A synthetic peptide corresponding to the extracellular loop 2 region of claudin-4 protects against Clostridium perfringens enterotoxin in vitro and in vivo.

Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since rclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

Development and application of a mouse intestinal loop model to study the in vivo action of Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of C. perfringens type A food poisoning, the second most commonly identified bacterial food-borne illness in the United States. CPE is produced by sporulating C. perfringens cells in the small intestinal lumen, where it then causes epithelial cell damage and villous blunting that leads to diarrhea and cramping. Those effects are typically self-limiting; however, severe outbreaks of this food poisoning, particularly two occurring in psychiatric institutions, have involved deaths. Since animal models are currently limited for the study of the CPE action, a mouse ligated intestinal loop model was developed. With this model, significant lethality was observed after 2 h in loops receiving an inoculum of 100 or 200 µg of CPE but not using a 50-µg toxin inoculum. A correlation was noted between the overall intestinal histological damage and lethality in mice. Serum analysis revealed a dose-dependent increase in serum CPE and potassium levels. CPE binding to the liver and kidney was detected, along with elevated levels of potassium in the serum. These data suggest that CPE can be absorbed from the intestine into the circulation, followed by the binding of the toxin to internal organs to induce potassium leakage, which can cause death. Finally, CPE pore complexes similar to those formed in tissue culture cells were detected in the intestine and liver, suggesting that (i) CPE actions are similar in vivo and in vitro and (ii) CPE-induced potassium release into blood may result from CPE pore formation in internal organs such as the liver.

Identification of a claudin-4 residue important for mediating the host cell binding and action of Clostridium perfringens enterotoxin.

The 24-member claudin protein family plays a key role in maintaining the normal structure and function of epithelial tight junctions. Previous studies with fibroblast transfectants and naturally sensitive Caco-2 cells have also implicated certain claudins (e.g., Claudin-4) as receptors for Clostridium perfringens enterotoxin (CPE). The present study first provided evidence that the second extracellular loop (ECL-2) of claudins is specifically important for mediating the host cell binding and cytotoxicity of native CPE. Rat fibroblast transfectants expressing a Claudin-4 chimera, where the natural ECL-2 was replaced by ECL-2 from Claudin-2, exhibited no CPE-induced cytotoxicity. Conversely, CPE bound to, and killed, CPE-treated transfectants expressing a Claudin-2 chimera with a substituted ECL-2 from Claudin-4. Site-directed mutagenesis was then used to alter an ECL-2 residue that invariably aligns as N in claudins known to bind native CPE but as D or S in claudins that cannot bind CPE. Transfectants expressing a Claudin-4(N149D) mutant lost the ability to bind or respond to CPE, while transfectants expressing a Claudin-1 mutant with the corresponding ECL-2 residue changed from D to N acquired CPE binding and sensitivity. Identifying carriage of this N residue in ECL-2 as being important for native CPE binding helps to explain why only certain claudins can serve as CPE receptors. Finally, preincubating CPE with soluble recombinant Claudin-4, or Claudin-4 fragments containing ECL-2 specifically blocked the cytotoxicity on Caco-2 cells. This result opens the possibility of using receptor claudins as therapeutic decoys to ameliorate CPE-mediated intestinal disease.

Noncytotoxic Clostridium perfringens enterotoxin (CPE) variants localize CPE intestinal binding and demonstrate a relationship between CPE-induced cytotoxicity and enterotoxicity.

Clostridium perfringens enterotoxin (CPE) causes the symptoms of a very common food poisoning. To assess whether CPE-induced cytotoxicity is necessary for enterotoxicity, a rabbit ileal loop model was used to compare the in vivo effects of native CPE or recombinant CPE (rCPE), both of which are cytotoxic, with those of the noncytotoxic rCPE variants rCPE D48A and rCPE(168-319). Both CPE and rCPE elicited significant fluid accumulation in rabbit ileal loops, along with severe mucosal damage that starts at villus tips and then progressively affects the entire villus, including necrosis of epithelium and lamina propria, villus blunting and fusion, and transmural edema and hemorrhage. Similar treatment of ileal loops with either of the noncytotoxic rCPE variants produced no visible histologic damage or fluid transport changes. Immunohistochemistry revealed strong CPE or rCPE(168-319) binding to villus tips, which correlated with the abundant presence of claudin-4, a known CPE receptor, in this villus region. These results support (i) cytotoxicity being necessary for CPE-induced enterotoxicity, (ii) the CPE sensitivity of villus tips being at least partially attributable to the abundant presence of receptors in this villus region, and (iii) claudin-4 being an important intestinal receptor for CPE. Finally, rCPE(168-319) was able to partially inhibit CPE-induced histologic damage, suggesting that noncytotoxic rCPE variants might be useful for protecting against some intestinal effects of CPE.

Interactions between Clostridium perfringens enterotoxin and claudins.

Clostridium perfringens enterotoxin (CPE), a single polypeptide of approximately 35 kDa in size, is -associated with type A food poisoning and such non-foodborne gastrointestinal diseases as antibiotic-associated diarrhea and sporadic diarrhea. CPE action begins with binding of the toxin to a claudin -receptor, forming a ∼90 kDa small complex that then rapidly oligomerizes into a hexamer of ∼450 kDa termed CH-1 (CPE hexamer-1). CH-1 is essentially a pore through which calcium gains entry to the cytoplasm, altering cell permeability and resulting in cell death by oncosis or apoptosis. Additionally, tight junctions are disrupted, allowing CPE access to the basolateral membrane so it can produce additional CH-1 -complexes and also the CH-2 complex (∼600 kDa) that contains occludin. We have recently demonstrated the presence of claudins-3 and -4 in both the CH-1 and CH-2 CPE complexes formed after CPE treatment naturally sensitive Caco-2 cells. Interestingly, claudin-1, which binds CPE poorly (if at all), was also present in these complexes.

Evidence for a prepore stage in the action of Clostridium perfringens epsilon toxin.

Clostridium perfringens epsilon toxin (ETX) rapidly kills MDCK II cells at 37°C, but not 4°C. The current study shows that, in MDCK II cells, ETX binds and forms an oligomeric complex equally well at 37°C and 4°C but only forms a pore at 37°C. However, the complex formed in MDCK cells treated with ETX at 4°C has the potential to form an active pore, since shifting those cells to 37°C results in rapid cytotoxicity. Those results suggested that the block in pore formation at 4°C involves temperature-related trapping of ETX in a prepore intermediate on the MDCK II cell plasma membrane surface. Evidence supporting this hypothesis was obtained when the ETX complex in MDCK II cells was shown to be more susceptible to pronase degradation when formed at 4°C vs. 37°C; this result is consistent with ETX complex formed at 4°C remaining present in an exposed prepore on the membrane surface, while the ETX prepore complex formed at 37°C is unaccessible to pronase because it has inserted into the plasma membrane to form an active pore. In addition, the ETX complex rapidly dissociated from MDCK II cells at 4°C, but not 37°C; this result is consistent with the ETX complex being resistant to dissociation at 37°C because it has inserted into membranes, while the ETX prepore readily dissociates from cells at 4°C because it remains on the membrane surface. These results support the identification of a prepore stage in ETX action and suggest a revised model for ETX cytotoxicity, i) ETX binds to an unidentified receptor, ii) ETX oligomerizes into a prepore on the membrane surface, and iii) the prepore inserts into membranes, in a temperature-sensitive manner, to form an active pore.

Compositional and stoichiometric analysis of Clostridium perfringens enterotoxin complexes in Caco-2 cells and claudin 4 fibroblast transfectants.

Clostridium perfringens enterotoxin (CPE) binds to host cell receptors, forming a small complex precursor for two large complexes reportedly having molecular masses of approximately 155 or approximately 200 kDa. Formation of the approximately 155 kDa complex causes a Ca(2+) influx that leads to apoptosis or oncosis. CPE complex composition is currently poorly understood, although occludin was identified in the approximately 200 kDa complex. The current study used heteromer gel shift analysis to show both CPE large complexes contain six CPE molecules. Ferguson plots and size exclusion chromatography re-sized the approximately 155 and approximately 200 kDa complexes as approximately 425-500 kDa and approximately 550-660 kDa respectively. Co-immunoprecipitation and electroelution studies demonstrated both CPE-binding and non-CPE-binding claudins are associated with all three CPE complexes in Caco-2 cells and with small complex and approximately 425-500 kDa complex of claudin 4 transfectants. Fibroblast transfectants expressing claudin 4 or C-terminal truncated claudin 4 were CPE-sensitive and formed the approximately 425 kDa complex, indicating claudin-induced cell signalling is not required for CPE action and that expression of a single receptor claudin suffices for approximately 425-500 kDa CPE complex formation. These results identify CPE as a unique toxin that combines with tight junction proteins to form high-molecular-mass hexameric pores and alter membrane permeability.

Identification of a putative azadirachtin-binding complex from Drosophila Kc167 cells.

Cell-proliferation in Drosophila Kc167 cells was inhibited by 50% when cell cultures contained 1.7 x 10(-7) M azadirachtin for 48 h (a tertranortriterpenoid from the neem tree Azadirachta indica). Drosophila Kc167 cells exhibited direct nuclear damage within 6-h exposure to azadirachtin (5 x 10(-7) M and above) or within 24 h when lower concentrations were used (1 x 10(-9) M). Fractionation of an extract of Drosophila Kc167 cells combined with ligand overlay technique resulted in the identification of a putative azadirachtin binding complex. Identification of the members of this complex by Peptide Mass Fingerprinting (PMF) and N-terminal sequencing identified heat shock protein 60 (hsp60) as one of its components.