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.

CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers

“…In our model, we propose that differences in charge and length of the pore β-hairpins impact physical properties of the lipid environment that could stimulate activity of mechanosensors, such as caveolin-1 to facilitate MAC removal and recovery from MAC attack. Similarly, membrane repair mechanisms triggered in response to bacterial pore forming toxins may rely on changes in mechanochemical properties of the plasma membrane caused by oligomerized prepores 41 . Although pore forming hairpins of these bacterial homologues extend throughout the bilayer, we speculate that an auxiliary domain’s cholesterol recognition loops that interact with the outer leaflet may serve a similar role as the partially-inserted MAC precursor.…”

The ins and outs of lipid rafts: functions in intracellular cholesterol homeostasis, microparticles, and cell membranes

“…The primary cellular mechanism for assembling multivesicular bodies uses the Endosomal Sorting Complex Required for Transport (ESCRT) of intraluminal vesicles into the endosome (121,122). An ESCRTindependent pathway has been identified that may be ceramide-dependent (123) low, but these levels often increase in disease states or during muscle repair (126), and it has been suggested that their concentration and composition might be used for diagnosis and prognosis (125,127). EV from platelets participate in normal procoagulant activity and thrombin production and their production is crucial for physiologic coagulation (128).…”

Bacterial pore‐forming toxin pneumolysin: Cell membrane structure and microvesicle shedding capacity determines differential survival of immune cell types

“…17 In brief, 2 × 10 6 cells in Ca 2+ Tyrode's buffer were incubated with 20 μg/mL propidium iodide (Sigma-Aldrich, Munich, Germany). 17 In brief, 2 × 10 6 cells in Ca 2+ Tyrode's buffer were incubated with 20 μg/mL propidium iodide (Sigma-Aldrich, Munich, Germany).…”