Fine mapping of the N-terminal cytotoxicity region of Clostridium perfringens enterotoxin by site-directed mutagenesis

Abstract

Clostridium perfringens enterotoxin (CPE) has a unique mechanism of action that results in the formation of large, sodium dodecyl sulfate-resistant complexes involving tight junction proteins; those complexes then induce plasma membrane permeability alterations in host intestinal epithelial cells, leading to cell death and epithelial desquamation. Previous deletion and point mutational studies mapped CPE receptor binding activity to the toxin's extreme C terminus. Those earlier analyses also determined that an N-terminal CPE region between residues D45 and G53 is required for large complex formation and cytotoxicity. To more finely map this N-terminal cytotoxicity region, site-directed mutagenesis was performed with recombinant CPE (rCPE). Alanine-scanning mutagenesis produced one rCPE variant, D48A, that failed to form large complexes or induce cytotoxicity, despite having normal ability to bind and form the small complex. Two saturation variants, D48E and D48N, also had a phenotype resembling that of the D48A variant, indicating that both size and charge are important at CPE residue 48. Another alanine substitution rCPE variant, I51A, was highly attenuated for large complex formation and cytotoxicity, but rCPE saturation variants I51L and I51V displayed a normal large complex formation and cytotoxicity phenotype. Collectively, these mutagenesis results identify a core CPE sequence extending from residues G47 to I51 that directly participates in large complex formation and cytotoxicity.

FIG. 1.

Functional map of CPE. (A) A previous deletion mutagenesis study of CPE (19) identified two distinct functional domains, including (i) an ∼10-amino-acid region near the N terminus that is essential for cytotoxicity and large complex formation and (ii) a receptor binding region located at the extreme C terminus of the toxin. (B) The amino acid sequence of the N-terminal CPE 45-54 cytotoxicity region targeted for mutagenesis in this study.

FIG. 2.

Morphological damage of CaCo-2 cells by rCPE variants. To screen variant rCPE species for cytotoxicity, confluent CaCo-2 cultures were treated with 2.5 μg of the indicated rCPE species/ml for 60 min at 37°C. Treatment buffer alone was added to cells in the control panel, while buffer containing a mock affinity enrichment preparation of lysates from E. coli cells transformed with the pTrcHis A empty vector was used to treat cells in the vector panel. After treatment, cells were photomicrographed at 130× total magnification.

FIG. 3.

⁸⁶Rb release from CaCo-2 cells induced by rCPE variants. Confluent CaCo-2 cells in 24-well plates were labeled with 4 μCi of ⁸⁶Rb per well for 4 h and then treated with increasing concentrations of rCPE species. ⁸⁶Rb released into the culture medium after 15 min of rCPE treatment was collected. To correct for background release, the data were converted to the percentage of maximal ⁸⁶Rb release (see Materials and Methods). (A) Depicted are the following rCPE species: rCPE (♦), D45A (▴), D48A (○), I51A (□), and L52A (•). (B) ⁸⁶Rb release experiments with rCPE saturation variants with the following rCPE species: rCPE (♦), D48A (○), D48E (▵), D48N (⋄), I51A (□), I51L (•), and I51V (▪). All data points represent means of three independent experiments, and error bars represent the standard deviations.

FIG. 4.

Competitive binding of rCPE variants. BBMs were preincubated with increasing concentrations (0.01 to 50 μg/ml) of each rCPE species before the addition of [¹²⁵I]CPE (2.5 μg/ml). The amount of [¹²⁵I]CPE bound after treatment was quantified and converted to the percentage of total binding ([¹²⁵I]CPE bound to BBMs in absence of a competitor). rCPE species shown are as follows: rCPE (♦), D48A (○), D48E (▴), D48N (⋄), I51A (□), the previously described (18) binding-deficient rCPE random point variant W226Stop (▪), and a mock affinity enrichment preparation with lysates from E. coli cells transformed with the pTrcHis A empty vector (•). Data points represent the average of duplicate independent experiments run in triplicate, and error bars represent the standard deviations.

FIG. 5.

Small complex formation by rCPE variants. BBMs were treated at 4°C with 5 μg of rCPE species per sample; Triton X-100 extracts of these samples were then separated on a 6% acrylamide native gel. After electrotransfer, small complex formation by each rCPE variant was evaluated by Western immunoblotting with rabbit polyclonal anti-CPE antiserum preabsorbed with BBMs. The migration of 20 ng of rCPE (∼39 kDa) is denoted by the arrow labeled free rCPE, while the migration of the classic ∼90-kDa small complex is depicted with the small complex arrow. The untreated lane represents BBMs treated with PBS only, while the empty vector lane corresponds to BBMs treated with a mock affinity enrichment preparation with lysates from E. coli cells transformed with the pTrcHis A empty vector.

FIG. 6.

Large complex formation by rCPE variants. Confluent CaCo-2 cells harvested from 100-mm culture dishes were treated with 2.5 μg of each rCPE species/ml. After 45 min, the treated cells were extracted with 1% SDS and loaded onto a 4% acrylamide SDS-PAGE gel. Complexes were then electrotransferred and Western immunoblotted with rabbit polyclonal anti-CPE serum. “Cells only” and “empty vector” lanes represent, respectively, CaCo-2 cells treated with either HBSS or mock affinity enrichment preparations of lysates from E. coli cells transformed with pTrcHis A.

FIG. 7.

Assessment of gross conformational changes in rCPE variants. rCPE species (400 ng) were incubated with trypsin (1 ng) at 25°C for the time periods indicated below each lane. Digestions were stopped by the addition of Laemmli buffer and boiling for 5 min. Samples from each reaction mixture were loaded on 10% acrylamide gels and separated by SDS-PAGE, followed by Western blotting with rabbit anti-CPE polyclonal antibody. The blot depicted in this figure represents a trypsin digestion of rCPE, a nontoxic affinity enrichment preparation of W50A (W50A¹), a toxic affinity enrichment preparation of W50A (W50A²), and D48A (see Results for explanation of W50A activity variation).