Stepwise degradation of membrane sphingomyelin by corynebacterial phospholipases

Abstract

The mechanism of in vitro synergistic lysis of sheep erythrocytes by Corynebacterium ovis [C. pseudotuberculosis] and C. equi was investigated. Hemolysis required the action of phospholipase D from C. ovis, the action of an extracellular protein of C. equi and Mg2+. Maximum lysis required imposition on the system of a 4th condition such as chilling. The extracellular protein of C. equi was purified to homogeneity and was a phospholipase C capable of hydrolyzing ceramide phosphate, phosphatidic acid and all of the isolated major phospholipids of mammalian erythrocyte membranes. The principal features of the synergistic hemolytic system could be reproduced in experiments involving liposomes containing sphingomyelin or ceramide phosphate and trapped [14C]glucose. Sphingomyelin of sheep erythrocytes may be first converted to ceramide phosphate by C. ovis phospholipase D. The ceramide phosphate may be converted to ceramide by C. equi phospholipase C. The resulting in situ ceramide then may undergo dislocation by chilling and perhaps by virtue of an affinity between ceramide and C. equi phospholipase C. The dislocation of ceramide presumably disorganizes the lipid bilayer sufficiently to result in cell lysis.