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Examination of toxicity of Clostridium perfringens -toxin in the MDCK cell line
C D Lindsay1, J L Hambrook, D G Upshall
1Chemical and Biological Defence Establishment, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
Abstract:
The epithelial Madin Darby canine kidney (MDCK) cell line was examined as a model to study the toxicity of Clostridium perfringens -toxin. The MDCK cell line was used because it is a monolayer cell line sensitive to -toxin. Using the neutral red (NR) retention assay (an indicator of lysosomal integrity), the concentration of toxin causing death in 50% of the cell population (LC(50)) was 900 pM, although this was found to vary between production batches. -Toxin was found to act rapidly but with a lag phase of 1 hr (NR assay). Pulsing the cultures with toxin (up to 4800 pM) indicated that the duration of exposure required to exert an effect was potentially very short (2.5 min). Increasing the duration of exposure beyond 3 hr did not decrease cell viability any further. Experiments with protease inhibitors failed to inactivate the toxin. Ethylenediaminetetraacetic acid (EDTA) was found to potentiate the lethality of the toxin by 90% This may be due to non-specific chaotropic effects such as membrane destabilization. By exposing cultures of MDCK cells to -toxin for a second time, resistance to the effects of the toxin was increased by 43%. The factor(s) controlling resistance to the toxin may have a heritable component.
Insights
Clostridium perfringens alpha-toxin (toxin) rapidly damages epithelial cells, with toxicity influenced by production batches and EDTA. Pre-exposure can increase cell resistance to this potent toxin.
Area of Science:
- Cell Biology
- Toxicology
- Microbiology
Background:
- Clostridium perfringens alpha-toxin is a key virulence factor.
- Understanding its cellular mechanisms is crucial for developing countermeasures.
- Epithelial cells are primary targets for toxin-induced damage.
Purpose of the Study:
- To evaluate the Madin Darby canine kidney (MDCK) cell line as a model for alpha-toxin toxicity.
- To characterize the kinetics and factors influencing alpha-toxin's cytolytic effects.
- To investigate potential methods for mitigating alpha-toxin-induced cell damage.
Main Methods:
- Utilized the neutral red (NR) retention assay to assess lysosomal integrity and cell viability.
- Determined the lethal concentration 50% (LC50) of alpha-toxin.
- Investigated the effects of exposure duration, protease inhibitors, EDTA, and repeated toxin exposure on MDCK cells.
Main Results:
- The LC50 of alpha-toxin in MDCK cells was determined to be 900 pM, with batch variability observed.
- Alpha-toxin exhibited rapid action with a 1-hour lag phase, requiring only 2.5 minutes of exposure for a significant effect.
- Ethylenediaminetetraacetic acid (EDTA) potentiated toxin lethality by 90%, while repeated exposure increased cell resistance by 43%.
Conclusions:
- MDCK cells serve as a sensitive model for studying alpha-toxin cytotoxicity.
- Toxin exposure kinetics are rapid, and cell viability is not further reduced after 3 hours of exposure.
- EDTA may enhance toxicity through membrane destabilization, and acquired resistance suggests a heritable component.
