Phospholipase C and sphingomyelinase activities of the Clostridium perfringens alpha-toxin.
Alpha-toxin is a major pathogenic determinant of Clostridium perfringens, the causative agent of gas gangrene. Alpha-toxin has been known for long to be a phospholipase C, but up to now its hydrolytic properties have been studied only through indirect methods, e.g. release of cell contents, or under non-physiological conditions, e.g., in micelles, or with soluble substrates. In this report we characterize the phospholipase C and sphingomyelinase activities of alpha-toxin using a direct assay method (water-soluble phosphorous assay) with phospholipids in bilayer form (large unilamellar vesicles) in the absence of detergents. The simplest bilayer compositions allowing measurable activities under these conditions were DOPC:Chol (2:1 mol ratio) and SM:PE:Chol (2:1:1 mol ratio) for the PLC and SMase activities respectively. PLC activity was five times higher than SMase activity. Both activities gave rise to vesicle aggregation, after a lag time during which ca. 10% of the substrate was hydrolyzed. Vesicle aggregation, measured as an increase in light scattering, was a convenient semi-quantitative method for estimating the enzyme activities. The optimum pH for the combined PLC and SMase activities was in the 5-7 range, in agreement with the proposed role of alpha-toxin in aiding the bacterium to escape the fagosome and survive within the cytosol.
Urbina
Patricia
P
Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), Departamento de Bioquímica, Universidad del Pais Vasco, Bilbao, Spain.
Flores-Díaz
Marietta
M
Alape-Girón
Alberto
A
Alonso
Alicia
A
Goni
Félix M
FM
D001427:Bacterial Toxins / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D003016:Clostridium perfringens / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D008081:Liposomes; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase; C040649:alpha toxin, Clostridium perfringens