The chaperonin TRiC/CCT is essential for the action of bacterial glycosylating protein toxins like Clostridium difficile toxins A and B

Abstract

Various bacterial protein toxins, including Clostridium difficile toxins A (TcdA) and B (TcdB), attack intracellular target proteins of host cells by glucosylation. After receptor binding and endocytosis, the toxins are translocated into the cytosol, where they modify target proteins (e.g., Rho proteins). Here we report that the activity of translocated glucosylating toxins depends on the chaperonin TRiC/CCT. The chaperonin subunits CCT4/5 directly interact with the toxins and enhance the refolding and restoration of the glucosyltransferase activities of toxins after heat treatment. Knockdown of CCT5 by siRNA and HSF1A, an inhibitor of TRiC/CCT, blocks the cytotoxic effects of TcdA and TcdB. In contrast, HSP90, which is involved in the translocation and uptake of ADP ribosylating toxins, is not involved in uptake of the glucosylating toxins. We show that the actions of numerous glycosylating toxins from various toxin types and different species depend on TRiC/CCT. Our data indicate that the TRiC/CCT chaperonin system is specifically involved in toxin uptake and essential for the action of various glucosylating protein toxins acting intracellularly on target proteins.

Keywords: Clostridium difficile toxins; TRiC/CCT; chaperonin; glycosyltransferase toxins; toxin uptake.

Fig. 1.

CCT4/5 mediates recovery of TcdB^GT activity after heat treatment. (A) Western blot analysis of CCT5 pulldown experiments from HeLa cells using GST-TcdB^GT–, GST-TcsL^GT–, and GST-TcnA^GT–coupled beads. GST-coupled beads served as control. Bound CCT5 was detected by Western blot analysis using anti-CCT5 antibody. GST and GST-TcdB^GT were detected by anti-GST antibody. (B) Heat-treated TcdB^GT was incubated in the presence of CCT4/5 (100 nM), BSA (100 nM), or HSP90 (100 nM) in a buffer containing 0.5 mM ATP for 1 h at 30 °C. Subsequently, recovery of TcdB^GT activity was determined by glucosylation of GST-RhoA using UDP-[¹⁴C]glucose. (C) Quantification of the recovery of TcdB^GT by CCT4/5 shown in B. Error bars indicate ± SD (n = 3) (D) In vitro [¹⁴C]glucosylation of GST-RhoA by heat-treated TcdB^GT after the indicated recovery times in the presence of CCT4/5, BSA, HSP70, or HSP90. (E, Left) Autoradiograph and Coomassie gel of the [¹⁴C]glucosylation of GST-RhoA by TcdB^GT (200 nM) after heat treatment (48 °C, 15 min) of the toxin with ATP or ADP (0.5 mM each) in the presence of CCT4/5 (200 nM). (E, Right) Quantification of four experiments. Values are average ± SD. Student’s t test was applied for statistical comparisons. ***P < 0.001.

Fig. 2.

HSF1A inhibits TcdB-mediated intoxication of HeLa cells. (A) HeLa cells were pretreated with the indicated concentrations of HSF1A or DMSO as control for 1 h before intoxication with TcdB (5 pM). Pictures were taken after 90 min. (Scale bar: 100 µm.) (Insets) Magnifications of dotted areas. (B) Quantification of HeLa cell intoxication with TcdB (5 pM) after 90 min with pretreatment of DMSO or HSF1A (100 µM). The percentage of rounded cells per picture (>500 cells) is given as mean ± SD (n = 4). Student’s t test was applied for statistical comparison. ***P < 0.001. (C, Left) HeLa cells, pretreated for 1 h with DMSO (control) or HSF1A (100 µM), were incubated with or without TcdB (5 pM) for 60 or 90 min. Cell lysates were analyzed by Western blot with the specific anti-Rac1 antibody (MAB102), which does not recognize glucosylated Rac1. Anti-Rac1 (23A8) antibody served as input control. (C, Right) Quantification of three experiments. Values are average ± SD. Student’s t test was applied for statistical comparison. n.s., not significant (P > 0.05); *P < 0.05; **P < 0.01. (D, Left) CCT4/5-mediated recovery assay of heat-treated (48 °C) TcdB^GT with the addition of DMSO or HSF1A (50 or 100 µM). Shown is the [¹⁴C]glucosylation of Rac1. (D, Right) Quantification of four experiments. Values are average ± SD. Student’s t test was applied for statistical comparisons. ***P < 0.001.

Fig. 3.

HSF1A does not inhibit C. botulinum C2-mediated intoxication of HeLa cells. (A) HeLa cells were pretreated with 50 µM HSF1A or with 50 µM of the HSP90 inhibitor radicicol (Rad) for 1 h at 37 °C before intoxication. DMSO served as control. The cells were intoxicated with TcdB (5 pM) or C2 toxin (50 ng/mL C2I plus 100 ng/mL C2IIa). Pictures were taken after 100 min. (Scale bar: 100 µm.) (Insets) Magnifications of dotted areas. (B) The percentage of rounded cells per picture (>200 cells) is given as mean ± SD (n = 8). Student’s t test was applied for statistical comparison. ***P < 0.001; n.s., not significant (P > 0.05). (C) Autoradiograph and Western blot analysis of lysates of C2 toxin-intoxicated cells, treated with additional C2I (40 nM) and radiolabeled [³²P]NAD (1 mM) after 100 min of intoxication. (D) Quantification of the autoradiograph shown in C. Values are average ± SD (n = 3). Student’s t test was applied for statistical comparison. **P < 0.01; n.s., not significant (P > 0.05).

Fig. 4.

Knockdown of CCT5 via siRNA inhibits intoxication by TcdB but not by C2 toxin. (A) siRNA (NT, nontargeting siRNA; CCT5 siRNA)-treated HeLa cells were intoxicated with TcdB (1 pM) or C2 toxin (50 ng/mL C2I plus 100 ng/mL C2IIa). Pictures were taken after 150 min for TcdB and after 100 min for C2-intoxicated cells. (Scale bar: 100 µm.) (Insets) Magnifications of dotted areas. (B) Percentage of rounded cells per picture (>400 cells) is given as mean ± SD (n = 8). Student’s t test was applied for statistical comparisons. ***P < 0.001; n.s., not significant (P > 0.05). (C) Cell lysates of TcdB-treated HeLa cells were analyzed after the indicated incubation times (90 and 150 min) by Western blot with the specific anti-Rac1 antibody (MAB102), which does not recognize glucosylated Rac1. Anti-Rac1 (23A8) antibody served as input control, and anti-CCT5 antibody was used to confirm CCT5 knockdown. (D) Modification of C2 toxin-intoxicated cells was analyzed via ADP ribosylation of actin in cell lysates with additional C2I (40 nM) and radiolabeled [³²P]NAD (1 mM) after intoxication of intact cells for the indicated times. Shown is an autoradiograph of SDS/PAGE and a Western blot of the cell lysates with anti-CCT5 antibody to confirm CCT5 knockdown. (E) Quantification of the autoradiograph of the [³²P]ADP ribosylation of actin shown in D (n = 3). Student’s t test was applied for statistical comparison. n.s., not significant (P > 0.05).