pH-enhanced cytopathic effects of Clostridium sordellii lethal toxin

Abstract

Clostridium sordellii lethal toxin (TcsL) is a large clostridial toxin (LCT) that glucosylates Ras, Rac, and Ral. TcsL differs from other LCTs because it modifies Ras, which does not cycle from cytosol to membrane. By using a suite of inhibitors, steps in cell entry by TcsL were dissected, and entry appears to be dependent on endosomal acidification. However, in contrast to TcdB, TcsL was substantially slower in its time course of entry. TcsL cytopathic effects (CPE) were blocked by bafilomycin A1 and neutralized by antiserum up to 2 h following treatment of cells with the toxin. The slow time course of intoxication and relatively high cytopathic dose were alleviated by exposing TcsL to acid pH, resulting in a time course similar to that of TcdB. The optimal pH range for activation was 4.0 to 5.0, which increased the rate of intoxication over 5-fold, lowered the minimal intoxicating dose by over 100-fold, and allowed complete substrate modification within 2 h, as shown by differential glucosylation. Fluorescence analysis of TcsL with 2-(p-toluidinyl) naphthalene-6-sulfonic acid as a probe suggested the acid pH stimulated a hydrophobic transition in the protein, a likely prelude to membrane insertion. Finally, acid entry by TcsL caused TcdB-like morphological changes in CHO cells, which suggesting that acid activation may impact substrate recognition profiles for TcsL.

FIG. 1

Time course of TcsL cytosolic entry. In a 96-well plate, CHO cells (5 × 10⁴ cells/well) were incubated with TcsL (1 pmol) in a final volume of 100 μl, and bafilomycin A1 (1 × 10⁻⁷ M) was added at 10-min intervals from 0 to 180 min. Each sample was tested in triplicate, and CPE were determined at 16 h. The error bars mark the standard deviation from the mean. Similar levels of inhibition were found in two subsequent repetitions of the same experiment. B, PBS control; LT, TcsL; I, bafilomycin.

FIG. 2

pH range of acid pulse-induced entry of TcsL. In a 96-well plate, CHO cells (5 × 10⁴ cells/well) were incubated with bafilomycin A1 (5 × 10⁻⁷ M) in a final volume of 100 μl for 30 min. The cells were then treated with TcsL (1 pmol) for 1 h at 37°C, subjected to a 10-min pH pulse across a range of pHs, and finally returned to pH 7.8 by addition of neutralized medium. Samples were observed for 11 h, and CPE were determined by visualization. Similar effects were determined for CHO, HeLa, and RAW cells. Curves: A, pH 4.0; B, pH 4.5; C, pH 5.0; D, TcsL (no pulse); E, pH 5.5; F, pH 6.0; G, pH 6.5; H, pH 7.0; I, pH 7.5.

FIG. 3

pH-enhanced rate of CHO cell intoxication by TcsL. In a 96-well plate, CHO cells (5 × 10⁴ cells/well) were treated with either 1 pmol of TcsL or acid-pulsed TcsL (pH 4.0) in the presence of bafilomycin A1 (5 × 10⁻⁷ M) in a final volume of 100 μl and then observed for 11 h. CPE were determined by visualization. Curves: A, acid-pulsed TcsL; B, TcsL. Similar rates were obtained for HeLa and RAW cells.

FIG. 4

TNS fluorescent analysis of TcsL. The analysis of pH-induced conformational changes in TcsL was carried out with the fluorescent probe TNS. Each spectrum represents the experimental sample with background (TNS and buffer alone) subtracted. For the pH shift condition, the toxin was treated with TNS at pH 7.5. The solution was then titrated to pH 4.0 by gradual addition of 1 N HCl and returned to neutrality by addition of 1 N NaOH. (A) TcsL hydrophobicity across a range of pHs. TcsL was incubated with TNS at pHs 4.0, 5.0, 6.0, 7.0, and 8.0, and the fluorescent spectrum of each sample was obtained. TNS fluorescence for samples above pH 5.0 were not above background levels. Emission from pHs 6.0, 7.0, and 8.0 were not detectable above background levels. (B) TNS analysis of TcsL hydrophobicity following pH shift.

FIG. 5

Time course of TcsL extracellular neutralization. In a 96-well plate, CHO cells (5 × 10⁴ cells/well) were treated with 1 pmol of TcdB, TcsL, or acid-pulsed TcsL in a final volume of 100 μl. At the indicated time points, cells were then subjected to neutralizing antiserum or wash treatments. (A) Treated cells were washed vigorously with PBS four times at the indicated time points. (B) Neutralizing antiserum (10 μl) was added to cells at the indicated time points. In both experiments, cells were then observed for 11 h, and CPE were determined by visualization. Curves: solid triangles, TcdB; solid circles, TcsL.

FIG. 6

SEM analysis of TcdB-, TcsL-, and acid-pulsed TcsL-treated CHO cells. CHO cells were grown on coverslips and treated with 1 pmol of TcdB, TcsL, or acid-pulsed TcsL in a final volume of 100 μl. After detection of changes in morphology, cells were fixed, dried, and mounted for SEM analysis and visualization. (A) PBS control (magnification, ×1,600). (B) TcsL (magnification, ×3,300). (C) TcdB (magnification, ×7,500). (D) Acid-pulsed TcsL (magnification, ×7,500).

FIG. 7

TcsL differential glucosylation of extracts from TcsL- and acid-pulsed TcsL-treated cells. Extracts from CHO cells that had been treated with 1 pmol of TcsL or acid-pulsed TcsL were used in the glucosylation assay to determine if pretreatment under these conditions blocks substrate. Glucosylation assays were carried out with [¹⁴C]UDP-glucose and TcsL with the extracts as target substrates. Incorporation of the radiolabeled glucose was determined by autoradiography. (A) Glucosylation of extracts from PBS-treated CHO cells. (B) Glucosylation of extracts from TcsL-treated CHO cells. (C) Glucosylation of extracts from TcsL-treated (acid pulsed; pH 4.0) CHO cells.