Effects of bilayer composition and physical properties on the phospholipase C and sphingomyelinase activities of Clostridium perfringens α-toxin.
α-Toxin, a major determinant of Clostridium perfringens toxicity, exhibits both phospholipase C and sphingomyelinase activities. Our studies with large unilamellar vesicles containing a variety of lipid mixtures reveal that both lipase activities are enhanced by cholesterol and by lipids with an intrinsic negative curvature, e.g. phosphatidylethanolamine. Conversely lysophospholipids, that possess a positive intrinsic curvature, inhibit the α-toxin lipase activities. Phospholipids with a net negative charge do not exert any major effect on the lipase activities, and the same lack of effect is seen with the lysosomal lipid bis (monoacylglycero) phosphate. Ganglioside GT1b has a clear inhibitory effect, while the monosialic ganglioside GM3 is virtually ineffectual even when incorporated at 6mol % in the vesicles. The length of the lag periods appears to be inversely related to the maximum (post-lag) enzyme activities. Moreover, and particularly in the presence of cholesterol, lag times increase with pH. Both lipase activities are sensitive to vesicle size, but in opposite ways: while phospholipase C is higher with larger vesicles, sphingomyelinase activity is lower. The combination of our results with previous structural studies suggests that α-toxin lipase activities have distinct, but partially overlapping and interacting active sites.
Urbina
Patricia
P
Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.
Flores-Díaz
Marietta
M
Alape-Girón
Alberto
A
Alonso
Alicia
A
Goñi
Félix M
FM
D001427:Bacterial Toxins / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005732:Gangliosides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008027:Light; D008051:Lipid Bilayers / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D008246:Lysophospholipids / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry*; D010738:Type C Phospholipases / Q000737:chemistry*
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D005732:Gangliosides; D008051:Lipid Bilayers; D008055:Lipids; D008246:Lysophospholipids; D010710:Phosphates; D011994:Recombinant Proteins; C016481:trisialoganglioside GT1; D002784:Cholesterol; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase; C040649:alpha toxin, Clostridium perfringens