Technical Reference #1860

1860.

Rapid Cytopathic Effects of C. perfringens Beta-Toxin on Porcine Endothelial Cells. Corinne Gurtner; Francesca Popescu; Marianne Wyder; Esther Sutter; Friederike Zeeh; Joachim Frey; Conrad von Schubert; Horst Posthaus, University of Bern, Infection & Immunity, 78(1860), (2010)
Link To Paper

Abstract:
Clostridium perfringens type C isolates cause fatal segmental necro-hemorrhagic enteritis in animals and humans. Typically acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C -toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton cell border retraction and cell shrinkage leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

Keywords:
Clostridium perfringens type C; necrotizing enteritis; beta-toxin; endothelial 21 cell; vascular thrombosis.

Materials & Methods:
Confluent PAEC cultures in collagen coated glass bottom 6-well plates (MatTek) 19 and confluent porcine fibroblasts were incubated with SFM containing 5 μg/ml propidium 20 iodide (33) and 32 ng/ml rCPB or culture supernatants of JF 3721diluted to the same 21 concentration of CPB. Cells were placed on a TE2000E-PFS microscope (Nikon) 22 equipped with a CFI Plan Fluor ELWD 40x objective (Nikon) Orca-ER CCD camera 23 (Hamamatsu) and incubation chamber (Life Imaging Services) and recorded at 37°C and 24 5% CO2 for 20 h at 5 min intervals using NIS Elements Software (Nikon).

Microscopic Technique
Light and Fluorescent Microscopy

Cell Type(s)
PAEC cultures