Abstract
A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated. We now show that the mutant cell is 10(5) times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell. To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C. perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA. Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector. Exposure of these two types of transfectant clones to C. perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase. Further experiments with genetically engineered C. perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells.
Highlights
Clostridium perfringens phospholipase C (PLC1), called ␣-toxin, is the major virulence factor in the pathogenesis of gas gangrene (1–3)
Don Q Is Hypersensitive to the Cytotoxic Effect of C. perfringens PLC—As previously shown, Don Q and wt cells exhibit the same sensitivity to phospholipases A2, B, and D, whereas Don Q is hypersensitive to the cytotoxic effect of C. perfringens PLC (13)
The B. cereus PLC showed the same cytotoxicity to the three cell lines (Fig. 1A) whereas the C. bifermentans phospholipase C was not cytotoxic to any of the cells
Summary
Clostridium perfringens phospholipase C (PLC1), called ␣-toxin, is the major virulence factor in the pathogenesis of gas gangrene (1–3). It is lethal, cytotoxic, hemolytic, necrotizing, and induces platelet aggregation (4, 5). A spontaneous revertant cell (Don QR) was isolated from Don Q and showed to have the same sensitivity to TcdB as Don wt and a partially compensated UDP-Glc level (15, 16). UDP-Glc deficiency occurs in cells cultured under hypoxia or in low glucose-containing media (17–20). Since these conditions are two of the hallmarks of ischemia, it is likely that a low UDP-Glc level occurs in ischemic tissues (16). The aim of this work was to determine whether a cellular UDP-Glc deficiency affects the sensitivity to the cytotoxic effect of C. perfringens PLC as well as to map which parts of this toxin are needed for its cytotoxicity
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