Recovery of fibroblasts from membrane attack by S. aureus α-toxin does not depend on acid sphingomyelinase but involves macropinocytosis

Damage of the plasma membrane by mechanical stress or pore forming proteins, like streptolysin O, may trigger Ca2+ influx-dependent repair mechanisms. Ca2+ influx-dependent lysosomal exocytosis, leading to release of acid sphingomyelinase, remodeling of the plasma membrane and caveolar endocytosis of membrane lesions, is reportedly involved in membrane repair after both mechanical damage or perforation by streptolysin O. Although the small {beta}-barrel pore forming S. aureus -toxin may also increase cytosolic Ca2+ concentration in certain cell types, and reportedly lead to release of acid sphingomyelinase from endothelial cells, evidence for a role of this response for membrane repair after attack by -toxin is lacking. We exploited fibroblasts expressing dysfunctional acid sphingomyelinase to investigate whether this enzyme is required for membrane repair after S. aureus -toxin attack. Because -toxin-dependent loss of cellular ATP and externalization of phosphatidylserine were reversible, membrane damage by this pore former triggered an effective repair response in these cells. Although -toxin depolarized the plasma membrane, it did not cause a simultaneous increase of [Ca2+]i. Consistently, there was no release of {beta}-hexosaminidase, a marker of lysosomal exocytosis. Acid sphingomyelinase-deficient fibroblasts internalized -toxin, which however did not co-localize with caveolin-1, but with FITC-dextran 70kDa, a cargo of macropinosomes. Inhibition of actin polymerization or sterol synthesis prevented recovery from -toxin-dependent membrane damage. Therefore, we conclude that defense of fibroblasts against -toxin does not depend on rapid calcium-influx, lysosomal exocytosis, and functional acid sphingomyelinase, but involves ongoing cholesterol synthesis and macropinocytosis.