protein bands were identified as diphtheria toxin-binding molecules. These results show clearly that the 14.5-kDa protein is the diphtheria toxin receptor, or at least the major diphtheria toxin-binding molecule. When partially purified receptor was applied to a Sephacryl S-300 column in the presence of detergent, the receptor was eluted in the fractions corresponding to the 60-90-kDa size range. This suggests that the protein forms a complex with itself or with another protein.

Animals↗

PubMed1984

Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro.

Diphtheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. We report here that resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [3H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to gamma-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication, which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [3H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium.

Animals↗

PubMed1990

Detection of diphtheria toxin in culture supernates of Corynebacterium diphtheriae and C. ulcerans by immunoassay with monoclonal antibody.

Monoclonal antibody (MAb) to diphtheria toxin was produced in mouse hybridomas, and shown by ELISA to be of sub-class IgG1. Hybridomas were inoculated into mice to produce ascitic fluid from which MAb was purified by caprylic acid. The MAb was shown by immunoblotting to be directed against the A fragment of the toxin and also against the intact toxin molecule. After conjugation with fluorescein isothiocyanate, it was used in an immunoassay to detect toxin in culture supernates of Corynebacterium diphtheriae and C. ulcerans. The assay correlated well with the Elek test and with virulence in guinea-pigs; but it gave occasional false positive results, probably by binding of MAb to defective toxin.

Animals↗

PubMed1999

Anion-coordinating residues at binding site 1 are essential for the biological activity of the diphtheria toxin repressor.

The homodimeric diphtheria toxin repressor (DtxR) uses Fe2+ as a corepressor, binds to iron-regulated promoters, and negatively regulates the syntheses of diphtheria toxin, corynebacterial siderophore, and several other Corynebacterium diphtheriae products. The crystal structure of DtxR shows that the second domain of each monomer has two binding sites for Fe2+ or certain other divalent metal ions. In addition, site 1 binds a sulfate or phosphate anion, suggesting that phosphate may function intracellularly as a co-corepressor. The effects of alanine substitutions for selected residues in sites 1 and 2 were determined by measuring the beta-galactosidase activities of a tox operator/promoter-lacZ reporter construct in Escherichia coli strains expressing each DtxR variant. Our studies demonstrated that single alanine substitutions for the anion-binding residues in site 1 (R80A, S126A, or N130A) caused severely decreased DtxR activity, similar to the effects of alanine substitutions for metal-binding residues in site 2 (C102A, E105A, or H106A) and greater than the effects of alanine substitutions for metal-binding residues in site 1 (H79A, E83A, or H98A) reported previously by other investigators. Various combinations of alanine substitutions for site 1 and site 2 residues were also analyzed to further elucidate the roles of these cation- and anion-binding ligands in DtxR activity. Furthermore, the interaction between residue E20 in the DNA binding domain and R80 in anion/cation binding site 1 was analyzed, and the E20A variant of DtxR was shown to have a phenotype indistinguishable from that of the R80A variant. Our data demonstrated for the first time that the anion-binding residues R80, S126, and N130 at site 1 are essential for DtxR activity. The data also showed that the interaction of E20 in domain 1 with R80 in domain 2, first revealed by X-ray crystallography in apo-DtxR and holo-DtxR, is a structural feature of DtxR that is important for its repressor activity.

Amino Acid Substitution↗

PubMed1995

The effect of receptor rapid-internalization signals on diphtheria toxin endocytosis and cell sensitivity.

Diphtheria toxin enters toxin-sensitive mammalian cells by receptor-mediated endocytosis employing the heparin-binding EGF-like growth factor precursor as its receptor. We reported previously (Almond and Eidels, 1994) that cytoplasmic domain mutants of the toxin receptor and cells expressing wild-type receptor internalize toxin slowly, the rate being approximately that of normal turnover of the plasma membrane. To determine whether it was possible to increase toxin sensitivity by increasing the rate of toxin internalization, we constructed diphtheria toxin cytoplasmic domain mutant cell lines containing rapid-internalization signals from either the low density lipoprotein receptor or from the lysosomal acid phosphatase precursor. Although cells transfected with mutant receptor genes internalized toxin at a faster rate than those expressing the wild-type receptor, they showed a decrease in toxin sensitivity. This decreased sensitivity may be accounted for by an observed decrease in the number of toxin-binding sites and by an increased rate of toxin internalization and degradation. These results suggest that the rate of toxin internalization may not be the rate-limiting step in the cytotoxic process.

Acid Phosphatase↗

PubMed1995

Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain.

We report the expression of fragment C of tetanus toxin (FC) fused to the eukaryotic cell binding domain (the carboxyl-terminus) of diphtheria toxin (FC-bDt fusion) in attenuated Salmonella typhi live vector vaccine strain CVD 908. The FC-bDt protein fusion was constructed using plasmid pTETnir15 which carries the gene encoding FC under control of the nirB promoter (nirBP). The open reading frame for FC was modified to incorporate an in-frame glycine-proline hinge region and a set of four restriction sites at the 3' end of the FC gene. A 482 bp DNA fragment encoding the eukaryotic cell binding domain of diphtheria toxin was then inserted at the 3' end of the modified FC gene to create an in-frame FC-bDt fusion gene. The resulting plasmid, pOG215, was able to express the FC-bDt fusion protein in both Escherichia coli DH5a and S. typhi CVD 908, as evidenced by Western immunoblots using anti-FC and anti-C-terminal diphtheria toxin monoclonal antibodies. Maximum expression of the FC-bDt fusion protein was achieved by growing CVD 908(pOG215) at the low oxidation-reduction potential of thioglycollate broth, i.e. in conditions that activate nirBP and drive transcription of the FC-bDt fusion gene. Whereas maximum expression of FC alone was also observed using thioglycollate broth, expression of bDt alone was unsuccessful using a variety of growth conditions. FC fusions constitute one strategy to "rescue" expression of proteins which are otherwise difficult to express.

Antigens, Bacterial↗

PubMed1998

Expression of mutant dynamin protects cells against diphtheria toxin but not against ricin.

Diphtheria toxin is believed to enter sensitive mammalian cells via receptor-mediated endocytosis from clathrin-coated pits, while ricin can enter via both clathrin-dependent and clathrin-independent endocytosis. The present study has confirmed this by determining the toxin sensitivity of COS-7y cells which were transiently overexpressing a trans dominant negative mutant of dynamin, a GTPase required for the budding of clathrin-coated vesicles from the plasma membrane. Cells overexpressing wild-type dynamin showed normal receptor-mediated endocytosis of transferrin and remained sensitive to both diphtheria toxin and ricin. Cells overexpressing a mutant dynamin defective in GTP binding and hydrolysis were unable to endocytose transferrin and were protected against diphtheria toxin, but they remained completely sensitive to ricin intoxication. Treating non-transfected cells or cells overexpressing mutant dynamin with nystatin caused a redistribution of the caveolae membrane marker protein VIP21-caveolin from the cell surface to intracellular locations, but did not affect their sensitivity to ricin. The redistribution of caveolin seen after nystatin treatment may reflect the disappearance of caveolae. If this is the case, caveolae are not responsible for the endocytosis of ricin. An alternative clathrin-independent route may operate for ricin, since cellular uptake, intracellular transport, and translocation into the cytosol remain unaffected when clathrin-dependent endocytosis is effectively blocked.

Animals↗