Toxin Insertion Research Articles

Characterization of an internal permissive site in the cholera toxin B-subunit and insertion of epitopes from human immunodeficiency virus-1, hepatitis B virus and enterotoxigenic Escherichia coli

We previously described the construction of novel hybrid proteins based on the B-subunit of cholera toxin (CTB) [Bäckström et al., Gene 149 (1994) 211-217], in which a neutralizing B-cell epitope from the third variable (V3) loop in the envelope glycoprotein gpl20 from human immunodeficiency virus type 1 (HIV-1) was inserted within a surface-exposed region between amino acids (aa) 55 and 64. The resulting protein retained properties of native CTB and could induce strong anti-CTB antibody (Ab) responses, but the inserted gpl20 epitope was only modestly immunogenic. In this study, the potential use of this internal permissive site in CTB for the insertion of heterologous epitopes has been further investigated. Six additional plasmids were constructed encoding HIV::CTB hybrid proteins with ten to fourteen aa from the V3 loop of gpl20 genetically inserted at different positions between aa 52 and 65, with deletions of different CTB aa. Plasmids encoding proteins with peptides inserted between aa 53 and 64 in CTB gave rise to stable proteins which reacted with CTB-specific monoclonal antibodies (mAb) and bound to GM 1 gangliosides (GM1), indicating that insertions between these positions do not drastically alter the conformation or the receptor-binding properties of native CTB. Plasmids were also constructed encoding CTB hybrid proteins which had either an 11-aa peptide from hepatitis B virus (HBV) pre-S(2) or one of two peptides related to the heat-stable toxin (ST a) of enterotoxigenic Escherichia coli inserted between aa 55 and 64 of CTB. This resulted in the production of HBV::CTB or ST::CTB hybrid proteins and illustrated that the internal permissive site can be used for insertion of peptides of varying aa composition. The reactivity of the inserted epitopes with epitope-specific mAb in GM 1-ELISA and immunoblots varied greatly between hybrid proteins and depended on the position in CTB and the as composition of the inserted peptides. Despite these differences, all the HIV::CTB, ST::CTB and HBV::CTB hybrid proteins could induce low, but significant, levels of serum Ab in mice against gp120, ST a or pre-S(2), in addition to strong serum Ab responses against CTB. The Ab response against the internally inserted gp120 peptide was similar to that against the same peptide fused to the N-terminus of CTB, indicating that internally placed peptides had similar immunogenicity to the same peptides added terminally.

Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers

Previous studies in our laboratory have shown that CryIC, a lepidopteran-specific toxin from Bacillus thuringiensis, triggers calcium and chloride channel activity in SF-9 cells (Spodoptera frugiperda, fall armyworm). Chloride currents were also observed in SF-9 membrane patches upon addition of CryIC toxin to the cytoplasmic side of the membrane. In the present study the ability of activated CryIC toxin to form channels was investigated in a receptor-free, artificial phospholipid membrane system. We demonstrate that this toxin can partition in planar lipid bilayers and form ion-selective channels with a large range of conductances. These channels display complex activity patterns, often possess subconducting states and are selective to either anions or cations. These properties appeared to be pH dependent. At pH 9.5, cation-selective channels of 100 to 200 pS were most frequently observed. Among the channels recorded at pH 6.0, a 25-35 pS anion-selective channel was often seen at pH 6.0, with permeation and kinetic properties similar to those of the channels previously observed in cultured lepidopteran cells under comparable pH environment and for the same CryIC toxin doses. We conclude that insertion of CryIC toxin in SF-9 cell native membranes and in artificial planar phospholipid bilayers may result from an identical lipid-protein interaction mechanism.

The role of tryptophan in structural and functional properties of equinatoxin II

A pore-forming, cytolytic and lethal polypeptide, equinatoxin II, from the sea anemone Actinia equina, was subjected to oxidation with N-bromosuccinimide to study the role of five present tryptophan residues in structure-function relationships. In the folded toxin molecule, 1-2 tryptophan residues were readily susceptible to oxidation with N-bromosuccinimide, whereas modification of a single residue resulted in complete impairment of the toxin lethal and hemolytic activities as well as the ability of an oxidized toxin to precipitate with serum lipoproteins. CD and fluorescence spectra indicated a slight alteration of a toxin secondary structure following N-bromosuccinimide treatment. Incubation with sphingomyelin of the toxin prior to oxidation did not prevent subsequent modification with N-bromosuccinimide and loss of its activities, indicating that the modified tryptophan residue is not directly involved in toxin binding and insertion into lipid membranes. It was concluded that the modified tryptophan residue is essential for the structure of equinatoxin II.

Early changes in cytosolic calcium and membrane potential induced by Actinobacillus actinomycetemcomitans leukotoxin in susceptible and resistant target cells.

Actinobacillus actinomycetemcomitans produces a cytolytic peptide leukotoxin which kills susceptible target cells, including human neutrophils, monocytes, lymphocytes, and HL-60 promyelocytic leukemia cells. Cell death occurs as a consequence of colloid osmotic lysis. In the present investigation early leukotoxin-induced changes in membrane permeability were studied by flow cytometry and quantitative spectrofluorimetry in leukotoxin-susceptible and resistant targets. Within 5 s toxin-susceptible cells exhibited concentration-dependent, sustained increases in systolic free Ca2+, and this was rapidly followed by a progressive fall in membrane potential. These early manifestations of membrane injury occurred approximately 10-15 min before cell death, as reflected by flow cytometric analysis of propidium iodide stained cells. The rise in cytosolic Ca2+ was almost entirely due to an influx of extracellular Ca2+. The results of Hill plots for the action of leukotoxin on Ca2+ permeability in human neutrophils or HL-60 cells suggested that two or more toxin molecules participate in the assembly of an ion conducting pore in the plasma membrane. Changes in membrane permeability or cell viability were not observed in response to heat-inactivated toxin. Under appropriate conditions toxin-induced membrane abnormalities were inhibited by leukotoxin-neutralizing mAb or relatively high concentrations (greater than or equal to 2.5 mM) of extracellular Ca2+. Leukotoxin-resistant target cells showed no evidence of membrane injury even when exposed to high concentrations of leukotoxin for prolonged periods of time. These included resistant human K562 erythroleukemia cells and murine SP2 myeloma cells which have previously been shown to adsorb the toxin, suggesting that they possess a protective mechanism(s) which impedes toxin insertion or assembly in the lipid bilayer. These data support the concept that A. actinomycetemcomitans leukotoxin acts as a cell-specific, pore-forming protein which permeabilizes the plasma membrane of susceptible target cells.

PH-Dependent membrane interactions of diphtheria toxin: A genetic approach

A genetic approach is described for exploring the mechanism by which diphtheria toxin undergoes pH-dependent membrane insertion and transfer of its enzymic A fragment into the cytoplasm of mammalian cells. The cloned toxin expressed inEscherichia coli is secreted to the periplasmic space, where it is processed normally and folds into a native structure. When bacteria synthesizing the toxin are exposed to pH 5, they die rapidly. The toxin undergoes a conformational change that is believed to allow it to be inserted into the bacterial inner membrane and form channels, which proves lethal for the cell. The membrane insertion event mimics the process by which the toxin inserts into the endosomal membrane of mammalian cells, leading to release of the enzymic A fragment into the cytoplasm. The observation of pH-dependent bacterial lethality provides the basis for a positive genetic selection method for mutant forms of the toxin that are altered in ability to undergo membrane insertion or pore formation.

Characterization of Diphtheria Toxin-induced Lesions in Liposomal Membranes: An Evaluation of the Relationship between Toxin Insertion and “channel” Formation

Abstract Diphtheria toxin interaction with membranes has been studied by following the release of a fluorescent dye (calcein) encapsulated within large unilamellar vesicles. Results showed that diphtheria toxin induced temperature- as well as pH-dependent permeability changes in these model membranes. Interestingly, insertion of the channel-forming B domain was not sufficient for calcein release, since dye release from vesicles composed of dimyristoyllecithin:cholesterol:dicetylphosphate 4:3:0.8) was completely inhibited at low temperatures which permitted B insertion. Rather, the temperature dependence of calcein release from and A domain insertion into dimyristoyllecithin:cholesterol:dicetyl phosphate vesicles suggest some relationship between formation and fragment A translocation across membranes. However, the nature of the toxin is called into question by our observations that size, in addition to activity, was pH-dependent. On the basis of these experiments, it is proposed that the toxin channel is the result of localized perturbations in the lipid bilayer at the interface between lipids and inserted toxin molecules that are sufficiently large in fluid membranes at low pH to allow the translocation of fragment A across the bilayer.

The acid-triggered entry pathway of Pseudomonas exotoxin A

In this study we examined the pH requirements and reversibility of early events in the Pseudomonas toxin entry pathway, namely, membrane binding, insertion, and translocation. At pH 7.4, toxin binding to vesicles and insertion into the bilayer are very inefficient. Decreasing the pH greatly increases the efficiencies of these processes. Acid-treated toxin exhibits pH 7.4 binding and insertion levels. This indicates that hydrophobic regions that become exposed upon toxin acidficiation become buried again when the pH is raised to 7.4. In contrast, the change in toxin conformation that occurs upon membrane binding is irreversible. Returning samples to pH 7.4, incubation with excess toxin, or dilution with buffer up to 1000-fold leads to very little loss of bound toxin. Bound toxin exhibits an extremely high susceptibility to trypsin compared to free toxin (at both pH 4 and pH 7.4). At pH 4, membrane-associated toxin slowly proceeds to a trypsin-protected state; neutralization halts this process. At low pH, toxin was found to bind and insert into DMPC vesicles very efficiently at temperatures both above and below 23 degrees C, the lipid melting point. With fluid targets, the proportion of bound toxin that was photolabeled from within the bilayer peaked rapidly and then decreased with time. With frozen targets, the efficiency of photolabeling peaked but then remained fairly constant.(ABSTRACT TRUNCATED AT 250 WORDS)

An endosomal model for acid triggering of diphtheria toxin translocation.

An endosomal model system was developed for studying the effects of pH on vesicle-entrapped diphtheria toxin. The "endosomes" were prepared from dioleoylphosphatidylcholine (1 mg), diphtheria toxin (0.25 mg), and lysozyme (2.25 mg) in water at pH 8.4. The method used for preparing large unilamellar vesicles was adapted from the procedure of Shew and Deamer (Shew, R. L., and Deamer, D. W. (1985) Biochim. Biophys. Acta 816, 1-8). Efficiencies of trapping (typically 45-75%) and separation from untrapped proteins (typically 95-100%) were assessed by fluorescamine assays conducted before and after column chromatography and in the presence and absence of Tergitol Nonidet P-40. Intramembranous photolabeling revealed that diphtheria toxin inserts into the vesicle bilayer when the pH is dropped to 4; surface labeling revealed that the same treatment leads to exposure of diphtheria toxin at the trans surface of the vesicles. Release of toxin to the solution was not detected under the experimental conditions employed (i.e. with nicked or unnicked toxin, +/- exogenous trypsin, pH 4 or 8.4). Preliminary results indicate that this model system will be a valuable tool for elucidating the pathway by which the ADP ribosyltransferase domain of diphtheria toxin gains access to the cytoplasmic compartment of cells after endosomal uptake.

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 2. Hydrophobic photolabelling and cell intoxication.

The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half-maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate-controlling step in the process of membrane translocation of diphtheria toxin. crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid-treated cells, suggesting that the 12-kDa COOH-terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin. It is suggested that crm 1001 is non-toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.

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.

Evidence for direct insertion of fragments A and B of diphtheria toxin into model membranes.

The entry of diphtheria toxin into model membranes was studied using a membrane-restricted photoprobe to monitor insertion. The results provided direct evidence that the A and B domains of diphtheria toxin can both insert into such membranes. Optimal binding was achieved with negatively charged liposomes at pH 3.6. Under these conditions, the A and B domains of nicked as well as unnicked toxin inserted. At 0 degrees C, only fragment B inserted although toxin was still optimally bound. At neutral pH, there was little binding of toxin, and fragment B inserted preferentially. Brief exposure of the toxin to pH 3.6 followed by adjustment of the pH to 7 and subsequent incubation of the toxin with vesicles at neutral pH greatly facilitated toxin binding as well as insertion of both A and B domains, indicating also that a pH gradient was not required for these processes. Data obtained from the tryptic fragmentation patterns and circular dichroism spectra indicated that exposure to acidic pH had induced a conformational change in the toxin which may have exposed hydrophobic regions. A similar conformational change may occur in vivo after acidification of cytoplasmic vesicles into which toxin is delivered by receptor-mediated endocytosis. This could facilitate insertion of toxin into the vesicle membranes and subsequent translocation of fragment A to the cytosol, where it causes inhibition of protein synthesis.

Mechanism of insertion of diphtheria toxin: peptide entry and pore size determinations.

Diphtheria toxin ( DTx ) is an extremely potent inhibitor of protein synthesis. It is secreted as a linear polypeptide, which is cleaved to produce disulfide-linked A and B fragments. Fragment A, the inhibitor of protein synthesis, requires fragment B, the recognition subunit, for entry into intact cells. Fragment B has been proposed to form a transmembrane channel through which A gains access to the cytosol. If it were demonstrated that the B subunit had an exclusive association with membrane lipid acyl chains, this might indicate that A is secluded in a proteinaceous B channel. However, our results from intramembranous photolabeling studies show that both subunits of DTx enter the hydrocarbon domain of the bilayer. Toxin cleavage is not required for penetration. Decreasing pH leads to increased binding and hence indirectly to increased penetration. Parallel permeability studies indicate that cleaved DTx does indeed form pores (24 A in diameter) and they are larger than those previously reported (5 A) with native toxin. The data suggest that these are dimeric structures. Cleaved DTx is much more effective than intact DTx at pore formation. Thus, we conclude that, while pore formation is a feature of toxin-membrane interaction, the pore structure does not protect A from contact with lipid side chains and may in fact consist of both the A and B domains in a dimeric configuration, (AB)2.

Lipid-protein interactions during ricin toxin insertion into membranes. Evidence for A and B chain penetration.

Lipid-protein interactions during ricin toxin insertion into membranes. Evidence for A and B chain penetration.

Insertion of diphtheria toxin into and across membranes: role of phosphoinositide asymmetry.

Diphtheria toxin (DT), a 63,000-molecular weight soluble protein, is toxic to most mammalian cells. The mechanism of intoxication involves a step in which one part of the molecule inserts into a membrane, facilitating the transport of the enzymatic fragment of the protein into the cytoplasm1,2. This event requires an acidic environment and seems to occur at the membrane of an endocytic vesicle3,4. Different cell lines and species differ in their sensitivities to DT—this is due at least in part to differences in the number of DT surface receptors on the cells5, but may also arise from differences in the membrane transport step. It is generally recognized that protein–lipid interactions are of critical importance in membrane transport phenomena (see ref. 6). Thus, it is of interest to determine whether the interaction of DT with membranes is modulated by their composition. We have shown previously that DT interacts with planar lipid bilayers at acidic pH to produce voltage-dependent channels7. We report here that this interaction of DT with bilayers depends on membrane phospholipid composition; the interaction is optimal when inositides are present within the membrane, and the inositides are required on the side of the membrane opposite to that in which DT is introduced.

Lipid insertion of cholera toxin after binding to GM1-containing liposomes.

The technique of hydrophobic photolabeling with photoreactive lipids was used to study the topology of interaction of cholera toxin with liposomes containing galactosyl-N-acetylgalactosaminyl-[N-acetyl neuraminyl]-galactosyl glucosyl ceramide (GM1). The toxin appears to locate itself superficially on the lipid bilayer. The interaction is mediated only by the gamma beta 5 part. On reduction of the disulfide bridge joining the alpha and gamma subunits, the alpha subunit penetrates deeply into the lipid bilayer. The mere binding of cholera toxin to GM1 is not sufficient to allow the insertion of the enzymatically active alpha subunit in the membrane. Some processing, which may involve a modification of covalent bonds of the toxin molecule (such as that caused by reduction) appears to be necessary. The specific reduction of the alpha-gamma disulfide bond on the external surface of the membrane as a prerequisite for the membrane penetration of the alpha subunit is discussed.

Diphtheria toxin forms transmembrane channels in planar lipid bilayers.

When exposed to a lipid bilayer, diphtheria toxin binds to it and forms transmembrane, voltage-dependent, anion-selective channels. The mean (+/- SD) conductance of these channels in asolectin membranes is 6.2 +/- 0.7 pmho (pS) in 0.2 M NaCl and 20 +/- 2 pmho in 1.0 M NaCl. The rate of channel formation depends on the pH in the toxin-containing compartment; it is very low at pH greater than 5.0 and increases abruptly as the pH decreases from 4.9 to 4.0. Binding of toxin to the membrane is also pH dependent, being unmeasurable at pH 7 and increasing monotonically with decreasing pH. The rate of channel formation depends upon membrane potential as well; channels form only at negative potentials. These channels are permanent in the time scale of the experiments (about 1 hr). The membrane conductance caused by the channels is also voltage dependent, being constant at positive potentials and decreasing at negative potentials. Hence, the current-voltage curve is linear at positive potentials and sublinear at negative potentials. The conditions necessary for insertion of toxin into the bilayer and formation of channels are similar to those that prevail inside the lysosome. Thus, these results lend credence to the idea that toxin enters the cytoplasm from the lysosomal compartment.