Clostridium perfringens Epsilon-Toxin Impairs the Barrier Function in MDCK Cell Monolayers in a Ca2+-Dependent Manner
Masahiro Nagahama1, Soshi Seike2, Sadayuki Ochi3
1Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan.
Abstract:
Epsilon-toxin produced by Clostridium perfringens significantly contributes to the pathogeneses of enterotoxemia in ruminants and multiple sclerosis in humans. Epsilon-toxin forms a heptameric oligomer in the host cell membrane, promoting cell disruption. Here, we investigate the effect of epsilon-toxin on epithelial barrier functions. Epsilon-toxin impairs the barrier integrity of Madin-Darby Canine Kidney (MDCK) cells, as demonstrated by decreased transepithelial electrical resistance (TEER), increased paracellular flux marker permeability, and the decreased cellular localization of junctional proteins, such as occludin, ZO-1, and claudin-1. U73122, an endogenous phospholipase C (PLC) inhibitor, inhibited the decrease in TEER and the increase in the permeability of flux marker induced by epsilon-toxin. The application of epsilon-toxin to MDCK cells resulted in the biphasic formation of 1,2-diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). U73122 blocked the formation of DAG and IP3 induced by the toxin. Epsilon-toxin also specifically activated endogenous PLC-γ1. Epsilon-toxin dose-dependently increased the cytosolic calcium ion concentration ([Ca2+]i). The toxin-induced elevation of [Ca2+]i was inhibited by U73122. Cofilin is a key regulator of actin cytoskeleton turnover and tight-junction (TJ) permeability regulation. Epsilon-toxin caused cofilin dephosphorylation. These results demonstrate that epsilon-toxin induces Ca2+ influx through activating the phosphorylation of PLC-γ1 and then causes TJ opening accompanied by cofilin dephosphorylation.
Insights
Clostridium perfringens epsilon-toxin disrupts epithelial barrier function by activating phospholipase C (PLC) and increasing calcium ion concentration, leading to tight-junction opening. This mechanism is crucial for understanding toxin-induced diseases.
Area of Science:
- Microbiology
- Cell Biology
- Toxicology
Background:
- Epsilon-toxin from *Clostridium perfringens* is implicated in ruminant enterotoxemia and human multiple sclerosis.
- The toxin forms oligomers in cell membranes, leading to cell disruption.
Purpose of the Study:
- To investigate the impact of epsilon-toxin on epithelial barrier functions.
- To elucidate the molecular mechanisms underlying epsilon-toxin-induced epithelial barrier disruption.
Main Methods:
- Madin-Darby Canine Kidney (MDCK) cell model used to assess epithelial barrier integrity via transepithelial electrical resistance (TEER) and paracellular flux.
- Measurement of junctional protein localization, phospholipase C (PLC) activity, 1,2-diacylglycerol (DAG), inositol-1,4,5-triphosphate (IP3), and intracellular calcium ion concentration ([Ca2+]i).
- Analysis of cofilin phosphorylation status.
Main Results:
- Epsilon-toxin significantly impaired MDCK cell barrier integrity, reducing TEER and increasing permeability.
- The toxin induced biphasic formation of DAG and IP3, activated PLC-γ1, and increased [Ca2+]i, effects inhibited by U73122.
- Epsilon-toxin treatment led to cofilin dephosphorylation, indicating actin cytoskeleton disruption.
Conclusions:
- Epsilon-toxin compromises epithelial barrier function by activating PLC-γ1 and inducing calcium influx.
- The toxin-mediated tight-junction opening is associated with cofilin dephosphorylation and actin cytoskeleton alterations.
- Understanding this mechanism provides insights into epsilon-toxin pathogenesis in various diseases.
