ROLE OF ALPHA-TOXIN IN CLOSTRIDIUM-PERFRINGENS INFECTION DETERMINED BY USING RECOMBINANTS OF CLOSTRIDIUM-PERFRINGENS AND BACILLUS-SUBTILIS

Clostridium perfringens type A strains which differed in alpha-toxin (phospholipase C [PLC]) productivity were inoculated intraperitoneally or intravenously into mice, and then their 50% mouse lethal doses (LD(50)) were determined. Strain NCTC 8237 produced ninefold higher PLC activity than strain 13. The mean LD(50) for the former was 1 log unit lower than that for the latter. Two isogenic strains were constructed from strain 13: strain 13(pJIR418 alpha) (pJIR418 alpha contains the plc gene), which produced ninefold higher PLC activity than strain 13; and strain 13 PLC(-), which showed no PLC productivity at all because of transformation-mediated gene disruption. The mean LD(50) for strain 13(pJIR418 alpha) was 1 log unit lower than those for strain 13 PLC(-) and strain 13. These results indicate that PLC functions as a virulence-determining factor when it is produced in a sufficient amount. Such a difference in LD(50) was also observed between Bacillus subtilis with and without the cloned plc gene. Inoculation of B. subtilis PLC(+) intravenously into mice caused marked thrombocytopenia and leukocytosis. Mice inoculated with B. subtilis at 2 LD(50) died because of circulatory collapse. Histological examination revealed that intravascular coagulation and vascular congestion occurred most prominently in the lungs. These results suggest that PLC plays a key role in the systemic intoxication of clostridial myonecrosis, probably by affecting the functions of platelets and phagocytes.