title: Intrinsic repair protects cells from pore-forming toxins by microvesicle shedding.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, TX, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Romero
authors.affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, TX, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Keyel
authors.affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, TX, USA.
authors.forename: Guilan
authors.initials: G
authors.lastname: Shi
authors.affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, TX, USA.
authors.forename: Pushpak
authors.initials: P
authors.lastname: Bhattacharjee
authors.affiliation: Department of Cell Biology and Physiology, Washington University of Saint Louis, St. Louis, MO, USA.
authors.forename: Robyn
authors.initials: R
authors.lastname: Roth
authors.affiliation: Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan.
authors.forename: John E
authors.initials: JE
authors.lastname: Heuser
authors.affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, TX, USA.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Keyel
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000494:pharmacology; D001427:Bacterial Toxins / Q000737:chemistry / Q000494:pharmacology*; D001430:Bacteriocins / Q000737:chemistry / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D055252:Cell-Derived Microparticles / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D004705:Endocytosis; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006460:Hemolysin Proteins / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D061251:Primary Cell Culture; D015202:Protein Engineering; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013301:Streptolysins / Q000737:chemistry / Q000494:pharmacology; D000074084:THP-1 Cells
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001430:Bacteriocins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; D013301:Streptolysins; C403910:intermedilysin protein, Streptococcus intermedius; C025964:streptolysin O; C020452:Clostridium perfringens theta-toxin; D002118:Calcium