Recombinant <italic>Clostridium difficile</italic> toxin B induces endoplasmic reticulum stress in mouse colonal carcinoma cells

Abstract

Clostridium difficile is the main cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans and animals. Its pathogenicity is primarily linked to the secretion of two exotoxins (TcdA and TcdB). Although great progress in the toxic mechanism of TcdA and TcdB has been achieved, there are many conflicting reports about the apoptotic mechanism. More importantly, apoptotic endoplasmic reticulum (ER) stress has been reported in cells treated with Shiga toxins-another kind of cytotoxins that can cause diarrhea and colitis. Herein we checked whether TcdB can induce ER stress. The results showed that recombinant TcdB (rTcdB) activated molecular markers of unfolded protein response, suggesting that rTcdB induced ER stress in CT26 cells. However, rTcdB did not induce the up-regulation of C/EBP homologous protein (CHOP), a classic mediator of apoptotic ER stress, but it activated the precursor of cysteine aspartic acid-specific protease 12 (caspase-12), a controversial mediator of apoptotic ER stress. Besides, glucosyltransferase activity-deficient mutant recombinant TcdB induced ER stress, though it has no cytotoxic or cytopathic effect on CT26 cells. Altogether, these data demonstrated that ER stress induced by rTcdB is glucosyltransferase-independent, indicating that ER stress induced by rTcdB is non-apoptotic. This work also offers us a new insight into the molecular mechanism of CHOP protein expression regulation and the role of CHOP expression in ER stress.

Salubrinal protects against toxin B-induced CT26 cell death

“…5B). Unexpectedly, the protein expression levels of CHOP in the CT26 cells exposed to TcdB only were significantly decreased at every time point, which was consistent with our previous work [30]. Furthermore, the addition of salubrinal did not alter the decrease of CHOP induced by TcdB.…”

“…Clostridium difficile TcdB is one of the major virulence factors of CDI and has been proved to induce apoptotic cell death through ER stress pathway in CT26 cell line according to our previous work [30].…”

“…Annexin V-FITC apoptosis detection and sub-G1 events determination were performed as previously described [30,34]. The results were analyzed by a flow cytometer (Beckman, Brea, USA) and Win-MDI software (Scripps Research Institute, Jupiter, USA).…”

“…These results demonstrated that the protective effect of salubrinal against TcdB-mediated cell apoptosis is associated with caspase-9-dependent apoptotic cell death pathway. Our group has previously revealed that TcdB can induce ER stress in CT26 cell line via PERK-eIF2α signaling pathway [30]. Based on the assumption that salubrinal could selectively elevate the phosphorylation level of eIF2α and permit cells recover from apoptosis caused by ER stress, whether 20 µM of salubrinal could affect the phosphorylation level of eIF2α in TcdB-intoxicated CT26 cells was assessed.…”

“…Until now, the reported roles of salubrinal include acceleration of bone wounds healing in mice [24], stimulation of osteoblast differentiation [25,26], attenuation of nuclear factor κB-mediated molecular signals [27], protection against cell apoptosis [28,29], and so on. Recently, upregulation of phosp-eIF2α was found in TcdB-exposed CT26 cell line (BALB/C mouse colon tumor cells) [30] and in mice cecum and colon tissues receiving C. difficile VPI10463 vegetative cells [31], which seemed to protect host cells and animals. These raise the prospect that salubrinal might have protective effect against TcdB-induced cell death.…”