The effect of a Clostridium perfringens 8-6 enterotoxin on viability and macromolecular synthesis in Vero cells

Clostridium perfringens type A 8-6 enterotoxin causes gross morphological damage to Vero cells grown in tissue culture. Damage was observed to occur after only 30 minutes exposure at concentrations as low as 20 ng/ml, and within 60 minutes 95% of the cells had detached. Concentrations as low as 0.01 ng were able to cause detectable inhibition of plating efficiency. The enterotoxin inhibited DNA, RNA and protein synthesis and caused the reversal of glucose transport. Heat inactivated enterotoxin had no effect on cell function or morphology.     

Binding versus biological activity of Clostridium perfringens enterotoxin in Vero cells.

Cells resistant to $\text{Clostridium perfringens}$ enterotoxin were selected from cultures of highly sensitive Vero (African green monkey kidney) cells. Studies were done with the sensitive and resistant cells to determine the relationship between binding and biological activity. Binding studies using ¹²⁵I-enterotoxin revealed the apparent existence of high and low affinity binding sites for the enterotoxin on both cell types. The binding site density on resistant cells was found to be $\text{1}\text{10}$ that of sensitive cells. It was found that, even with high doses of enterotoxin, only partial affect upon DNA synthesis, membrane permeability, and plating efficiency was noted in resistant cells. It is concluded that without specific binding there is little or no ability of the enterotoxin to effect biological activity in cells.     

The effects of Clostridium perfringens enterotoxin on intracellular levels or transport of uridine, thymidine and leucine do not fully explain enterotoxin-induced inhibition of macromolecular synthesis in Vero cells

Clostridium perfringens type A enterotoxin (CPE) has been shown previously to inhibit the incorporation of radiolabeled precursors into acid-insoluble material but the mechanism of inhibition is unknown. It has also been shown that extracellular calcium is required for some CPE effects. In this report, it is shown that CPE completely and virtually simultaneously inhibits incorporation of precursors into RNA, DNA and protein in either the presence or absence of extracellular divalent cations and that changes in intracellular precursor levels did not consistently correlate with this CPE-induced inhibition of incorporation. These results strongly suggest that CPE can inhibit macromolecular synthesis, not just inhibit precursor transport. It is inferred from this that CPE can affect DNA and RNA synthesis, and possibly protein synthesis, by altering other cellular processes besides, or in addition to, precursor transport and these effects then lead to a shutdown of macromolecular synthesis.     

Calcium-independent and dependent steps in action of Clostridium perfringens enterotoxin on HeLa and Vero cells.

Purified enterotoxin (20–200 ng/ml) of $\text{Clostridium}$$\text{perfringens}$ rapidly induced bled and balloon formation on HeLa and Vero cells in the presence, but not the absence, of Ca²⁺. The action of the toxin involved two, sequential, temperature-dependent steps: The first was Ca²⁺-independent and included binding of toxin and the bound toxin after 30–60 sec could no longer be removed by washing. The second step was Ca²⁺-dependent and eventually led to bled and balloon formation. On adding Ca²⁺ to cells pretreated with toxin in Ca²⁺-free medium, bled and balloon formation started immediately. The ionophore A23187 mimicked the action of toxin. The effects of sucrose (0.2 M), trypsin-treatment of the cells and various pretreatments of the toxin on the action of enterotoxin were studied.     

Characterization of membrane permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin

Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were alpha-amino[14C]isobutyric acid (Mr 103), 3H-labeled nucleotide (Mr approx. 300), 51Cr label (Mr approx. 3000) and [3H]RNA (Mr>25000). Over a 2h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and 51Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of Mr 3000 but not 25000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional 'holes' of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and nonconfluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional 'holes' of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death.     

Identification and isolation of the binding substance for Clostridium perfringens enterotoxin on Vero cells

Abstract To identify the binding substance for Clostridium perfringens enterotoxin (CPE), the CPE-binding substances metabolically labelled with [³H]leucine on CPE-susceptible (Vero) and resistant (L-929) cells were analyzed by solubilization, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The CPE-binding substance was found on Vero cells, but not on L-929 cells. The molecular weight of the CPE-binding substance was found to be 60 000 on SDS-PAGE. The CPE-binding substances were isolated from Vero cells and Balb/c mouse intestinal brush border membranes by affinity chromatography on CPE-coupled Sepharose 4B. They were homogeneous substances with molecular weights of 60 000 on SDS-PAGE and inhibited to the same extent the binding reaction of ¹²⁵I-labeled CPE with Vero cells. These results suggests that the CPE-binding substances are the receptors of CPE on these cells.     

Protective effects of osmotic stabilizers on morphological and permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin

Culture medium made hypertonic by the addition of osmotic stabilizers such as sucrose, poly(ethylene glycol), dextran and bovine serum albumin protected against changes in morphology and plasma membrane permeability induced by Clostridium perfringes enterotoxin. The protection did not appear to be due to binding inhibition. Results of these studies support an osmotic disruption mechanism for the action of the enterotoxin. A comprehensive model of the enterotoxin's action based on an osmotic disruption mechanism is proposed.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin.

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

Comparative biochemical and immunocytochemical studies reveal differences in the effects of Clostridium perfringens enterotoxin on polarized CaCo-2 cells versus Vero cells.

Since most in vitro studies exploring the action of Clostridium perfringens enterotoxin (CPE) utilize either Vero or CaCo-2 cells, the current study directly compared the CPE responsiveness of those two cell lines. When CPE-treated in suspension, both CaCo-2 and Vero cells formed SDS-resistant, CPE-containing complexes of approximately 135, approximately 155, and approximately 200 kDa. However, confluent Transwell cultures of either cell line CPE-treated for 20 min formed only the approximately 155-kDa complex. Since those Transwell cultures also exhibited significant (86)Rb release, approximately 155-kDa complex formation is sufficient for CPE-induced cytotoxicity. Several differences in CPE responsiveness between the two cell lines were also detected. (i) CaCo-2 cells were more sensitive when CPE-treated on their basal surface, whereas Vero cells were more sensitive when CPE-treated on their apical surface; those sensitivity differences correlated with CPE binding the apical versus basolateral surfaces of these two cell lines. (ii) CPE-treated Vero cells released (86)Rb into both Transwell chambers, whereas CaCo-2 cells released (86)Rb only into the CPE-containing Transwell chamber. (iii) Vero cells express the tight junction (TJ) protein occludin but (unlike CaCo-2 cells) cannot form TJs. The ability of TJs to affect CPE responsiveness is supported by the similar effects of CPE on Transwell cultures of CaCo-2 cells and Madin-Darby canine kidney cells, another polarized cell forming TJs. Confluent CaCo-2 Transwell cultures CPE-treated for >1 h formed the approximately 200-kDa CPE complex (which also contains occludin), exhibited morphologic damage, and had occludin removed from their TJs. Collectively, these results identify CPE as a bifunctional toxin that, in confluent polarized cells, first exerts a cytotoxic effect mediated by the approximately 155-kDa complex. Resultant damage then provides CPE access to TJs, leading to approximately 200-kDa complex formation, internalization of some TJ proteins, and TJ damage that may increase paracellular permeability and thereby contribute to the diarrhea of CPE-induced gastrointestinal disease.     

Spore coat protein and enterotoxin synthesis in Clostridium perfringens.

Polyacrylamide gel profiles of Clostridium perfringens spore coat protein revealed four and occasionally five components. Pulse-chase experiments indicated that synthesis of coat protein polypeptide and enterotoxin was an early sporulation event. However, maximum synthesis occurred coincident with the onset of heat resistance.     

Osmotic stabilizers differentially inhibit permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin

Using a sensitive Vero (African green monkey kidney) cell model system, studies were performed to further investigate whether Clostridium perfringens enterotoxin acts via disruption of the colloid-osmotic equilibrium of sensitive cells. Enterotoxin was shown to cause a rapid loss of intracellular 86Rb+ (Mr approx. 100) with time- and dose-dependent kinetics. The enterotoxin-induced release of intracellular 86Rb+ preceded the loss of two larger labels, 51Cr label (Mr approx. 3500) and 3H-labeled nucleotides (Mr less than 1000). The osmotic stabilizers, sucrose and poly(ethylene glycol), differentially inhibited enterotoxin-induced larger label loss versus 86Rb+ loss. Further, enterotoxin was shown to cause a rapid influx of 24Na+ that was not significantly inhibited by osmotic stabilizers. Additional studies demonstrated that lysosomotropic agents were not protective against characteristic enterotoxin-induced membrane permeability alterations or morphological damage. Taken collectively, these results are consistent with an action for enterotoxin which involves a disruption of the osmotic equilibrium.     

Inhibition of macromolecular synthesis by caffeine in Clostridium perfringens

Caffeine (2 mg/mL) inhibited the incorporation of [14C]adenine into actively growing cells of Clostridium perfringens NCTC 8679 in a dose-dependent manner. Also reduced by caffeine was incorporation of [14C]thymidine and 14C-labeled amino acids. No effect on guanine, uracil, adenosine, guanosine, or uridine was detected. Actual incorporation of [14C]caffeine or [14C]thymine in control cultures did not occur.     

Evidence that alterations in small molecule permeability are involved in the Clostridium perfringens type A enterotoxin-induced inhibition of macromolecular synthesis in Vero cells

The mechanism by which Clostridium perfringens enterotoxin (CPE) simultaneously inhibits RNA, DNA, and protein synthesis is unknown. In the current study the possible involvement of small molecule permeability alterations in CPE-induced inhibition of macromolecular synthesis was examined. Vero cells CPE-treated in minimal essential medium (MEM) completely ceased net precursor incorporation into RNA and protein within 15 minutes of CPE treatment. However, RNA and protein synthesis continued for at least 30 minutes in Vero cells CPE-treated in buffer (ICIB) approximating intracellular concentrations of most ions. Addition of intracellular concentrations of amino acids to ICIB (ICIB-AA) caused a further small but detectable increase in protein synthesis in CPE-treated cells. ICIB did not affect CPE-specific binding levels or rates. Similar small molecule permeability changes (i.e., 86Rb-release) were observed in cells CPE-treated in either ICIB or in Hanks' balanced salt solution. Collectively these findings suggest that CPE-treatment of cells in ICIB-AA ameliorates CPE-induced changes in intracellular concentrations of ions and amino acids and permits the continuation of RNA and protein synthesis. These results are consistent with and support the hypothesis that permeability alterations for small molecules are involved in the CPE-induced inhibition of precursor incorporation into macromolecules in Vero cells.     

Clostridium perfringens type A: in vitro system for sporulation and enterotoxin synthesis.

Polysomes were isolated from an enterotoxigenic strain of Clostridium perfringens during vegetative growth and at 1-h intervals after transfer into Duncan-Strong sporulation medium. During vegetative growth, about 67% of the ribosomes were in polysomal complexes. This proportion decreased to about 20% during the first 2 h in sporulation medium and then gradually increased to a maximum of 45% at 6 h. Ribosomes isolated from cells in vegetative or in sporulation phase could equally translate vegetative, sporulation, and natural viral R17 messenger ribonucleic acid with either vegetative or sporulation initiation factors. When polysomes were allowed to complete their nascent chains with labeled amino acids in vitro, most of the polypeptides synthesized by the vegetative phase and by the sporulation phase polysomes appeared to be identical. There were, however, notable differences upon further investigation. Specifically, when antiserum against the enterotoxin was reacted with the completed polypeptides, no counts were precipitated from the vegetative products. On the other hand, up to 12% of the total labeled protein was precipitated from the products obtained with the sporulation phase polysomes. Upon electrophoresis on sodium dodecyl sulfate, the putative enterotoxin synthesized in vitro ran as a major band with a molecular weight of 35,000, and as two minor bands with molecular weights of 17,000 and 52,000, respectively.     

Binding of enterotoxin from Clostridium perfringens type A to liver cells in vivo and in vitro. The enterotoxin causes membrane leakage

Enterotoxin from Clostridium perfringens was shown to retain its biological activity after labelling with 125I. When injected intravenously into mice and rats, most of the radioactivity in the organs was present in the form of intact toxin. Studies of the tissue distribution of labelled enterotoxin showed the largest amounts in the liver, where the activity reached a maximum 10--15 min after administration. The highest concentration per g tissue was found in liver and kidneys. The radioactivity was excreted in the urine as a mixture of intact labelled toxin and low molecular weight degradation products. In vitro studies with purified parenchymal liver cells showed rapid release of lactate dehydrogenase (LDH) during treatment with enterotoxin, thus indicating severe membrane damage.     

Anomalous aggregation of Clostridium perfringens enterotoxin under dissociating conditions.

Polyacrylamide gel electrophoresis of highly purified Clostridium perfringens enterotoxin revealed electrophoretic microheterogeneity of the enterotoxin, apparently because of slight charge differences in the peptides. Detergent gel electrophoresis showed that purified enterotoxin formed high molecular weight aggregates in the presence of both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide. No conditions capable of inhibiting this phenomenon were found. Although a molecular weight of 35 000 daltons has been reported in the literature, the experimentally determined molecular weight values in the presence of detergents corresponded to multiples of a theoretical subunit molecular weight of 17 500 daltons. Binding studies performed by equilibrium dialysis and ultracentrifugation methods revealed that the enterotoxin bound very small amounts of SDS per gram of protein. The evidence presented indicates possible detergent induced structural alterations of the protein.