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.
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Abbreviations
- TcdA:
-
Toxin A of Clostridium difficile
- TcdB:
-
Toxin B of Clostridium difficile
- EGCs:
-
Enteric glial cells
- ENS:
-
Enteric nervous system
- GDNF:
-
Glial cell-derived neurotrophic factor
- NGF:
-
Nerve growth factor
- TNF-α:
-
Tumour necrosis factor-alpha
- IFN-γ:
-
Interferon-gamma
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- FBS:
-
Foetal bovine serum
- PI:
-
Propidium iodide
- IL-1β :
-
Interleukin-1beta
- U:
-
Densitometric units
- CDKs:
-
Cyclin-dependent kinases
- Cdc2:
-
Cell division cycle 2
- GFAP:
-
Glial fibrillary acidic protein
- PARP:
-
Poly (ADP-ribose) polymerase
- BAF:
-
Boc-Asp(OMe)-fluoromethylketone
- Z-DEVD-FMK:
-
Z-Asp-Glu-Val-Asp-fluoromethylketone
- ROCK1:
-
Rho-associated coiled-coil containing kinase 1
- PAK1:
-
p21-activated kinase 1
- PI3K:
-
Phosphatidylinositide 3-kinase
- JNK:
-
c-Jun N-terminal kinase
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Acknowledgements
The authors thank Springer Nature Author Services for editing of English language. We are greatly indebted to Alfa Wasserman, Sofar, and Almirall companies for their generous unrestricted support. This work was supported by a Project from the Department of Experimental Medicine for Basic Research (bando 2014) awarded to Doctor Katia Fettucciari, a Grant from Fondazione Cassa di Risparmio di Perugia, Italy (bando 2015-2015.0327.021 Ricerca Scientifica e Tecnologica) to Professor Lanfranco Corazzi, and a grant from “AIGO fa RICERCA” awarded to Professor Gabrio Bassotti by the Italian Society of Hospital Gastroenterologists and Endoscopists (AIGO).
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Fettucciari, K., Ponsini, P., Gioè, D. et al. Enteric glial cells are susceptible to Clostridium difficile toxin B. Cell. Mol. Life Sci. 74, 1527–1551 (2017). https://doi.org/10.1007/s00018-016-2426-4
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DOI: https://doi.org/10.1007/s00018-016-2426-4