Studies of Clostridium perfringens enterotoxin action at different temperatures demonstrate a correlation between complex formation and cytotoxicity.

The cytotoxicity of Clostridium perfringens enterotoxin (CPE) was completely blocked in Vero cells continuously CPE treated at 4 degrees C. [125I]CPE-specific binding to either Vero cells or isolated rabbit intestinal brush border membranes (BBMs) was lower at 4 degrees C than at 24 or 37 degrees C, but reduced enterotoxin binding could not totally explain the loss of cytotoxicity at low temperature. Insertion of enterotoxin into Vero cell membranes or BBMs was temperature independent. However, CPE complex formation (A. P. Wnek and B. A. McClane, Infect. Immun. 57:574-581, 1989) in BBMs and Vero cells was blocked at 4 degrees C. When Vero cells were CPE treated at 4 degrees C, washed to remove unbound toxin, and then shifted to 37 degrees C, complex formation and cytotoxicity were rapidly detected. When CPE binding and complex formation were permitted for 2 min at 37 degrees C, and the Vero cells were then shifted to 4 degrees C, cytotoxicity was detectable at 4 degrees C. These results are consistent with complex formation, rather than complex activity, being the temperature-sensitive step in CPE action which is blocked at 4 degrees C. These studies demonstrate a strong correlation between complex formation and cytotoxicity and are consistent with complex involvement in CPE cytotoxicity. These studies also strongly suggest that CPE insertion precedes both complex formation and induction of small-molecule permeability changes.

The size and heterogeneity of the messenger RNA associated with 70 S monosomes from down-shifted Escherichia coli.

After an energy source shift-down, Escherichia coli accumulates 70 S ribosome-mRNA complexes ("70 S monosomes"). The monosome mRNA strands are predominantly primary transcription products with purine nucleoside 5'-triphosphate and 5'-diphosphate termini present at a 1:2 ratio. The number-average chain length is 564 +/- 30 nucleotides, indicating that the population represents primarily monocistronic mRNAs. Digestions with endonucleases and exonucleases indicate that the ribosomes lie near the 5' ends of the mRNA strands and that the majority of the mRNA strands contain 5'-proximal "leader" sequences (average 10 nucleotides) outside the protective boundary of the ribosome. These data are consistent with the hypothesis that the increased functional stability of mRNA in down-shifted cells may result from protection by bound ribosomes of endonuclease-susceptible site(s) near the 5' ends of the mRNA strands.

Comparison of receptors for Clostridium perfringens type A and cholera enterotoxins in isolated rabbit intestinal brush border membranes.

The rabbit intestinal brush border membrane (BBM) receptors for Clostridium perfringens type A (CPE) and cholera (CT) enterotoxins were compared. Initial studies characterized binding of 125I-CPE to isolated BBMs as specific, saturable, and irreversible. BBMs appear to contain a single type of CPE binding site. Protease pretreatment of BBMs strongly reduced subsequent specific binding of 125I-CPE but not 125I-CT, while neuraminidase pretreatment had little effect on binding of either enterotoxin. Proteases did not significantly release pre-bound 125I-CPE. Preincubation of CPE with an affinity-purified preparation containing a previously identified (Biochem. Biophys. Res. Commun. 112, 1099-105) CPE-binding protein resulted in reduced specific binding of 125I-CPE and an inhibition of CPE biologic activity. Similar experiments showed that CPE-binding protein had no effect on CT binding or biologic activity. Gangliosides had no significant effect on specific binding or biologic activity of CPE but reduced CT binding and biologic activity. Lipids, including gangliosides, separated by thin layer chromatography specifically bound CT but not CPE. Preincubation of BBMs with CT did not reduce subsequent binding of 125I-CPE; conversely, prebound CPE did not affect subsequent 125I-CT binding. These results strongly suggest that CPE does not share the CT BBM receptor ganglioside GM1, and support a role for the CPE-binding protein in CPE binding.

Preliminary evidence that Clostridium perfringens type A enterotoxin is present in a 160,000-Mr complex in mammalian membranes.

Clostridium perfringens type A 125I-enterotoxin (125I-CPE) was bound to rabbit intestinal brush border membranes (BBMs) or Vero cells and then solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS). Solubilized radioactivity was analyzed by gel filtration chromatography on a Sepharose 4B column or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without sample boiling and autoradiography. Specifically bound 125I-CPE extracted from either BBMs or Vero cells was primarily associated with a complex of approximately 160,000 Mr. The CPE complex was partially purified by gel filtration or SDS-PAGE without sample boiling. SDS-PAGE analysis with sample boiling of the partially purified 125I-CPE complex from Vero cells or BBMs suggested that CPE complex contains both a 50,000-Mr protein and a 70,000-Mr protein in approximately equimolar amounts. This result is supported by affinity chromatography with CPE immobilized on Sepharose 4B, which showed the specific interaction of similar size proteins with CPE. The simplest explanation for these results is that CPE (Mr 35,000) interacts with 50,000-Mr and 70,000-Mr eucaryotic proteins to form a membrane-dependent complex of approximately 160,000 Mr. These results suggest that the receptor or target site(s) or both for CPE are similar in both BBMs and Vero cells. The significance of these findings in terms of CPE binding, insertion, and biologic action is discussed.

Identification of a 50,000 Mr protein from rabbit brush border membranes that binds Clostridium perfringens enterotoxin.

A protein that binds Clostridium perfringens enterotoxin was extracted with NP-40 from rabbit intestinal brush border membranes. This protein was partially purified by affinity chromatography on enterotoxin-coupled CNBr-activated Sepharose 4B. The molecular weight of this protein was approximately 50,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity-purified samples containing this protein specifically inhibited biological activity of the enterotoxin on Vero (African green monkey kidney) cells. These studies suggest that this protein may be involved in the binding of the enterotoxin to rabbit intestinal epithelial cells.

Clostridium perfringens enterotoxin.

Current knowledge of CPE action is briefly summarized in Figure 1. After specific binding to a protein receptor(s), the entire CPE molecule rapidly inserts into membranes forming a complex of 150,000 Mr. Almost simultaneously with insertion, there is a sudden change in ion fluxes. The molecular events behind the induction of ion flux changes remain undefined, but might involve either direct formation of membrane pores by CPE or activation of pre-existing membrane pores. As intracellular ion levels change, cellular metabolism is affected and processes such as macromolecular syntheses are inhibited. One of the ion flux effects resulting from CPE treatment involves increased Ca2+ influx; as more Ca2+ enters the cell, morphologic damage and permeability alterations for larger molecules occur. It remains to be determined if both morphologic damage and larger permeability alterations are necessarily linked but, for example, it could be envisioned that CPE-induced Ca2+ influx causes a cytoskeletal collapse leading to altered membrane permeability. The cytoskeleton has been shown to be sensitive to intracellular Ca2+ levels and is important in normal membrane structure/function relationships. As the cumulative effects of CPE inhibit cellular metabolism, cell death occurs. The precise irreversible CPE lethal action still must be identified. As CPE-treated intestinal epithelial cells die in vivo, histopathologic damage appears. This damage results in loss of normal intestinal function causing secretion of fluids and electrolytes. This effect is clinically manifested as diarrhea. The strongly cytotoxic action of CPE clearly distinguished the action enterotoxin from STa or CT.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between translational initiation and messenger RNA inactivation in down-shifted Escherichia coli.

The parameters of protein synthesis and functional inactivation of global messenger RNA (mRNA) were examined in a Tic+ strain of Escherichia coli during the 30-min period following a shift-down from glucose-minimal to succinate-minimal medium. The rate of mRNA inactivation and the relative translational initiation frequency were both most severely depressed immediately after the shift-down and increased slowly thereafter. If glucose was restored to the medium at any time after shift-down, mRNA inactivation immediately resumed its normal (preshift) rate and the protein-forming capacity was increased. These changes in mRNA inactivation rate do not reflect an altered mRNA composition in the down-shifted cells. The relative rate of mRNA inactivation was linearly proportional to the relative translational initiation frequency over a 10-fold range of initiation frequencies. Low initiation frequencies represent increased "dwell" of the ribosomes at the initiation site before the commencement of polypeptide chain initiation. We propose that initiating ribosomes protect mRNA from an inactivating endonucleolytic cleavage at or near the ribosome binding site.

Interferon pretreatment enhances the sensitivity of Vero cells to Clostridium perfringens type A enterotoxin.

Treatment of Vero (African green monkey kidney) cells with interferon (IFN) before the addition of Clostridium perfringens type A enterotoxin (CPE) significantly increased the sensitivity of these cells to CPE. IFN pretreatment caused a subsequent two- to four-fold increase in CPE-induced membrane permeability alterations and also decreased the time of CPE treatment required before the onset of permeability alterations and morphologic damage. Enhancement of CPE activity was dependent on the amount of IFN added during pretreatment and on the duration of IFN pretreatment incubations. Potentiation of CPE activity was observed following pretreatment of Vero cells with natural human IFN-alpha or IFN-gamma or Roferon recombinant human IFN-alpha. However, pretreatment with mouse IFN did not affect CPE activity. IFN pretreatment did not grossly enlarge the size of the functional hole produced in plasma membranes by CPE. IFN pretreatment of Vero cells slightly increased CPE specific binding, but this effect occurred kinetically after the enhancement of CPE toxicity. These results suggest that IFN pretreatment enhances the action of CPE on IFN pretreated Vero cells by increasing the sensitivity of these cells to the action of CPE rather than by increasing CPE specific binding or by directly activating the CPE molecule. Additional studies are required to further clarify the mechanism by which IFN sensitized Vero cells to CPE.

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.

Molecular cloning of the 3' half of the Clostridium perfringens enterotoxin gene and demonstration that this region encodes receptor-binding activity.

Clostridium perfringens type A enterotoxin (CPE) causes the symptoms associated with C. perfringens food poisoning. To determine whether the C-terminal half of CPE contains receptor-binding activity, the 3' half of the cpe structural gene was cloned with an Escherichia coli expression vector system. E. coli lysates containing the expressed C-terminal CPE fragment (CPEfrag) were then assayed for CPE-like serologic, receptor-binding, and cytotoxic activities. CPEfrag was shown to contain an epitope located at or near the receptor-binding domain of the CPE molecule. Competitive-binding studies showed specific competition for CPE receptors between CPE and CPEfrag lysates. CPEfrag lysates did not cause cytotoxicity in Vero (African green monkey kidney) cells. However, preincubation with CPEfrag lysates specifically protected Vero cells from subsequent CPE challenge. This indicates that CPEfrag recognizes the physiologic receptor which mediates CPE cytotoxicity. Collectively, these studies indicate that the C-terminal half of CPE contains a receptor-binding domain but additional amino acid sequences appear to be required for CPE cytotoxicity.

Evidence that an approximately 50-kDa mammalian plasma membrane protein with receptor-like properties mediates the amphiphilicity of specifically bound Clostridium perfringens enterotoxin.

Previous studies suggest that Clostridium perfringens enterotoxin (CPE) inserts into mammalian membranes. Using Triton X-114 phase separation analysis and charge-shift electrophoresis, this study demonstrates that CPE exhibits the amphiphilicity required for membrane insertion, but this behavior develops only after exposure of CPE to membranes. This effect does not require proteolytic or covalent CPE modifications or formation of a previously reported 160-kDa CPE-containing complex. A novel 90-kDa CPE-containing complex with amphiphilic properties was detected in intestinal brush-border membranes and in CPE-sensitive, but not CPE-insensitive, cell lines using nondenaturing Triton X-100 electrophoresis. Immunoprecipitation analysis suggested that the 90-kDa complex is composed of CPE and a 45-50-kDa membrane protein. Since the 90-kDa complex is formed only in cells that bind and respond to CPE, these results are consistent with the 45-50-kDa protein mediating CPE amphiphilicity and serving as a functional CPE receptor. A four-step model for CPE action is proposed. 1) CPE binds to the 45-50-kDa protein to form a 90-kDa complex. 2) The 90-kDa complex undergoes some physical change corresponding to insertion or a conformational change. 3) The 90-kDa complex and a 70-kDa membrane protein interact to form a 160-kDa complex. 4) Formation of the 160-kDa complex leads to permeability alterations.

Production and characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin.

Hybridomas secreting monoclonal antibodies (MABs) specific for Clostridium perfringens type A enterotoxin were produced by fusion of P3X63Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with purified enterotoxin. Wells containing hybridomas secreting immunoglobulin G (IgG) antibodies against enterotoxin were specifically identified by an indirect enzyme-linked immunosorbent assay (ELISA), and 10 ELISA-positive hybridomas were selected and cloned twice by limiting dilution. All 10 hybridomas produced MABs containing immunoglobulin G1 heavy chains and kappa (kappa) light chains. These hybridomas were then grown as ascitic tumors in mice, and MABs were purified from the ascites fluids with DEAE Affi-gel blue. The specificity of the MABs for enterotoxin was demonstrated by immunoblotting and ELISA. Competitive radioimmunoassay with 125I-MABs suggests that these MABs recognized at least four epitopes on the enterotoxin molecule. The enterotoxin-neutralizing ability of MABs from both hybridoma culture supernatants and ascites fluids was assessed by using a 3H-nucleotide-release Vero (African green monkey kidney) cell assay. Only 2 of the 10 hybridomas produced MABs which completely (greater than 90%) neutralized the biologic activity of enterotoxin. Preincubation of 125I-enterotoxin with MABs demonstrated that MAB neutralizing ability correlated with MAB-specific inhibition of specific binding of enterotoxin to intestinal brush border membranes.

Divalent cation involvement in the action of Clostridium perfringens type A enterotoxin. Early events in enterotoxin action are divalent cation-independent.

Clostridium perfringens type A enterotoxin (CPE) is a membrane-active cytotoxin. There are a number of recognized early steps in CPE cytotoxicity including binding of CPE to a protein receptor, insertion of CPE into membranes, and CPE-mediated induction of changes in membrane permeability for small molecules such as ions and amino acids. Further support for the existence of these early steps and further characterization of these events are presented in this report. We now report that these early steps in CPE action are largely independent of extracellular divalent cations. It is also shown that 3H-nucleotide release, known to be a later CPE effect, is Ca2+-dependent. A model for CPE cytotoxicity is suggested involving CPE action as a two-step process with Ca2+-independent early steps and Ca2+-dependent late steps.