Irreversible versus repairable membrane poration: differences in permeabilization elicited by Bordetella Adenylate Cyclase Toxin and its hemolysin domain in macrophages

Abstract

Rapid plasma membrane repair in response to pore-forming toxins is crucial for cell survival, but the molecular mechanisms employed by eukaryotic nucleated cells to maintain membrane integrity and the specificities of such pathways remain poorly understood. Here, we have explored the permeabilization elicited by the Bordetella pertussis adenylate cyclase toxin, a 200-kDa protein toxin with a-helical pore-forming domain that forms pores of tunable size, and evaluated the response of target macrophages to such toxin poration. We show here that the response and the fate of target macrophages depend on toxin pore width. We find that the toxin 0 s hemolysin moiety induces a transient membrane permeabilization by forming wide enough pores allowing Ca 2+ influx into the target cell cytosol. This activates a Ca 2+ -dependent cellular response involving exocytosis and endocytosis steps eliminating toxin pores and restoring membrane integrity. In contrast, the full-length native toxin, at low concentrations, forms very small pores that cause insidious perturbation of cell ion homeostasis that escapes control by the macrophage membrane repair response, eventually leading to cell death. Our data reveal that permeability to Ca 2+ and ATP are key elements in the membrane repair pathway for eliminating a-helical pores of bacterial origin.Abbreviations AC, adenylate cyclase domain; ACT or CyaA, adenylate cyclase toxin-hemolysin; ASM, acid sphingomyelinase; BAPTA-AM, 1,2-Bis

Membrane Repair Mechanisms against Permeabilization by Pore-Forming Toxins

“…Recently, we explored the permeabilization elicited by sublytic doses of ACT on target macrophages and evaluated whether the permeabilized cell membrane was resealed or not, and we found that the full-length toxin induces an irreversible membrane permeabilization that eventually leads to cell death [ 84 ] (Etxaniz et al, manuscript in revision), which suggests that cellular repair mechanisms are not operative in the ACT-treated macrophages. Interestingly, an ACT mutant extending from amino acids 483 to 1706 (ACT-∆N482 hemolysin), which contains the entire ACT RTX hemolysin domain, induces by contrast a transient membrane permeabilization that is rapidly reverted in a time scale of minutes (≈30 min) by a repair pathway activated by extracellular Ca 2+ influx and that requires ATP [ 84 ] (Etxaniz et al, manuscript in revision).…”

“…Recently, we explored the permeabilization elicited by sublytic doses of ACT on target macrophages and evaluated whether the permeabilized cell membrane was resealed or not, and we found that the full-length toxin induces an irreversible membrane permeabilization that eventually leads to cell death [ 84 ] (Etxaniz et al, manuscript in revision), which suggests that cellular repair mechanisms are not operative in the ACT-treated macrophages. Interestingly, an ACT mutant extending from amino acids 483 to 1706 (ACT-∆N482 hemolysin), which contains the entire ACT RTX hemolysin domain, induces by contrast a transient membrane permeabilization that is rapidly reverted in a time scale of minutes (≈30 min) by a repair pathway activated by extracellular Ca 2+ influx and that requires ATP [ 84 ] (Etxaniz et al, manuscript in revision). The increase of intracellular Ca 2+ induced by the sublytic doses of ACT was found to be very limited [ 84 ] (Etxaniz et al, manuscript in revision), and translocation of the adenylate cyclase domain into the cytosol of the macrophages consumes cellular ATP; both factors might contribute to the incapability of the ACT-treated cells to reseal the injured membrane [ 84 ] (Etxaniz et al, manuscript in revision).…”

“…Interestingly, an ACT mutant extending from amino acids 483 to 1706 (ACT-∆N482 hemolysin), which contains the entire ACT RTX hemolysin domain, induces by contrast a transient membrane permeabilization that is rapidly reverted in a time scale of minutes (≈30 min) by a repair pathway activated by extracellular Ca 2+ influx and that requires ATP [ 84 ] (Etxaniz et al, manuscript in revision). The increase of intracellular Ca 2+ induced by the sublytic doses of ACT was found to be very limited [ 84 ] (Etxaniz et al, manuscript in revision), and translocation of the adenylate cyclase domain into the cytosol of the macrophages consumes cellular ATP; both factors might contribute to the incapability of the ACT-treated cells to reseal the injured membrane [ 84 ] (Etxaniz et al, manuscript in revision). Furthermore, we have found that the repair pathway acting in the ACT-∆N482 hemolysin-treated cells involves consecutive steps of exocytosis and endocytosis, likely initiated by lysosomal fusion with the damaged cell membrane, subsequent secretion to the extracellular medium of acid sphingomyelinase, and concomitant local generation of ceramide, all of which culminates in the endocytosis of the pore-ridden membrane ( Figure 4 ) [ 84 ] (Etxaniz et al, manuscript in revision).…”

“…The increase of intracellular Ca 2+ induced by the sublytic doses of ACT was found to be very limited [ 84 ] (Etxaniz et al, manuscript in revision), and translocation of the adenylate cyclase domain into the cytosol of the macrophages consumes cellular ATP; both factors might contribute to the incapability of the ACT-treated cells to reseal the injured membrane [ 84 ] (Etxaniz et al, manuscript in revision). Furthermore, we have found that the repair pathway acting in the ACT-∆N482 hemolysin-treated cells involves consecutive steps of exocytosis and endocytosis, likely initiated by lysosomal fusion with the damaged cell membrane, subsequent secretion to the extracellular medium of acid sphingomyelinase, and concomitant local generation of ceramide, all of which culminates in the endocytosis of the pore-ridden membrane ( Figure 4 ) [ 84 ] (Etxaniz et al, manuscript in revision). Given the high similarity among the ACT RTX-hemolysin moiety and the other toxins from the RTX family, it is enticing to surmise that a similar, Ca 2+ -dependent, membrane repair mechanism might also operate for those pore-forming toxins.…”

Four Cholesterol-Recognition Motifs in the Pore-Forming and Translocation Domains of Adenylate Cyclase Toxin Are Essential for Invasion of Eukaryotic Cells and Lysis of Erythrocytes

“…The “pore model” for AC translocation proposed here may thus explain why ACT is apparently weakly haemolytic relative to other RTX pore-forming toxins, such as Escherichia coli α-haemolysin, which do not possess an equivalent N-terminal domain [ 18 , 19 ]. Supporting this idea that in native conditions the presence of the AC domain somehow blocks the ion flux through the ACT pore, others previously observed that the elimination of the AC domain plus residues of the translocation region from the ACT polypeptide made the mutant toxins to exhibit a lytic potency comparable to the RTX toxins [ 52 , 53 ]. Future work will be needed to further prove this effect and map interactions between the AC domain and residues at the pore entrance.…”