Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum

Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assay and in-vitro Vero cell assay. The assay has also been found to be effective in determining the rising antibody titer in the equines inducted in DATS production. The present study indicated that although biological assays hold the key for final potency estimations till date but in the future scenario in-vitro Vero cell assay may be a good alternative to in-vivo biological assay.     

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

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

Antibodies introduced into living cells by red cell ghosts are functionally stable in the cytoplasm of the cells.

The function and fate of antibodies introduced into living cells by red cell ghosts were studied using CRM 176 (a mutant diphtheria toxin having lower toxicity than the wild-type) and antibody against fragment A of diphtheria toxin. IgG labeled with iodine and FITC was found in the cytoplasm of the recipient cells. When about 1500 molecules of anti-fragment A antibody (rabbit IgG) were introduced into diphtheria toxin-sensitive Vero cells or FL cells, these cells became resistant to the toxin and formed normal colonies. It was calculated from the survival of cells without anti-fragment A IgG under these conditions that about 300 molecules of fragment A-176 were transferred to the cells. These results showed that the antigen-antibody reaction took place in living cells as effectively as in a cell-free system. The functional stability of antibody IgG in cells was examined by exposing Vero cells containing a subminimal amount of anti-fragment A IgG (about 1000 molecules) to the toxin for 2 hr at various times after the introduction of anti-fragment A IgG. More than 50% of the initial activity of the antibody to neutralize toxin still remained even after incubation of the cells at 37°C for 20 hr. The same degree of stability was also demonstrated using iodine-labeled specific anti-fragment A IgG. The IgG recovered from the recipient cells after various times of incubation at 37°C retained its full ability to bind to fragment A-conjugated Sepharose 4B, although the total amount of IgG associated with the cells decreased about 50% in 24 hr.     

Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 μg/ml. ¹²⁵I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH₄Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cells were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells.     

Effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin

When Vero cells were depleted of potassium, the cells were protected against diphtheria toxin. Potassium depletion of Vero cells strongly reduced the binding of the toxin to cell surface receptors. Likewise, potassium depleted L-cells were protected against pseudomonas toxin. Diphtheria toxin binding was completely restored upon addition of potassium to the cells. This restoration was not prevented by inhibition of protein synthesis by cycloheximide. When cells were depleted of potassium in the presence of metabolic inhibitors, and then treated with diphtheria toxin, protein synthesis was reduced to the same extent as in cells with normal intracellular level of potassium. The results indicate that potassium depletion of Vero cells reduces the ability of the cells to bind diphtheria toxin by an ATP requiring process, and that binding, endocytosis and transfer of diphtheria fragment A across the membrane may occur at low intracellular levels of potassium.     

Affinity chromatography purification of diphtheria toxin

NAD was covalently linked to Sepharose-4B using a 6 carbon spacer. Sterile, dialyzed spent culture medium containing 100 Lf/ml of diphtheria toxin or material concentrated by (NH₄)₂SO₄ precipitation containing 1500 Lf/ml, was chromatographed on a column of NAD–Sepharose. Ultraviolet absorbing material which did not flocculate with diphtheria antitoxin was eluted with 0.02M phosphate buffer. When the elation buffer was changed to one containing 0.5M NaCl, purified toxin was eluted off the column.     

Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin

We have studied the cytotoxicity of ricin in cells treated with brefeldin A (BFA), which dramatically disrupts the structure of the Golgi apparatus causing Golgi content and membrane to redistribute to the ER. BFA inhibits the cytotoxicity of ricin in Chinese hamster ovary, normal rat kidney, and Vero cells and abolishes the enhancement of ricin cytotoxicity by NH4Cl, nigericin, swainsonine, and tunicamycin or by a mutation in endosomal acidification. BFA protects cells from the cytotoxicities of modeccin and Pseudomonas toxin, but has no effect on the intoxication by diphtheria toxin. Pretreatment of BFA does not protect cells from ricin treatment in the absence of BFA. Our results suggest that ricin, modeccin, and Pseudomonas toxin share a common pathway of intracellular transport from endosomes to the Golgi region where they are released into the cytosol. In contrast, the lack of protection of Vero cells from diphtheria toxin by BFA indicates that diphtheria toxin is released from acidified endosomes without involving the Golgi region.     

Enhancement of diphtheria toxin-induced apoptosis in Vero cells by combination treatment with brefeldin A and okadaic acid

In the present study, we compared the abilities of ricin and diphtheria toxin to induce apoptosis in Vero cells. The cytolysis and DNA fragmentation by ricin paralleled its protein synthesis inhibitory activity. However, unlike ricin, diphtheria toxin could induce neither cytolysis nor DNA fragmentation in Vero cells up to very high concentration, in spite of the fact that Vero cells were even more sensitive to protein synthesis inhibition by diphtheria toxin than ricin. Interestingly, coexistence of brefeldin A (BFA) and okadaic acid (OA) significantly enhanced diphtheria toxin-mediated cytolysis and DNA fragmentation without affecting the activity of protein synthesis inhibition. Ammonium chloride almost completely abolished the ability of diphtheria toxin to induce apoptosis in the presence of BFA and OA as well as the protein synthesis inhibitory activity. The mutant CRM 197, which does not catalyze the ADP ribosylation of elongation factor-2 (EF-2), failed to induce apoptosis in Vero cells even in the presence of BFA and OA. Thus, translocation of diphtheria toxin into the cytosol and subsequent enzymatic inactivation of EF-2 may be necessary steps to induce apoptosis. Taken together our results suggest that protein synthesis inhibition by toxins is not sufficient to induce apoptosis, and underlying mechanisms of apoptosis induction may be distinct between ricin and diphtheria toxin. Since a morphological change in the Golgi complex was observed in Vero cells treated with BFA and OA, modulation of the Golgi complex by these reagents may be partly responsible for enhanced apoptosis induction by diphtheria toxin.     

Interactions between diphtheria toxin entry and anion transport in vero cells. III. Effect on toxin binding and anion transport of tumor-promoting phorbol esters, vanadate, fluoride, and salicylate

When Vero cells were incubated with TPA (12-O-tetradecanoylphorbol 13-acetate) and related tumor promoters, their ability to bind diphtheria toxin in a functional way was rapidly reduced to less than 1% of the normal value. Upon further incubation with TPA, the cells recovered their ability to bind the toxin, apparently because they became resistant to TPA. Treatment with Na3VO4 reduced the ability of the cells to bind diphtheria toxin to approximately the same extent as treatment with TPA, but the reduction required longer time to develop and it persisted upon prolonged incubation with Na3VO4. ATP depletion of the cells prevented the reduction in binding capability. Such treatment also prevented the reduction in toxin binding induced by treatment with salicylate or fluoride. Treatment with TPA, fluoride, vanadate, and salicylate altered the ability of the cells to carry out anion transport and interfered with their ability to regulate the transport. The results indicate that the binding sites for diphtheria toxin on Vero cells are modulated by TPA, Na3VO4, salicylate, and fluoride by a process which requires ATP. The possibility is discussed that the modulation consists in phosphorylation of the toxin binding sites, which may be identical with, or closely linked to, the anion antiporter in the cells.     

Expression and Immunogenicity of a Recombinant Diphtheria Toxin Fragment A in Streptococcus gordonii

A nontoxic mutant diphtheria toxin fragment A (DTA) was genetically fused in single, double, or triple copy to the major surface protein antigen P1 (SpaP) and surface expressed in Streptococcus gordonii DL-1. The expression was verified by Western immunoblotting. Mouse antisera raised against the recombinant S. gordonii recognized the native diphtheria toxinm suggesting the recombinant DTA was immunogenic. When given intranasally to mice with cholera toxin subunit B as the adjuvant, the recombinant S. gordonii expressing double copies of DTA (SpaP-DTA₂) induced a mucosal immunoglobulin A response and a weak systemic immunoglobulin G response. S. gordonii SpaP-DTA₂ was able to orally colonize BALB/c mice for a 15-week period and elicited a mucosal response, but a serum immunoglobulin G response was not apparent. The antisera failed to neutralize diphtheria toxin cytotoxicity in a Vero cell assay.     

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.     

Entry of diphtheria toxin linked to concanavalin A into primate and murine cells

Diphtheria toxin linked by a disulfide bridge to concanavalin A was highly toxic to HeLa S3 and Vero cells, as well as to murine L cells. The cells could be protected with alpha-methyl mannoside, indicating that the conjugate binds mainly through its concanavalin A moiety. Treatment of Vero cells with phospholipase C, TPA (12-O-tetradecanoylphorbol-13-acetate), and vanadate, which strongly reduce the ability of the cells to bind free diphtheria toxin, had little protective effect against the conjugate, whereas SITS (L-acetamido-4'-isothiocyano-stilbene-2,2'disulfonic acid), which inhibits diphtheria toxin binding, as well as the subsequent entry, protected Vero cells, but not L cells. Both types of cells are protected against the conjugate by NH4Cl and monensin, indicating that an acidified compartment is necessary for entry into the cytosol. Exposure of cells, bound with surface conjugate, to low pH induced entry of the toxin into Vero cells, but not into L Cells. Phospholipase C, TPA, and vanadate did not protect L cells against the conjugate. It is concluded that toxin in the conjugate enters L cells by a route which involves low pH, but which is not identical to that in Vero cells.     

Entry of diphtheria toxin-protein A chimeras into cells

Fusion proteins consisting of diphtheria toxin and a duplicated Fc-binding domain of protein A were made in vitro after amplification of the DNA template by the polymerase chain reaction. The fusion proteins bound avidly to Vero cells coated with antibodies. A fusion protein containing full-length diphtheria toxin was toxic at lower concentrations than diphtheria toxin alone, apparently due to more efficient binding. The enzymatic part of the fusion protein was translocated across the surface membrane upon exposure to low pH. Like authentic diphtheria toxin, the fusion protein formed cation selective channels at low pH. Excess amounts of unlabeled diphtheria toxin inhibited formation of pronase-protected fragments derived from radiolabeled fusion protein. Furthermore, conditions that down-regulate the diphtheria toxin receptors reduced the sensitivity of the cells to the fusion protein, supporting the notion that authentic diphtheria toxin receptors are required. At temperatures below 18 degrees C the toxicity of the fusion protein was strongly reduced, whereas there was no temperature block for authentic diphtheria toxin. Brefeldin A protected Vero cells against the fusion protein but not against diphtheria toxin. The results indicate that the diphtheria toxin receptor is required for efficient toxin translocation even under conditions where the toxin is bound by an alternate binding moiety, and they suggest that the intracellular routing of the fusion protein is different from that of diphtheria toxin.     

Evidence that membrane phospholipids and protein are required for binding of diphtheria toxin in Vero cells

Treatment with phospholipase C strongly protected monkey kidney (Vero) cells against diphtheria toxin and reduced the ability of the cells to bind 125I-labelled toxin. Treatment with phospholipase D and with trypsin also protected the cells, although to a lesser extent. Phospholipase A2 had no protective effect. Phospholipase C also protected fetal hamster kidney cells against the toxin. After removal of the enzymes, as well as after treatment of the cells with 4-acetamide 4'-isothiocyanostilbene 2,2'-disulfonic acid, diphtheria toxin binding capability was restored slowly, apparently by a process requiring protein synthesis, since cycloheximide blocked the restoration. The data indicate that both phospholipids and protein are involved in the binding sites for diphtheria toxin.     

Action of diphtheria toxin does not depend on the induction of large,stable pores across biological membranes

Summary Vero cells exposed to diphtheria toxin at pH 4.5 leak monovalent cations but not amino acids or phosphorylated metabolites; affected cells do not take up trypan blue. Monovalent cation leakage is inhibited by 1mmCd²⁺, but not by 1mmZn²⁺ or Ca²⁺. Cd²⁺ blocks calcein leakage from liposomes and closes diphtheria toxin-induced channels in lipid bilayers. It is concluded that translocation of the A fragment of diphtheria toxin across biological membranes does not depend on the formation of large stable pores, but that small Cd²⁺-sensitive pores may play a role.     

Studies on the MgSO4-induced cytoplasmic uptake of proteins by cells in culture

When healthy HeLa cells were exposed to an intracellular acting cytocidal protein extracted from vaccinia virus-infected cells, or non-toxic diphtheria A fragment, there was no detectable drop in cell viability. However, when the cells were treated with vaccinia cytocidal protein, low non-toxic doses of whole diphtheria toxin, or high doses of non-toxic diphtheria A fragment in the presence of 0.85 M MgSO₄ for 15 min, to induce intra-cytoplasmic uptake, 25–30% of the cells died.This partial killing effect was shown to be due to a heterogeneous cytoplasmic uptake of the cytotoxic protein; induced protein uptake occurred in only 25–30% of the cell population. It was not found possible to correlate this cell sub-population with a genetically determined cell sub-population, cells in a specific stage of the active cell division cycle or, inactive G0-like cells. Electron microscopic studies of cells after 0.85 M MgSO₄ treatment showed increased vacuolation and uptake of horse-radish peroxidase into such newly formed vacuoles was demonstrated. Light microscope studies indicated that this induced vacuolation was also not homogeneous within the population, which led to the possibility that excessive induced vacuolation and eventual cytoplasmic entry of macromolecules were linked phenomena. This hypothesis was supported by studies on rabbit kidney (RK), and baby hamster kidney (BHK) cells, which showed similar degrees of vacuole inducibility and toxin susceptibilities as HeLa cells, and by studies on HEp-2 cells, Chang conjunctival cells (W-K derivative), and McCoy cells, which showed no MgSO₄-induced vacuolation and were totally resistant to vaccinia cytocidal protein and diphtheria toxin A fragment in the presence of hypertonic MgSO₄. Evidence was obtained which indicated that excessive vacuolation is also linked with aberrant vacuolar fusion events within the cell after treatment with hypertonic MgSO₄.     

Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments

Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment.     

A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly.

We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L. monocytogenes utilizes a cellular rho dependent mechanism in this process.     

Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain

CRM197, CRM176, and CRM228 are products of single or multiple missense mutations in the diphtheria toxin gene. CRM197 differs from wild-type toxin in 1 amino acid residue of the fragment A region, and also CRM176 and CRM228 have amino acid substitution(s) in fragment A. We compared the binding properties of CRM197 to toxin-sensitive Vero cells with those of diphtheria toxin and other CRMs. Nicked CRM197 is about 50 times more effective than intact CRM197 in inhibiting the action of diphtheria toxin on sensitive cells, as shown by inhibition of diphtheria toxin cytotoxicity or inhibition of binding of 125I-diphtheria toxin. The binding of native toxin or other CRMs was not significantly affected by nicking. Moreover, the binding of CRM197 to cells was unaffected by ATP, although ATP clearly inhibits binding of diphtheria toxin, CRM176, and CRM228. Two kinds of hybrid protein were formed using fragment B of CRM197: one with fragment A of diphtheria toxin and one with fragment A of CRM228. ATP inhibited the binding of these hybrid proteins. Furthermore, the affinities of these hybrid proteins for diphtheria toxin-sensitive cells were the same as that of native toxin. Thus, it was concluded that the altered binding properties of CRM197 were due to alteration of fragment A and what the interaction of diphtheria toxin with ATP involves both fragments. The results also suggest that fragment A plays a role in diphtheria toxin-receptor interaction.     

Proline-Rich Peptide from the Coral Pathogen Vibrio shiloi That Inhibits Photosynthesis of Zooxanthellae

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29°C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH₄Cl, pure natural or synthetic toxin P (10 μM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH₄Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH₄Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH₃ into the cell. It is known that uptake of NH₃ into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.