Identification of a prepore large-complex stage in the mechanism of action of Clostridium perfringens enterotoxin

Abstract

Clostridium perfringens enterotoxin (CPE) is the etiological agent of the third most common food-borne illness in the United States. The enteropathogenic effects of CPE result from formation of large CPE-containing complexes in eukaryotic cell membranes. Formation of these approximately 155- and approximately 200-kDa complexes coincides with plasma membrane permeability changes in eukaryotic cells, causing a Ca2+ influx that drives cell death pathways. CPE contains a stretch of amino acids (residues 81 to 106) that alternates markedly in side chain polarity (a pattern shared by the transmembrane domains of the beta-barrel pore-forming toxin family). The goal of this study, therefore, was to investigate whether this CPE region is involved in pore formation. Complete deletion of the CPE region from 81 to 106 produced a CPE variant that was noncytotoxic for Caco-2 cells and was unable to form CPE pores. However, this variant maintained the ability to form the approximately 155-kDa large complex. This large complex appears to be a prepore present on the plasma membrane surface since it showed greater susceptibility to proteases, increased complex instability, and a higher degree of dissociation from membranes compared to the large complex formed by recombinant CPE. When a D48A mutation was engineered into this prepore-forming CPE variant, the resultant variant was unable to form any prepore approximately 155-kDa large complex. Collectively these findings reveal a new step in CPE action, whereby receptor binding is followed by formation of a prepore large complex, which then inserts into membranes to form a pore.

FIG. 1.

Functional map of CPE. Previous analysis of CPE identified two major functional regions (10, 17). Within the 5-amino-acid core cytotoxicity sequence at CPE's N terminus, the aspartic acid residue at position 48 (inverted triangle) is essential for cytotoxicity (32). At the C terminus, binding activity maps to the last 30 amino acids. The putative TMD examined in this study exists between residues 81 to 106 and contains alternating hydrophobic (asterisks) and hydrophilic amino acids.

FIG. 2.

Constructs used in the present study. (A) Each of the four constructs in this study contains an N-terminal His₆ for affinity enrichment from E. coli lysates. The D48A construct is a single point mutant generated previously (32). TM1 and TM1-D48A both have an internal deletion of 25 amino acids corresponding to the putative TMD; the TM1-D48A construct also contains the D48A point mutation described above. (B) Affinity-enriched samples (100 ng) of affinity enrichments of these constructs were electrophoresed on an SDS-containing 12% acrylamide gel and Western blotted with rabbit polyclonal anti-CPE antiserum to assess their stability. Arrows to the left of the blot represent the migration of molecular mass standards run with these samples.

FIG. 3.

Caco-2 morphological damage assay. Constructs were initially screened for cytotoxic activity by treating confluent Caco-2 cell monolayers with 2.5 μg/ml of each toxin for 60 min at 37°C. The control panel represents cells treated with HBSS²⁺ alone, while the pTrcHis panel depicts cells after treatment with a mock enrichment from E. coli transformed with an empty vector. After treatment, Caco-2 cells were photomicrographed at 200× total magnification.

FIG. 4.

⁸⁶Rubidium release from Caco-2 cells. Caco-2 cells grown to confluence in 24-well plates were radiolabeled with 4 μCi/well of ⁸⁶Rb and, after a washing, treated with the indicated concentration of toxin for 15 min at 37°C. After treatment, ⁸⁶Rb released into the culture supernatant was collected and gamma radiation was quantitated. Background release was corrected for by treating cells with HBSS²⁺ alone, and data were converted to percent maximal release (see Materials and Methods). All data points are the statistical means of three independent experiments, with error bars representing the standard deviations.

FIG. 5.

Formation of SDS-resistant large complexes in Caco-2 cells. To assay for large-complex formation in Caco-2 cells, cultures were harvested from 100-mm plates by gentle scraping and treated with 2.5 μg/ml of toxin for 45 min at 37°C with inversion. After a washing, the treated cells were lysed and Western blotted with rabbit polyclonal anti-CPE antiserum. Lanes labeled “Cells only” and “pTrcHis” represent cells treated with Hanks' balanced salt solution without Ca²⁺ or Mg²⁺ alone or a mock enrichment from empty vector transformants, respectively. Arrows to the left of the blot represent the migration of molecular mass standards run with these samples.

FIG. 6.

Pronase resistance of the CPE large complex. Isolated rabbit BBMs were treated with either rCPE or TM1 for 20 min and then washed to remove unbound toxin. Membranes were treated with the indicated concentrations of pronase and incubated at 4°C for the denoted amounts of time. After digestion, membranes were pelleted and resuspended in Laemmli buffer before separation and Western blotting with rabbit polyclonal anti-CPE antiserum. Arrows to the left of the blot represent the migration of molecular mass standards run with these samples.

FIG. 7.

Dissociation of the large complex from membranes. BBMs were treated with rCPE or TM1 (2.5 μg/ml) for 20 min at 37°C to allow formation of the ∼155-kDa large complex. After a washing, the BBMs were incubated in PBS-PI at 4°C for the indicated periods of time (numbers at top of gel). After microcentrifugation, samples from both the supernatant and pellet were Western blotted with rabbit polyclonal anti-CPE antiserum. P, samples from the pellet; S, samples from the supernatants of the microcentrifugation; OE, overnight exposures of lanes containing the 20-h supernatant for either rCPE- or TM1-treated BBMs. Arrows to the left of the blot represent the migration of molecular mass standards run with these samples, along with the migration of free rCPE and rCPE-treated BBMs.

FIG. 8.

Heat denaturation of extracted large complexes. Heat sensitivity of the large complexes was assayed by treating BBMs with rCPE or TM1 (2.5 μg/ml) for 20 min at 37°C to allow formation of the ∼155-kDa large complex. After a washing, the BBMs were extracted with Laemmli buffer and heated at 90°C for the indicated time periods. After heat denaturation, samples were Western blotted with rabbit polyclonal anti-CPE antiserum. Arrows to the left of the blot represent the migration of molecular mass standards run with these samples, along with the migration of free rCPE.

FIG. 9.

Updated model of CPE action. Stage 1 is binding. CPE action begins when CPE binds certain claudins, transmembrane tight junction proteins, using sequences at the CPE C terminus. Stage 2 is prepore large-complex formation. After membrane localization via receptor binding, CPE joins with other proteins (and/or additional molecules of CPE) to form an SDS-resistant complex. Formation of this complex is dependent upon the presence of an intact N terminus, particularly the aspartic acid residue at position 48. However, without the presence of CPE's putative TMD, this complex is trapped at the prepore stage and is unable to resist degradation by proteases. Stage 3 is membrane insertion/pore formation. With an intact N terminus and putative TMD, the CPE large complex can undergo the conformational changes necessary for insertion into the membrane. This stage not only protects the complex from proteases but also allows the penetration of the phospholipid bilayer of enterocytes, causing the influx of Ca²⁺ into the cell. Check marks and “x” indicate rCPE species which can or cannot, respectively, undergo this stage in CPE action.