Assessment of aspects of the toxicity of Clostridium perfringens E-toxin using the MDCK cell line

Abstract

1 The epithelial Madin Darby Canine Kidney (MDCK) cell line was used to study the toxicity of ∈-toxin from Clostridium perfringens. The epithelial MDCK cell line is known to be sensitive to ∈-toxin of Clostridium perfringens and to investigate its mechanism of action, the neutral red assay has been used to determine the viability of cultures of this cell line. 2 Comparison of the LC 50s obtained at 34°C and 0°C showed that the lethality of ∈-toxin was reduced by 18- fold at the lower temperature. The effect of tempera ture on ∈-toxin lethality is unlikely to be due to reductions in membrane fluidity for the addition of Ca2+ or Mg2+ (2 mM) to buffer containing toxin was without effect. Varying the pH of the toxin-containing buffer from 6.9 to 8.7 did not increase the lethality of the toxin, though the most acidic pH used (5.8) was found to potentiate its action on MDCK cells. 3 The effect of inhibiting endocytosis on the lethality ofe toxin was also investigated by incubating cultures of MDCK cells with and without sodium azide over a range of concentrations of toxin. The co-administra tion of sodium azide did not reduce the toxicity of ∈ toxin, suggesting that energy-dependent uptake pro cesses such as endocytosis were unlikely to be involved in its mechanism of action. The results are, however, consistent with known receptor-based me chanisms of uptake and with other mechanisms of internalisation across the plasma membrane. ∈-toxin thus interacts with cell surfaces by a temperature sensitive mechanism potentiated by low pH.