Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol

Pseudomonas exotoxin (PE) was incubated with cells and extracts analyzed for processed fragments. PE was proteolytically cleaved to produce a N-terminal 28-kDa and a C-terminal 37-kDa fragment, the latter being composed of a portion of domain II and all of domain III (the ADP-ribosylating domain). Cleavage was evident at 10 min after toxin addition and endosome preparations contained the processed fragments. Initially, the two fragments were linked by a disulfide bond. Subsequently, the 37-kDa fragment was reduced and translocated to the cytosol where it inactivated protein synthesis. Cytosol from toxin-treated cells was greatly enriched in the 37-kDa fragment. The 37-kDa fragment appears to be essential for toxicity since mutant PE molecules that do not produce this fragment, or cannot deliver it to the cytosol, fail to kill cells.     

Reduction of furin-nicked Pseudomonas exotoxin A: an unfolding story

Upon entering mammalian cells, Pseudomonas exotoxin A (PE) is proteolytically processed by furin to produce an N-terminal fragment of 28 kDa and a C-terminal fragment of 37 kDa. Cleavage is followed by the reduction of a key disulfide bond (cysteines 265-287). This combination of proteolysis and reduction releases the 37 kDa C-terminal fragment, which then translocates to the cytosol where it ADP-ribosylates elongation factor 2 and inhibits protein synthesis. To investigate toxin reduction, furin-nicked PE or a hypercleavable mutant, PEW281A, was subjected to various treatments and then analyzed for fragment production. Reduction was evident only when unfolding conditions and a reducing agent were applied. Thermal unfolding of PE, as evidenced by changes in alpha-helical content and increased sensitivity to trypsin, rendered nicked toxin susceptible to protein disulfide isomerase- (PDI-) mediated reduction. When subcellular fractions from toxin-sensitive cells were incubated with nicked PE, toxin unfolding and reducing activities were present in the membrane fraction but not the soluble fraction. These data indicate that PE reduction is a two-step process: unfolding that allows access to the Cys265-287 disulfide bond, followed by reduction of the sulfur-sulfur bond by PDI or a PDI-like enzyme. With regard to cellular processing, we propose that the toxin's three-dimensional structure retains a "closed" conformation that restricts solvent access to the Cys265-287 disulfide bond until after a cell-mediated unfolding event.     

Analysis of sequences in domain II of Pseudomonas exotoxin A which mediate translocation

Pseudomonas exotoxin (PE) contains 613 amino acids that are arranged into 3 structural domains. PE exerts its cell-killing effects in a series of steps initiated by binding to the cell surface and internalization into endocytic vesicles. The toxin is then cleaved within domain II near arginine-279, generating a C-terminal 37-kDa fragment that is translocated into the cytosol where it ADP-ribosylates elongation factor 2 and arrests protein synthesis. In this study, we have focused on the functions of PE which are encoded by domain II. We have used the chimeric toxin TGF alpha-PE40 to deliver the toxin's ADP-ribosylating activity to the cell cytosol. Deletion analysis revealed that sequences from 253 to 345 were essential for toxicity but sequences from 346 to 364 were dispensable. Additional point mutants were constructed which identified amino acids 339 and 343 as important residues while amino acids 344 and 345 could be altered without loss of cytotoxic activity. Our data support the idea that domain II functions by first allowing PE to be processed to a 37-kDa fragment and then key sequences such as those identified in this study mediate the translocation of ADP-ribosylation activity to the cytosol.     

Receptor-mediated transport of the hybrid protein ricin-diphtheria toxin fragment A with subsequent ADP-ribosylation of intracellular elongation factor II.

A hybrid protein of ricin and the enzymatically active fragment A of diphtheria toxin (toxin A) has been synthesized and purified. The diphtheria toxin A fragment of the hybrid protein is shown to enter the cytosol compartment of HeLa cells, its presence assayed by the fall of intracellular elongation factor II (EF-2) and the rise of ADP-ribosylated EF-2. Hybrid entrance to HeLa cells is blocked by lactose which blocks receptor-mediated entry of ricin but not by NH4Cl which blocks the transport of diphtheria toxin. It is concluded that the diphtheria toxin fragment A moiety of the hybrid enters the cell cytosol via the ricin receptor-mediated transport system. The kinetics of intracellular ADP-ribosylation of EF-2 by diphtheria toxin have also been studied. Ribosylation is preceded by a toxin dose-dependent lag period. The data suggest that the time constant responsible for the lag period is in the transport step. Models consistent with these data are discussed.     

Requirement of a transmembrane pH gradient for the entry of diphtheria toxin into cells at low pH

The effects of acidification of the cytosol and of electrical depolarization on the entry of diphtheria toxin were studied. Entry of the toxin from the cell surface was induced by low pH, and the presence of the toxin in the cytosol was monitored as toxin-induced inhibition of protein synthesis. To reduce the membrane potential the cells were incubated in a buffer containing a high concentration of potassium. The cytosol was acidified either by incubating the cells with acetic acid, by incubating them with ammonium chloride which was subsequently removed in the presence of amiloride to prevent pH regulation by the Na+/H+ exchanger, or by incubating the cells in isotonic KCl in the presence of nigericin and valinomycin. The results showed that when the cytosol was acidified by either method toxin entry was inhibited, while a reduction in the membrane potential did not strongly interfere with the entry. A pH gradient across the membrane of at least 1 pH unit was required for entry. Possibly this gradient acts as a driving force for diphtheria toxin entry.     

Endosomes and toxin translocation

In this review we discuss data obtained by our group regarding the entry of toxins, especially ricin, diphtheria toxin (DT) and Pseudomonas exotoxin A (PE) into animal cells. We studied the translocation process of these toxins using endosomes purified from lymphocytes. This process is rate-limiting for toxicity and enables these toxins to reach the cytosol where they will inactivate the protein synthesis system and kill the cell. We could show that each of these toxins uses a different strategy to cross the endosome membrane. Whereas ricin transmembrane transport only relies on cytosolic ATP hydrolysis, PE first requires exposure to the low endosomal pH (pH-6), presumably to insert into the endosome membrane, before being translocated via a process which also requires cytosolic ATP hydrolysis. DT translocation is directly triggered and energized by the endosome-cytosol pH gradient. Using conjugates with dihydrofolate reductase we could indirectly show that ricin and PE require unfolding for translocation. A deletion approach enabled to produce a more cytotoxic PE mutant by increasing its translocation activity.     

Role of anions in low pH-induced translocation of diphtheria toxin

Previous work has shown that when Vero cells with surface-bound diphtheria toxin are exposed to low pH, toxin entry across the plasma membrane is induced and that this entry involves two steps, insertion of the B-fragment of the toxin into the membrane and translocation of the enzymatically active A-fragment to the cytosol. Here we have studied the role of permeant anions in this process. It was found that when the B-fragment was inserted into the membrane, part of it, a 25-kDa polypeptide, was shielded from externally added Pronase. This insertion did not require permeant anions. The translocation of the A-fragment was monitored by measuring either its ability to inhibit protein synthesis in the cells or the appearance of radioactively labeled 21-kDa fragment after treatment of the cells with externally applied Pronase. The translocation of the A-fragment was dependent on the presence of permeant anions in the medium. However, when the cells were depleted of Cl- by incubation in Cl- free buffer at high pH, translocation of the A-fragment did not require permeant anions in the medium. The possibility that translocation of the A-fragment is inhibited by an outward directed chloride gradient rather than by the absence of chloride is discussed.     

Shiga toxin regulates its entry in a Syk-dependent manner

Shiga toxin (Stx) is composed of an A-moiety that inhibits protein synthesis after translocation into the cytosol, and a B-moiety that binds to Gb3 at the cell surface and mediates endocytosis of the toxin. After endocytosis, Stx is transported retrogradely to the endoplasmic reticulum, and then the A-fragment enters the cytosol. In this study, we have investigated whether toxin-induced signaling is involved in its entry. Stx was found to activate Syk and induce rapid tyrosine phosphorylation of several proteins, one protein being clathrin heavy chain. Toxin-induced clathrin phosphorylation required Syk activity, and in cells overexpressing Syk, a complex containing clathrin and Syk could be demonstrated. Depletion of Syk by small interfering RNA, expression of a dominant negative Syk mutant (Syk KD), or treatment with the Syk inhibitor piceatannol inhibited not only Stx-induced clathrin phosphorylation but also endocytosis of the toxin. Also, Golgi transport of Stx was inhibited under all these conditions. In conclusion, our data suggest that Stx regulates its entry into target cells.     

Increasing stability and toxicity of Pseudomonas exotoxin by attaching an antiproteasic Peptide

Trypsin-like activities are present within the endocytic pathway and allow cells to inactivate a fraction of incoming toxins, such as Pseudomonas exotoxin (PE), that require endocytic uptake before reaching the cytosol to inactivate protein synthesis. PE is a favorite toxin for building immunotoxins. The latter are promising molecules to fight cancer or transplant rejection, and producing more active toxins is a key challenge. More broadly, increasing protein stability is a potentially useful approach to improve the efficiency of therapeutic proteins. We report here that fusing an antiproteasic peptide (bovine pancreatic trypsin inhibitor, BPTI) to PE increases its toxicity to human cancer cell lines by 20-40-fold. Confocal microscopic examination of toxin endocytosis, digestion, and immunoprecipitation experiments showed that the fused antiproteasic peptide specifically protects PE from trypsin-like activities. Hence, the attached BPTI acts as a bodyguard for the toxin within the endocytic pathway. Moreover, it increased the PE elimination half-time in mice by 70%, indicating that the fused BPTI stabilizes the toxin in vivo. This BPTI-fusion approach may be useful for protecting other circulating or internalized proteins of therapeutic interest from premature degradation.     

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.     

Requirement for prolonged action in the cytosol for optimal protein synthesis inhibition by diphtheria toxin

Diphtheria toxin A-fragment enters the cytosol of target cells, where it inhibits protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibition in single cells by autoradiography and compared it with inhibition of protein synthesis in the whole cell culture. The data show that half-maximal protein synthesis inhibition in the whole cell population after a short incubation time is achieved by partially inhibiting protein synthesis in basically all the cells, while half-maximal protein synthesis inhibition after a long incubation time is due to a complete protein synthesis block in about half the cells in the population. We have also compared stable and unstable A-fragment mutants with respect to the kinetics of cell intoxication. While the toxicity of the stable mutants increased with time, the unstable mutants showed a similar toxicity at early and late time points. When studying the kinetics of cell intoxication by toxins with short cytosolic half-life, we could not detect any recovery of protein synthesis at late time points when all the mutant A-fragments should be degraded. This indicates that the ADP-ribosylation of EF-2 cannot be reversed by an endogenous activity in the cells. The data indicate that entry of toxin into a cell is not associated with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations.     

Entry of lethal doses of abrin, ricin and modeccin into the cytosol of HeLa cells

In attempts to assess how many molecules of the toxic lectins abrin, ricin and modeccin are needed in the cytosol to kill HeLa cells the effect of these toxins on protein synthesis and plating efficiency was studied. The incubation time of the cells after a 1 h exposure to the toxins influenced strongly the extent of inhibition of protein synthesis. The full toxic effect was expressed about 20 h of incubation after the exposure. On further incubation, protein synthesis again increased at a rate comparable to that in the control cells. After exposure to increasing concentrations of toxins the inhibition of cellular protein synthesis measured after 20 h showed excellent agreement with the inhibition of plating efficiency, indicating that the inhibition of protein synthesis can be used as a measure of cell killing. The inhibition of protein synthesis by toxins was found to follow first order kinetics, indicating that the cells are killed by an all- or none-effect. Autoradiographic studies indicated that after exposure to intermediate toxin concentrations protein synthesis was completely abolished in some cells, whereas it appeared to proceed at a normal rate in the remaining cells. The results provide evidence that penetration of one molecule of abrin, ricin or modeccin into cytosol is lethal to HeLa cells and that the efficiency of toxin entry into the cytoplasm is very low compared to the rate of bulk toxin uptake.     

Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.     

Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro , furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5–10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli , purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0–5.5. When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.     

Pathways followed by ricin and Shiga toxin into cells

The plant toxin ricin and the bacterial toxin Shiga toxin belong to a group of protein toxins that inhibit protein synthesis in cells enzymatically after entry into the cytosol. Ricin and Shiga toxin, which both have an enzymatically active moiety that inactivates ribosomes and a moiety that binds to cell surface receptors, enter the cytosol after binding to the cell surface, endocytosis by different mechanisms, and retrograde transport to the Golgi apparatus and the endoplasmic reticulum (ER). The toxins can be used to investigate the various transport steps involved, both the endocytic mechanisms as well as pathways for retrograde transport to the ER. Recent studies show that not only do several endocytic mechanisms exist in the same cell, but they are not equally sensitive to removal of cholesterol. New data have revealed that there is also more than one pathway leading from endosomes to the Golgi apparatus and retrogradely from the Golgi to the ER. Trafficking of protein toxins along these pathways will be discussed in the present article.     

Structural Complementation of the Catalytic Domain of Pseudomonas Exotoxin A

The catalytic moiety of Pseudomonas exotoxin A (domain III or PE3) inhibits protein synthesis by ADP-ribosylation of eukaryotic elongation factor 2. PE3 is widely used as a cytocidal payload in receptor-targeted protein toxin conjugates. We have designed and characterized catalytically inactive fragments of PE3 that are capable of structural complementation. We dissected PE3 at an extended loop and fused each fragment to one subunit of a heterospecific coiled coil. In vitro ADP-ribosylation and protein translation assays demonstrate that the resulting fusions—supplied exogenously as genetic elements or purified protein fragments—had no significant catalytic activity or effect on protein synthesis individually but, in combination, catalyzed the ADP-ribosylation of eukaryotic elongation factor 2 and inhibited protein synthesis. Although complementing PE3 fragments are catalytically less efficient than intact PE3 in cell-free systems, co-expression in live cells transfected with transgenes encoding the toxin fusions inhibits protein synthesis and causes cell death comparably as intact PE3. Complementation of split PE3 offers a direct extension of the immunotoxin approach to generate bispecific agents that may be useful to target complex phenotypes.     

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen.

The biochemical and biologic properties of a purified disulfide conjugate of diphtheria toxin fragment A and human placental lactogen (toxin A-hPL) have been studied by (a) assaying the ADP-ribosyltransferase activity of the intact conjugate, (b) assaying the binding of the intact conjugate to mammary gland plasma membrane lactogenic receptors, and (c) assaying the effect of the conjugate on the rate of protein synthesis in rabbit mammary gland explants maintained in organ culture. The toxin A-hPL conjugate retains one-third of the NAD+:EF-2 ADP-ribosyltransferase activity of toxin A, and 26% of the hPL-binding activity to lactogenic receptors. Binding activity was demonstrated by radioreceptor assay and by assaying toxin A activity bound to membranes which was competitively displaced by excess hPL. Since the toxin A-hPL conjugate retained activities of its separate subunits, it could be regarded as a structural analogue of nicked diphtheria toxin with replacement of the original membrane-binding chain by another binding chain that is specific for lactogenic receptor. However, the conjugate failed to inhibit protein synthesis in organ-cultured mammary gland explants, although these were sensitive to native diphtheria toxin and could bind hPL. It is concluded from these results that the toxin A-hPL conjugate does not act as a functional analogue of diphtheria toxin with altered receptor specificity, and that the hPL receptor cannot mediate the entry of toxin A or toxin A-hPL from membrane-bound conjugate into the cytosol site of action of toxin A.     

Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells

Clostridium difficile toxin B (269 kDa) is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. Toxin B acts in the cytosol of eukaryotic target cells where it inactivates Rho GTPases by monoglucosylation. The catalytic domain of toxin B is located at the N terminus (amino acid residues 1-546). The C-terminal and the middle region of the toxin seem to be involved in receptor binding and translocation. Here we studied whether the full-length toxin or only a part of the holotoxin is translocated into the cytosol. Vero cells were treated with recombinant glutathione S-transferase-toxin B, and thereafter, toxin B fragments were isolated by affinity precipitation of the glutathione S-transferase-tagged protein from the cytosolic fraction of intoxicated cells. The toxin fragment (approximately 65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B. The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells. After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547-2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells. Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol. Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane. The data indicate that toxin B is proteolytically processed during its cellular uptake process.     

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.     

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.