Abstract
The previous model for the action of Clostridium perfringens enterotoxin (CPE) proposed that (i) CPE binds to host cell receptor(s), forming a small ( approximately 90 kDa) complex, (ii) the small complex interacts with other eucaryotic protein(s), forming a large ( approximately 160 kDa) complex, and (iii) the large complex triggers massive permeability changes, thereby inducing enterocyte death. In the current study, Western immunoblot analysis demonstrated that CPE bound to CaCo-2 human intestinal cells at 37 degrees C forms multiple large complex species, with apparent sizes of approximately 200, approximately 155, and approximately 135 kDa. These immunoblot experiments also revealed that occludin, an approximately 65-kDa tight junction protein, is present in the approximately 200-kDa large complex but absent from the other large complex species. Immunoprecipitation studies confirmed that occludin physically associates with CPE in large complex material and also indicated that occludin is absent from small complex. These results strongly suggest that occludin becomes associated with CPE during formation of the approximately 200-kDa large complex. A postbinding association between CPE and occludin is consistent with the failure of rat fibroblast transfectants expressing occludin to bind CPE in the current study. Those occludin transfectants were also insensitive to CPE, strongly suggesting that occludin expression is not sufficient to confer CPE sensitivity. However, the occludin-containing, approximately 200-kDa large complex may contribute to CPE-induced cytotoxicity, because nontoxic CPE point mutants did not form any large complex species. By showing that large complex material is comprised of several species (one containing occludin), the current studies indicate that CPE action is more complicated than previously appreciated and also provide additional evidence for CPE interactions with tight junction proteins, which could be important for CPE-induced pathophysiology.
Highlights
Clostridium perfringens is a Gram-positive, endospore-forming, anaerobic bacterium that produces a plethora of protein toxins, including a 35-kDa single polypeptide named C. perfringens enterotoxin (CPE).1 Considerable experimental and epidemiologic evidence [1,2,3] implicates CPE as the virulence factor responsible for the diarrheal and cramping symptoms of several important human gastrointestinal illnesses, which include C. perfringens type A food poisoning, the second most commonly reported foodborne disease in the United States, and non-foodborne diarrheal illnesses, such as antibiotic-associated diarrhea and sporadic diarrhea
Results from that CPE Western immunoblot analysis indicated that SDS extracts from CaCo-2 cells treated with CPE at 37 °C contain significant levels of material that reacts with CPE antibodies and migrates more slowly than free CPE (Fig. 1A, compare lanes 1– 4 with CPE lane)
Results shown in lanes 6 –9 of the left and right panels of Fig. 6a (i) indicate that CPE antibodies, but not occludin antibodies, can immunoprecipitate small complex and (ii) confirm that intact 125I-CPE is present in the small complex that was immunoprecipitated by CPE antibodies
Summary
Described methods [27] were used to purify native CPE to homogeneity from C. perfringens strain NCTC 8239 and to assay the biological activity of the resultant purified toxin. Aliquots (2 mg) of the purified native CPE were radioiodinated as described previously [5], using lactoperoxidase-glucose oxidase (Bio-Rad) and 2 mCi of Na125I (17 mCi/mg; ICN radiochemicals). Using previously described assays [5], the radiolabeled CPE preparation was determined to retain binding and cytotoxic activity. Rabbit polyclonal antibody raised against a fusion protein consisting of the C-terminal 150 amino acids of human occludin fused to glutathionine S-transferase was purchased from Zymed Laboratories Inc. NRS IgG was purchased from Sigma. Acrylic beads containing immobilized protein A were purchased from Sigma
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