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The development of tolerance to Clostridium perfringens type D epsilon-toxin in MDCK and G-402 cells
Dominic R Beal1, Richard W Titball, Christopher D Lindsay
1Biomedical Sciences Department, Dstl, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK.
Abstract:
The epithelial Madin Darby canine kidney (MDCK) cell line, Caucasian renal leiomyoblastoma (G-402) cells, human small airways epithelial (HSAE) cells, human bronchial epithelial (HBE) cells and human renal proximal tubule (HRPT) epithelial cells were examined for sensitivity to Clostridium perfringens biotype D epsilon-toxin. MDCK and G-402 cells were confirmed as being the only established cell lines that are sensitive to the toxin. HSAE, HBE and HRPT epithelial cells were only found to be sensitive to the toxin at concentrations of > 1 mg/ mL. Cultures of MDCK and G-402 cells, with increased resistance (tolerance) to the cytotoxic effects of epsilon-toxin, were developed by exposing these cultures to progressively higher concentrations of toxin. The greatest relative increase in tolerance to epsilon-toxin was developed in MDCK cells, in which the LC50 in control cultures was 2 microg/mL as determined by the MTS/PMS assay system; after selection for tolerance, this was raised to 100 microg/mL. This represents a 50-fold increase in tolerance as measured by this index. Using G-402 cells, it was possible to increase the LC50 by twofold from 290 to 590 microg/mL. Subsequent 2-D electrophoresis of membrane preparations from tolerant and control MDCK cells revealed that the expression of a discrete group of proteins found in control cells with a range of molecular weights from 32-36 kDa, all with acidic isoelectric points (IEPs), were either not expressed in epsilon-toxin tolerant cells or had undergone a shift in IEP to a more alkaline pH in tolerant cells. This suggests that epsilon-toxin lethality in MDCK cells may be mediated by membrane-located proteins. Their absence or alteration in toxin-resistant cells would, at least partly, explain the failure of most cell lines to demonstrate sensitivity to this toxin, despite being derived from tissues that are damaged by epsilon-toxin. This approach may have utility in the study of other toxin-cell interactions and could be used in the development of novel medical countermeasures by identifying cellular targets which mediate toxin lethality.
Insights
Clostridium perfringens epsilon-toxin primarily affects Madin Darby canine kidney (MDCK) and G-402 cells. Epsilon-toxin resistance in these cells is linked to altered expression of specific membrane proteins, suggesting their role in toxin-induced cell death.
Area of Science:
- Cell Biology
- Microbiology
- Toxicology
Background:
- Clostridium perfringens biotype D epsilon-toxin causes significant damage to various tissues.
- Understanding cell sensitivity and resistance mechanisms to epsilon-toxin is crucial for developing countermeasures.
- Previous studies have identified limited cell lines sensitive to epsilon-toxin.
Purpose of the Study:
- To determine the sensitivity of various epithelial cell lines to Clostridium perfringens epsilon-toxin.
- To develop epsilon-toxin-tolerant cell lines and investigate the underlying molecular changes.
- To identify cellular targets mediating epsilon-toxin lethality.
Main Methods:
- Exposure of Madin Darby canine kidney (MDCK), G-402, human small airways epithelial (HSAE), human bronchial epithelial (HBE), and human renal proximal tubule (HRPT) cells to epsilon-toxin.
- Development of toxin-tolerant cell lines through progressive exposure to increasing toxin concentrations.
- Quantification of cell viability using the MTS/PMS assay.
- Analysis of membrane protein expression in control and tolerant cells using 2-D electrophoresis.
Main Results:
- MDCK and G-402 cells were confirmed as the most sensitive cell lines to epsilon-toxin.
- HSAE, HBE, and HRPT cells showed sensitivity only at high toxin concentrations (> 1 mg/mL).
- MDCK cells developed a 50-fold increase in epsilon-toxin tolerance (LC50 increased from 2 to 100 µg/mL), while G-402 cells showed a twofold increase (LC50 from 290 to 590 µg/mL).
- 2-D electrophoresis revealed that a group of membrane proteins (32-36 kDa, acidic pI) present in sensitive MDCK cells were absent or altered in tolerant cells.
Conclusions:
- Epsilon-toxin lethality in MDCK cells is likely mediated by specific membrane-located proteins.
- The absence or alteration of these proteins in resistant cells explains their reduced sensitivity.
- This study provides a foundation for understanding toxin-cell interactions and developing novel medical countermeasures by identifying key cellular targets.
