Enteric glial cells are susceptible to Clostridium difficile toxin B

Abstract

Clostridium difficile causes nosocomial/antibiotic-associated diarrhoea and pseudomembranous colitis. The major virulence factors are toxin A and toxin B (TcdB), which inactivate GTPases by monoglucosylation, leading to cytopathic (cytoskeleton alteration, cell rounding) and cytotoxic effects (cell-cycle arrest, apoptosis). C. difficile toxins breaching the intestinal epithelial barrier can act on underlying cells, enterocytes, colonocytes, and enteric neurons, as described in vitro and in vivo, but until now no data have been available on enteric glial cell (EGC) susceptibility. EGCs are crucial for regulating the enteric nervous system, gut homeostasis, the immune and inflammatory responses, and digestive and extradigestive diseases. Therefore, we evaluated the effects of C. difficile TcdB in EGCs. Rat-transformed EGCs were treated with TcdB at 0.1-10 ng/ml for 1.5-48 h, and several parameters were analysed. TcdB induces the following in EGCs: (1) early cell rounding with Rac1 glucosylation; (2) early G2/M cell-cycle arrest by cyclin B1/Cdc2 complex inactivation caused by p27 upregulation, the downregulation of cyclin B1 and Cdc2 phosphorylated at Thr161 and Tyr15; and (3) apoptosis by a caspase-dependent but mitochondria-independent pathway. Most importantly, the stimulation of EGCs with TNF-α plus IFN-γ before, concomitantly or after TcdB treatment strongly increased TcdB-induced apoptosis. Furthermore, EGCs that survived the cytotoxic effect of TcdB did not recover completely and showed not only persistent Rac1 glucosylation, cell-cycle arrest and low apoptosis but also increased production of glial cell-derived neurotrophic factor, suggesting self-rescuing mechanisms. In conclusion, the high susceptibility of EGCs to TcdB in vitro, the increased sensitivity to inflammatory cytokines related to apoptosis and the persistence of altered functions in surviving cells suggest an important in vivo role of EGCs in the pathogenesis of C. difficile infection.

Enteric glial cells counteract Clostridium difficile Toxin B through a NADPH oxidase/ROS/JNK/caspase-3 axis, without involving mitochondrial pathways

“…Since we observed previously that TcdB induced apoptosis in EGCs17, and confirmed here apoptosis already at 18-hour treatment (Supplementary Fig. S1), we investigated whether ROS act as an upstream signal in this process.…”

“…JNK interacts with different pro- and anti-apoptotic proteins20 of Bcl-2 family members. We reported previously that TcdB did not induce changes in the expression/activation of Bax, Bak, Bcl-2, and Bcl-X L 17. One potential target of JNK is Bim.…”

“…In this context, the protective role of EGCs is essential. We found previously that TcdB exherts cytopathic and cytotoxic effect on EGCs, producing cell cycle arrest and apoptosis, and increases cell sensitivity to inflammatory cytokines17. To extend this study, we investigated the molecular mechanisms activated by EGCs as defense strategy in response to TcdB.…”

Clostridium difficile-related postinfectious IBS: a case of enteroglial microbiological stalking and/or the solution of a conundrum?

“…In fact, TcB in vitro caused in a dose-and time-dependent manner cytopathic and cytotoxic effects on EGC, correlated with Rac1 glucosylation, such as cytoskeleton disorganization, cell-cycle arrest, apoptosis, increased susceptibility to apoptosis induced by proinflammatory cytokines [54]. More importantly, in EGC surviving the cytotoxic effect of TcdB persistently impaired cell functions were observed, such as Rac1 glucosylation and cell-cycle arrest, even though there was evidence of a selfrescuing mechanism as suggested by an increased production of glial-derived neurotrophic factor [54]. Moreover, surviving EGC were more susceptible to TNFα and IFNγ action, which induces apoptosis to doses of cytokines not affecting control EGC.…”

“…Recently, we reported that the adverse effects of toxin B extend to EGC [54]. In fact, TcB in vitro caused in a dose-and time-dependent manner cytopathic and cytotoxic effects on EGC, correlated with Rac1 glucosylation, such as cytoskeleton disorganization, cell-cycle arrest, apoptosis, increased susceptibility to apoptosis induced by proinflammatory cytokines [54].…”