The development of tolerance to Clostridium perfringens type D o-toxin in MDCK and G-402 cells

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 o-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 o-toxin, were developed by exposing these cultures to progressively higher concentrations of toxin. The greatest relative increase in tolerance to o-toxin was developed in MDCK cells, in which the LC₅₀ in control cultures was 2mg/mL as determined by the MTS/PMS assay system; after selection for tolerance, this was raised to 100 mg/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 mg/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 o-toxin tolerant cells or had undergone a shift in IEP to a more alkaline pH in tolerant cells. This suggests that o-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 o-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.

Keywords

Clostridium perfringens 2-D electrophoresis G-402 MDCK MTS E-toxin

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