Microvesicle shedding and lysosomal repair fulfill divergent cellular needs during the repair of streptolysin O-induced plasmalemmal damage

Abstract

Pathogenic bacteria secrete pore-forming toxins that permeabilize the plasma membrane of host cells. Nucleated cells possess protective mechanisms that repair toxin-damaged plasmalemma. Currently two putative repair scenarios are debated: either the isolation of the damaged membrane regions and their subsequent expulsion as microvesicles (shedding) or lysosome-dependent repair might allow the cell to rid itself of its toxic cargo and prevent lysis. Here we provide evidence that both mechanisms operate in tandem but fulfill diverse cellular needs. The prevalence of the repair strategy varies between cell types and is guided by the severity and the localization of the initial toxin-induced damage, by the morphology of a cell and, most important, by the incidence of the secondary mechanical damage. The surgically precise action of microvesicle shedding is best suited for the instant elimination of individual toxin pores, whereas lysosomal repair is indispensable for mending of self-inflicted mechanical injuries following initial plasmalemmal permeabilization by bacterial toxins. Our study provides new insights into the functioning of non-immune cellular defenses against bacterial pathogens.

Figure 1. Plasmalemmal repair in SLO-damaged cells is accomplished by the expulsion of microvesicles.

Shedding of annexin A1-rich microvesicles by SLO-treated, annexin A1-YFP-expressing HEK 293 cells was recorded by laser-scanning confocal microscopy. Magnification bar = 5 µm.

Figure 2. Destabilization of the cortical cytoskeleton enhances microvesicle release by SLO-damaged cells and potentiates their survival.

(A) Enhanced release of microvesicles by SLO-damaged cells, which were treated with latrunculin A. (B) Latrunculin A protects HEK 293 cells from SLO-induced cell lysis. (C) Diminished release of microvesicles by SLO-damaged cells which were treated with jasplakinolide. (D) Treatment with jasplakinolide results in increased cell lysis. (E) Calpeptin reduces microvesicle release by SLO-damaged cells. (F) SLO induces an increased rate of lysis in calpeptin-treated cells. **p<0.001, *p<0.01. (G) Amounts of annexin A1 shed within microvesicles were analyzed by Western Blotting in culture supernatants of SLO-treated cells pre-treated with either latrunculin (Latr/SLO), jasplakinolide (Jasp/SLO), cells treated with latrunculin without SLO treatment (Latr) or cells treated with DTT only (Contr). **p<0.001.

Figure 3. Pinpoint shedding of microvesicles.

Two HEK 293 and two SH-SY5Y cells transfected with annexin A1-YFP are shown. SLO-pores are quarantined within compact plasmalemmal regions or within thin outward protrusions (arrows). An asterisk denotes the nucleus of a lysed cell. Magnification bars = 5 µm.

Figure 4. The plasmalemmal repair of SLO pores occurs in lysosome-free cellular protrusions.

(A,B) SLO-induced plasmalemmal translocation/cytoplasmic back-translocation of annexin A1-YFP marks the successful elimination of the SLO-pores in (A) a neurite of a SH-SY5Y cell or in (B) a cytoplasmic protrusion of a HEK 293 cell. Arrows denote the plasmalemmal translocation of annexin A1 (plasmalemmal permeabilization). Magnified images of the squared region are shown in (A). Magnification bars = 5 µm.

Figure 5. The plasmalemmal repair of SLO pores occurs in lysosome-free blebs.

SLO-induced plasmalemmal translocation/cytoplasmic back-translocation of annexin A1-YFP marks the successful elimination of the SLO-pores in blebs of HEK 293 cells. Arrows denote the plasmalemmal translocation of annexin A1 (plasmalemmal permeabilization). Magnification bars = 5 µm.

Figure 6. Self-inflicted mechanical damage in SLO-perforated cells.

(A–C) The SLO-perforated protrusions of SH-SY5Y or HEK 293 cells are wrenched apart by mechanical forces. (A) The global translocation of annexin A1-YFP manifests cell lysis. (B,C) The cytoplasmic localization of annexin A1-YFP within the cell body is evidence of successful resealing of the plasmalemma. The arrow in (B) points at the resealed base of the destroyed protrusion. The asterisks in (B,C) denote regions of clear demarcation between permeabilized (plasmalemmal localization of annexin A1-YFP) and non-permeabilized (cytoplasmic localization of annexin A1-YFP) cellular compartments. Magnification bars = 5 µm.

Figure 7. SLO-perforation inflicts mechanical damage and triggers lysosomal fusion.

(A) HEK 293 cells do not adhere extensively to the substratum at the cellular periphery while SH-SY5Y cells are firmly attached by multiple protrusions. Magnification bars = 2 µm. (B) Inhibition of myosin contraction does not protect from SLO induced lysis. (C) Lysosomal exocytosis (β-hexosaminidase release) after SLO-injury is more pronounced in SH-SY5Y cells compared to HEK 293 cells. (D) Vacuolin-1 does not increase the SLO-induced lysis in HEK 293 cells. In contrast, Vacuolin-1-treated SH-SY5Y cells are more prone to the SLO-induced lysis. *p<0.01.

Figure 8. Microvesicle shedding is responsible for the initial elimination of toxin pores whereas lysosomal repair mends secondary, self-inflicted mechanical injuries.

(A) Individual experiments in which SH-SY5Y cells either repaired efficiently (low damage; 72 cells, 3 independent experiments) or suffered from extensive plasmalemmal damage (high damage; 66 cells, 3 independent experiments) were analyzed for: (B) amount of microvesicles released per cell, (C) percentage of cells that suffered from secondary, self-inflicted mechanical damage (100% = total number of cells in each individual experiment), (D) percentage of cells that recovered after self-inflicted mechanical damage (100% = number of mechanically-damaged cells in each individual experiment), (E) contribution of lysosomal repair to total repair (100% = total number of repaired cells in each individual experiment). **p<0.001, *p<0.01.