Diphtheria toxin entry into cells is facilitated by low pH

At neutral pH, NH4Cl and chloroquine protected cells against diphtheria toxin. A brief exposure of the cells to low pH (4.5-5.5) at 37 degrees completely abolished this protection. When, to cells preincubated with diphtheria toxin and NH4Cl, neutralizing amounts of anti-diphtheria toxin were added before the pH was lowered, the toxic effect was considerably reduced, but it was not completely abolished. A much stronger toxic effect was seen when antibodies were added immediately after incubation at low pH. Upon a short incubation with diphtheria toxin at low pH, the rate of protein synthesis in the cells decreased much faster than when the normal pH was maintained. The data suggest that, at low pH, diphtheria toxin (or its A fragment) penetrates directly through the surface membrane of the cell. The possibility is discussed that, when the medium has a neutral pH, the entry of diphtheria toxin involves adsorptive endocytosis and reduction of the pH in the vesicles possibly by fusion with lysosomes. Low pH did not facilitate the entry of the closely related toxins abrin, ricin, and modeccin.     

Requirements for entry of poliovirus RNA into cells at low pH.

HeLa S3 cells were protected against infection by poliovirus type I by the presence of monensin and N,N'-dicyclohexylcarbodiimide (DCCD), compounds elevating the pH of acidic intracellular compartments. The protection was fully overcome by exposing the cells to pH 5.5 and lower, and at approximately pH 6.1 it was reduced by half. Measurements of the ability of the virus to enter the detergent phase under conditions where Triton X-114 was separated from water indicated that the virus is hydrophilic at neutral pH, and that it exposes hydrophobic regions at low pH. When the cells were pretreated with acetic acid, which reduces the intracellular pH, virus entry was inhibited, indicating that a pH gradient across the membrane is necessary for infection. Under all conditions which induced infection, the virus particles were altered to more slowly sedimenting material. Also, virus bound to aldehyde-fixed cells was altered when exposed to low pH at 37 degrees C. The data indicate that poliovirus bound to receptors on cells exposes hydrophobic regions at low pH, and that at physiological temperature it undergoes alteration. This alteration may be a necessary, but not sufficient requirement for infection.     

Cell-mediated reduction of the interfragment disulfide in nicked diphtheria toxin. A new system to study toxin entry at low pH

When 125I-labeled nicked diphtheria toxin bound to Vero cells was exposed to pH less than 5.0, a small fraction was reduced to yield A- and B-fragments. The pH required for reduction correlates well with that required to induce intoxication, and the amount of A-fragment released was of the same order as that required to intoxicate the cells. Conditions that protect cells against intoxication, such as acidification of the cytosol, treatment with anion transport inhibitors, or treatment with anti-diphtheria toxin antibodies, prevented the reduction of the interfragment disulfide in cell-bound toxin. In vitro, thioredoxin reduced nicked diphtheria toxin only at pH 5.0 and lower, and the reduction was inhibited by anti-toxin antibodies. This indicates that a conformational change in the toxin, necessary for reduction by the thioredoxin system, is prevented by the antibodies. Reduction by glutathione and cysteine was most efficient at neutral pH and was not inhibited by anti-toxin. The results are consistent with the possibility that cell-mediated reduction of the interfragment disulfide is a measure of the entry of fragment A into the cytosol.     

On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells.

Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed.     

Monoclonal antibody against diphtheria toxin. Effect on toxin binding and entry into cells

A number of monoclonal antibodies against diphtheria toxin were isolated. Some of their properties were determined. Antibody 2 reacts with the region of between 30 and 45 kDa from the NH2 terminus of toxin. Antibody 7 reacts with the COOH-terminal 17-kDa region of toxin. These two antibodies show sharp contrasts in their effects on toxin action in cultured cells. When antibody 2 or 7 and toxin were mixed, incubated at 37 degrees C, and then added to sensitive Vero cells, antibody 7 blocked toxin action, but antibody 2 did not. When antibody 2 or 7 was added to cells to which toxin had been prebound at 4 degrees C, and the cells were then shifted to 37 degrees C, antibody 7 did not block toxin action, but antibody 2 inhibited intoxication. Antibody 7 blocked binding of 125I-toxin to cells and did not block degradation of toxin associated with cells. Antibody 2 did not block binding of 125I-toxin to cells, and was able to bind to cells in the presence of toxin. The results obtained from the effect of antibody 2 on degradation of 125I-toxin associated with cells resemble those seen with amines, which block toxin action but do not inhibit binding of toxin to cells. These facts show that antibody 2 does not block binding of toxin to cell surfaces, but blocks the entry of toxin into the cytosol at a step after binding of toxin to the receptor. Antibodies 14 and 15 react with fragment A of diphtheria toxin, but have no effect on any activity of toxin. The other monoclonal antibodies have effects on toxin binding and entry intermediate between those of 2 and 7.     

Mutations in diphtheria toxin to improve immunotoxin selectivity and understand toxin entry into cells

Diphtheria toxin can be used to selectively kill target cells by coupling it to cell-type-specific binding moieties such as monoclonal antibodies. These reagents have important potential in treating diseases, selectively ablating cell populations in experimental systems and for understanding how proteins cross membranes. Point mutations and deletions in the diphtheria toxin gene have been used to identify and localize regions of diphtheria toxin involved in cell killing. Mutations have been identified that prevent binding of the toxin to a cell surface receptor yet these mutations do not inhibit the cell entry activity or the intracellular cytotoxicity of the toxin. Coupling of these mutant toxins to new, cell-type-specific binding moieties yields potent reagents with up to 200,000-fold selectivity between target and nontarget cells. Mutations and deletions in the membrane transport regions are beginning to explain how the toxin enters cells and may also help in the design of more effective therapeutic reagents.     

Insertion of diphtheria toxin B-fragment into the plasma membrane at low pH. Characterization and topology of inserted regions

When the enzymatically active A-fragment of diphtheria toxin is translocated to the cytosol, the B-fragment inserts into the membrane in such a way that a 25-kDa polypeptide becomes shielded from proteases added to the external medium. We have attempted to determine the boundaries of this polypeptide within the toxin B-fragment as well as the topology of the B-fragment in the membrane. Chemical cleavage of the 25-kDa polypeptide with hydroxylamine and o-iodosobenzoic acid yielded fragments of sizes indicating that the 25-kDa polypeptide starts at residue approximately 300 and extends to the COOH-terminal end. Experiments where the toxin was labeled with [35S]cysteine at distinct positions of the B-fragment supported this conclusion. Treatment of cells with inserted B-fragment with L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin and with V8 protease from Staphylococcus aureus yielded protected 27- and 30-kDa fragments in addition to 25 kDa, indicating that the region 240-264 is also at the outside. The topology of the inserted B-fragment is discussed.     

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.     

Quantal entry of diphtheria toxin to the cytosol

The rate-limiting step in diphtheria toxin (DT) intoxication of Vero cells has been determined utilizing cycloheximide as an inhibitor of the intoxication process. Cycloheximide is shown to inhibit the toxin catalyzed ADP-ribosylation of elongation factor 2 (EF-2). The inhibition is blocked by puromycin thus establishing the ribosome as the location of cycloheximide protection. Washing cells free of cycloheximide rapidly reverses the protective effect. The initial rates of protein synthesis inhibition observed after removal of cycloheximide from DT-intoxicated cells are 5 to 12-fold greater than rates observed in unprotected cells and are shown to reflect ADP-ribosylation of EF-2 by cytosolic DT. Ten to thirty minutes after cycloheximide removal, the rate of protein synthesis inhibition abruptly changes to values identical to those of unprotected cells. Both the initial rates and extent of the initial rapid inactivation are directly related to toxin concentration and time of incubation with DT in the presence of cycloheximide. We concluded that: the rate-limiting step in protein synthesis inhibition by DT is not the ADP-ribosylation of EF-2 by cytosolic toxin but rather the earlier entry step of DT into the cytosol. DT enters the cytosol as a bolus of sufficient size to rapidly inactivate all EF-2 in that cell. It is inferred from 1 and 2 that the first order inactivation rate exhibited by DT is the result of the probability of the release of a bolus of toxin to the cytosol of any cell in the population per unit time. Autoradiographic analysis of intoxicated cell populations support this two-population state model. The size of a single bolus or quantum of DT is calculated from data over the range of 10(-11) to 10(-9) M DT and is found to remain constant. We suggest that the cytosolic entry mechanism of DT results from a unique ability of the internalized toxin molecules to destabilize the vesicular membrane resulting in a random release of a bolus of toxin into the cytosol. Because the bolus size remains constant over a 50-fold change in receptor occupancy the possibility is raised that DT undergoes a post-receptor packaging process, package size remaining a constant and package number increasing with receptor occupancy.     

The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement

Lysosomotropic amines, such as ammonium chloride, are known to protect cells from the cytotoxic effects of diphtheria toxin. These drugs are believed to inhibit the transport of the toxin from a receptor at the cell exterior into the cytoplasm where a fragment of the toxin arrests protein synthesis. We studied the effects of lysosomotropic agents on the cytotoxic process to better understand how the toxin enters the cytoplasm. The cytotoxic effects of diphtheria toxin were not inhibited by antitoxin when cells were preincubated at 37 degrees C with toxin and ammonium chloride, exposed to antitoxin at 4 degrees C, washed to relieve the ammonium chloride inhibition, and finally warmed to 37 degrees C. The antigenic determinants of the toxin were, therefore, either altered or sheltered. It is likely that the combination of ammonium chloride and a low temperature trapped the toxin in an intracellular vesicle from which the toxin could proceed to the cytoplasm. Because lysosomotropic amines raise the pH within acidic intracellular vesicles, such as lysosomes, they could trap the toxin within such a vesicle if an acidic environment were necessary for the toxin to penetrate into the cytoplasm. We simulated acidic conditions which the toxin might encounter by exposing cells with toxin bound to their surface to acidic medium. We then measured the effects of lysosomotropic amines on the activity of the toxin to see if the acidic environment substituted for the function normally inhibited by the drugs. The drugs no longer protected the cells. This suggests that exposing the toxin to an acidic environment, such as that found within lysosomes, is an important step in the penetration of diphtheria toxin into the cytoplasm.     

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre‐loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site‐directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine‐rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild‐type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.     

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.     

Interactions between diphtheria toxin entry and anion transport in Vero cells. II. Inhibition of anion antiport by diphtheria toxin

When cells with surface-bound diphtheria toxin were exposed to pH 4.5, the toxin became shielded against lactoperoxidase-catalyzed radioiodination, indicating that the toxin was inserted into the membrane. Cells thus treated had strongly reduced ability to take up 36Cl-, 35SO4(2-), and [14C]SCN-. The reduction of chloride uptake was strongest at neutral pH, whereas that of sulfate was strongest at acidic pH. Lineweaver-Burk plots indicated that the toxin treatment reduced the Jmax but not the Km for the anions. The toxin also inhibited the NaCl-stimulated efflux of 35SO4(2-), indicating that the toxin inhibits the antiporter. No inhibition was found when toxin-treated cells were not exposed to low pH, whereas exposure to pH 4.5 for 20 s induced close to maximal inhibition. Half-maximal inhibition was obtained after exposure to pH 5.4. The concentration of diphtheria toxin required to obtain maximal inhibition (0.3 micrograms/ml) was sufficient to ensure close to maximal toxin binding to the cells. Even in ATP-depleted cells and in the absence of permeant anions, low pH induced inhibition of anion antiport in toxin-treated Vero cells. There was no measurable inhibition of anion antiport in cells with little or no ability to bind the toxin.     

Localization in diphtheria toxin fragment B of a region that induces pore formation in planar lipid bilayers at low pH

Like diphtheria toxin and the N-terminal (M_r 23 000) region of fragment B, CB1 (M_r 13 000), the cyanogen bromide peptide located in the middle region of fragment B is able to induce pore formation in lipid bilayer membrane at low pH. These two peptides (M_r 23 000 and 13 000) share a common segment (M_r 6300) containing the predicted amphipathic, -helical, transverse lipid-associating domain (M_r 2750) of fragment B[J. Cell Biol. (1980) 87, 837–840]. Therefore, we postulated this domain to be responsible for the pore formation ability of diphtheria toxin [Proc. Natl. Acad. Sci. USA (1981) 78, 172–176]. A relationship between the pH dependency of pore formation and the presence of a cluster of prolines in the C-terminal region of CB1 is proposed.     

pH dependent insertion of a diphtheria toxin B fragment peptide into the lipid membrane: a conformational analysis

A peptide of diphtheria toxin B fragment (residues 147-266) has been shown to induce pore formation in lipid bilayer membranes at low pH. Such an effect was obtained at a much lower extent or not at all at pH = 7. The region localized between residues 225 and 246 is highly hydrophobic (27.3% polarity) and characterized by a high concentration of proline residues. Since proline cis-trans isomerization is highly sensitive to the pH of the medium, we investigated the capability of the cis and trans isomers to penetrate into the lipid matrix. Obviously, the cis-trans isomerization of proline 242 and 245, assumed to be imposed by a low pH, uncovers the hydrophobic region and induces its insertion into a lipid layer of dipalmitoylphosphatidylcholine. The lipid matrix destabilization resulting from this process could promote the penetration into the lipid bilayer of an amphipatic structure (153-178) similar to the transverse lipid associating domains of membrane proteins.     

Liposomes as a means to introduce fragment A of diphtheria toxin into cells

The incorporation of fragment A of diphtheria toxin into liposomes is described. The intracellular delivery of the entrapped toxin, as evidenced by the inhibition of protein synthesis by a human lymphoblastoid cell line could be demonstrated with liposomes that contained phosphatidylethanolamine or phosphatidylserine in addition to phosphatidylcholine and cholesterol. Free fragment A, either alone or added to empty liposomes of any composition, did not affect protein synthesis, even when present in considerably higher concentrations than the liposome-entrapped form.     

Requirement for vacuolar H+ -ATPase activity and Ca2+ gradient during entry of rotavirus into MA104 cells

The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca(2+) concentration in the endosome. In this paper, we monitored rotavirus entry by measuring alpha-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca(2+) concentration on virus entry was studied by inhibiting the endosomal H(+)-ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-alpha-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca(2+)-dependent endocytosis model.     

Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells.

The role that endosomal acidification plays during influenza virus entry into MDCK cells has been analyzed by using the macrolide antibiotics bafilomycin A1 and concanamycin A as selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidification of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prevented the entry of influenza virus into cells when added during the first minutes of infection. Attachment of virion particles to the cell surface was not the target for the action of bafilomycin A1. N,N'-Dicyclohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also blocked virus entry, whereas elaiophylin, an inhibitor of the plasma-proton ATPase, had no effect. The inhibitory actions of bafilomycin A1 and concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the virus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potently blocked influenza virus infection, even in the absence of antibiotics. These results indicate that a pH gradient, rather than low pH, is necessary for efficient entry of influenza virus into cells.     

Uptake of aspartate aminotransferase into mitochondria in vitro depends on the transmembrane pH gradient.

1. The effects of various inhibitors of electron transport and of oxidative phosphorylation and the effects of ionophores on the uptake of native aspartate aminotransferase into mitochondria were investigated. 2. Both antimycin and cyanide completely inhibited the uptake of the enzyme. On the other hand, uptake was stimulated to ATP and by oligomycin; however, the stimulation by ATP is inhibited by oligomycin. 3. The effects of ionophores of the valinomycin type in media containing K+ ions depended on the conditions used. Valinomycin alone stimulated the uptake of the enzyme, but in the presence of phosphate ions uptake was abolished. Nonactin was without effect at a low K+ concentration, but was stimulatory at 100 mM-KCl. Gramicidin also stimulated the uptake process. 4. Nigericin completely abolished uptake of aspartate aminotransferase into mitochondria. 5. The uptake of te enzyme was decreased by 18% in the absence of inhibitors or ionophores when the external pH was increased from 6.9 to 7.6. 6. These results indicate that ATP is not directly involved in the uptake of aspartate aminotransferase into mitochondria, neither is there a requirement for a cation gradient. Rather the uptake depends on the maintenance of a pH gradient across the mitochondrial inner membrane.     

Mechanism of entry into the cytosol of poliovirus type 1: requirement for low pH

The effect of a number of drugs and culture conditions on the entry into cells of a strain of poliovirus 1 (Brunende) was tested. The cells were exposed in the dark to light-sensitive, neutral red-containing virus, in the presence of the drug to be tested. Then the cells were exposed to light, transferred to normal medium, and incubated overnight. Cytopathogenic effect was measured as inhibition of [3H]leucine incorporation. Compounds that dissipate proton gradients across membranes, like monensin, protonophores, and amines, and compounds that inhibit the acidification process, such as N,N'- dicyclohexylcarbodiimide (DCCD) and tributyltin, inhibited the entry of virus, but not virus binding. This was also the case with metabolic inhibitors that deplete cells for ATP. The same compounds also inhibited the cell-induced alteration of the virus particles. When cells with surface-bound virus were exposed to low pH, the virus entered efficiently, even in the presence of monensin and DCCD. The results indicate that acidification somehow facilitates the entry of the virus RNA into the cytosol and that under normal conditions the entry occurs from intracellular acidic vesicles.