Pseudomonas aeruginosa cytotoxin: the influence of sphingomyelin on binding and cation permeability increase in mammalian erythrocytes.

A cytotoxic protein isolated from Pseudomonas aeruginosa damages the plasma membranes of many mammalian cells by forming pores. We studied binding of the 125I-cytotoxin and the resulting increase of cation permeability in erythrocytes of various mammalian species. The sensitivity of red blood cells was inversely related to the relative sphingomyelin content in their external surface. Thus, erythrocytes with a sphingomyelin to phosphatidylcholine ratio below 1 (dog, rat, rabbit and man) were sensitive, whereas red blood cells with a ratio above 1 (pig, cattle and sheep) were not attacked even with 100-fold higher cytotoxin concentrations. At 37 degrees C 6.8 +/- 1.2 x 10(3) molecules of 125I-cytotoxin were bound per rabbit erythrocyte (KD = 59 nM), whereas no binding occurred to cattle cells. Cleavage of sphingomyelin by sphingomyelinase C from Bacillus cereus (EC 3.1.4.12) triggered a dose-dependent enhancement in binding and permeability increase, particularly in red blood cells with a high proportion of sphingomyelin. The KDs for all animal species investigated were 53-60 nM. Pretreatment with mainly phosphatidylcholine-hydrolyzing phospholipases D from Streptomyces chromofuscus and cabbage (EC 3.1.4.4) or phospholipase C from Bacillus cereus (EC 3.1.4.3) did not influence the cytotoxin effect. The negative correlation between susceptibility and the proportion of sphingomyelin in plasma membranes suggests a binding site close to sphingomyelin.