Cellular Uptake of Clostridium botulinum C2 Toxin Requires Acid Sphingomyelinase Activity

Abstract

Clostridium botulinum C2 toxin consists of an enzyme component (C2I) and a binding component (C2II). Activated C2II (C2IIa) binds to a cell receptor, giving rise to lipid raft-dependent oligomerization, and it then assembles with C2I. The whole toxin complex is then endocytosed into the cytosol, resulting in the destruction of the actin cytoskeleton and cell rounding. Here, we showed that C2 toxin requires acid sphingomyelinase (ASMase) activity during internalization. In this study, inhibitors of ASMase and lysosomal exocytosis blocked C2 toxin-induced cell rounding. C2IIa induced Ca 2ϩ influx from the extracellular medium to cells. C2 toxininduced cell rounding was enhanced in the presence of Ca 2ϩ . ASMase was released extracellularly when cells were incubated with C2IIa in the presence of Ca 2ϩ . Small interfering RNA (siRNA) knockdown of ASMase reduced C2 toxin-induced cell rounding. ASMase hydrolyzes sphingomyelin to ceramide on the outer leaflet of the membrane at acidic pH. Ceramide was detected in cytoplasmic vesicles containing C2IIa. These results indicated that ASMase activity is necessary for the efficient internalization of C2 toxin into cells. Inhibitors of ASMase may confer protection against infection.

Characterization of Plasma Membrane Ceramides by Super‐Resolution Microscopy

“…[29,30] Recently,the distribution of sphingolipids in the plasma membrane of cells has been investigated by super-resolution microscopy. [31,32] Furthermore,h igh-resolution imaging mass spectrometry has been used to map the distribution of 15 N-enriched ions from metabolically labeled 15 N-sphingolipids with about 50 nm lateral resolution and an analysis depth of < 5nmtoensure the selective investigation of membrane sphingolipids. [33] Thes tudy revealed the existence of micrometer-scale sphingolipid platforms with amean diameter of about 200 nm, independent of fixation conditions and temperature.…”

Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin

“…Recently, we demonstrated that C2 toxin needs the activity of acid sphingomyelinase (ASMase) during the initial step of endocytosis [ 41 ]. Several bacterial pore-forming cytotoxic proteins cause calcium entry and provoke the exocytosis of lysosomes, leading to the release of lysosomal ASMase to the extracellular medium [ 42 , 43 ].…”

“…Namely, the hydrolysis of sphingomyelin by ASMase secreted from the exocytosis of lysosomes produces plasma membrane microdomains enriched in ceramide, leading to endocytosis [ 44 , 45 ]. C2IIa causes the entry of extracellular calcium into sensitive cells, and the cytotoxic activity of C2 toxin is increased in calcium-containing medium [ 41 ]. Blockers of lysosomal exocytosis and ASMase inhibit the cytotoxic activity of C2 toxin.…”

“…Then, C2 toxin induces ceramide production in plasma membranes. On the basis of these findings, it is concluded that ASMase activity is required for C2 toxin entry into host cells [ 41 ]. In Figure 2 , we show a hallmark of the key role of ASMase in the endocytosis of C2 toxin in sensitive cells.…”

Characterization of Plasma Membrane Ceramides by Super‐Resolution Microscopy

“…[1,2] Moreover, SMases and ceramides have been shown to be critically involved in the internalization of pathogens. [10][11][12][13][14][15] The recent observation, that CRP formation is essential in controlling the metabolic activity of regulatory T cells demonstrates that ceramides play also an essential role in regulating immune functions and inflammation. [16,17] However, despite these important roles the molecular organization of ceramides in the plasma membrane remains elusive.…”