Intrinsic repair protects cells from pore-forming toxins by microvesicle shedding

• Published in: Cell death and differentiation Vol. 24; no. 5; pp. 798 - 808
• Main Authors: Romero, Matthew, Keyel, Michelle, Shi, Guilan, Bhattacharjee, Pushpak, Roth, Robyn, Heuser, John E, Keyel, Peter A
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-05-2017; Nature Publishing Group
• Subjects: 13/1; 13/31; 14/28; 631/326; 631/80; 82/29; Animals; Apoptosis; Bacterial Proteins - chemistry; Bacterial Proteins - pharmacology; Bacterial Toxins - chemistry; Bacterial Toxins - pharmacology; Bacteriocins - chemistry; Bacteriocins - pharmacology; Biochemistry; Biology; Biomedical and Life Sciences; Calcium - metabolism; Cell Biology; Cell Cycle Analysis; Cell death; Cell Membrane - chemistry; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Membrane Permeability - drug effects; Cell-Derived Microparticles - chemistry; Cell-Derived Microparticles - drug effects; Cell-Derived Microparticles - metabolism; Cholesterol; Cytotoxicity; Endocytosis; Female; HEK293 Cells; HeLa Cells; Hemolysin Proteins - chemistry; Hemolysin Proteins - pharmacology; Homeostasis; Humans; Hypotheses; Immune system; Life Sciences; Lipids; Macrophages - cytology; Macrophages - drug effects; Macrophages - metabolism; Male; Mice; Mice, Inbred C57BL; Mutation; Original Paper; Pathogens; Primary Cell Culture; Protein Engineering; Protein Multimerization; Recombinant Proteins - chemistry; Recombinant Proteins - pharmacology; Stem Cells; Streptolysins - chemistry; Streptolysins - pharmacology; THP-1 Cells; Toxins; Lubbock Texas; United States > US; Texas
• ISSN: 1350-9047; 1476-5403
• DOI: 10.1038/cdd.2017.11

Pore-forming toxins (PFTs) are used by both the immune system and by pathogens to disrupt cell membranes. Cells attempt to repair this disruption in various ways, but the exact mechanism(s) that cells use are not fully understood, nor agreed upon. Current models for membrane repair include (1) patch formation (e.g., fusion of internal vesicles with plasma membrane defects), (2) endocytosis of the pores, and (3) shedding of the pores by blebbing from the cell membrane. In this study, we sought to determine the specific mechanism(s) that cells use to resist three different cholesterol-dependent PFTs: Streptolysin O, Perfringolysin O, and Intermedilysin. We found that all three toxins were shed from cells by blebbing from the cell membrane on extracellular microvesicles (MVs). Unique among the cells studied, we found that macrophages were 10 times more resistant to the toxins, yet they shed significantly smaller vesicles than the other cells. To examine the mechanism of shedding, we tested whether toxins with engineered defects in pore formation or oligomerization were shed. We found that oligomerization was necessary and sufficient for membrane shedding, suggesting that calcium influx and patch formation were not required for shedding. However, pore formation enhanced shedding, suggesting that calcium influx and patch formation enhance repair. In contrast, monomeric toxins were endocytosed. These data indicate that cells use two interrelated mechanisms of membrane repair: lipid-dependent MV shedding, which we term ‘intrinsic repair’, and patch formation by intracellular organelles. Endocytosis may act after membrane repair is complete by removing inactivated and monomeric toxins from the cell surface.