Missing elimination via membrane vesicle shedding contributes to the diminished calcium sensitivity of listeriolysin O

Abstract

The lytic capacity of cholesterol-dependent cytolysins is enhanced in the extracellular calcium-free environment through a combination of limited membrane repair and diminished membrane toxin removal. For a typical neurotoxin of the group, pneumolysin, this effect has already been observed at reduced (1 mM) calcium conditions, which are pathophysiologically relevant. Here, we tested another neurotoxin of the group, listeriolysin O from L. monocytogenes, active in the primary vacuole after bacterium phagocytosis in host cells. Reduced calcium did not increase the lytic capacity of listeriolysin (in contrast to pneumolysin), while calcium-free conditions elevated it 2.5 times compared to 10 times for pneumolysin (at equivalent hemolytic capacities). To clarify these differences, we analyzed membrane vesicle shedding, known to be a calcium-dependent process for toxin removal from eukaryotic cell membranes. Both pneumolysin and listeriolysin initiated vesicle shedding, which was completely blocked by the lack of extracellular calcium. Lack of calcium, however, elevated the toxin load per a cell only for pneumolysin and not for listeriolysin. This result indicates that vesicle shedding does not play a role in the membrane removal of listeriolysin and outlines a major difference between it and other members of the CDC group. Furthermore, it provides new tools for studying membrane vesicle shedding.

Membrane Binding, Cellular Cholesterol Content and Resealing Capacity Contribute to Epithelial Cell Damage Induced by Suilysin of Streptococcus suis

“…In case of SLO, the archetype of a CDC produced by S. pyogenes, pores are repaired by Ca 2+ -dependent and annexin-mediated fusion of the plasma membrane and shed as microvesicles into the extracellular space (ectocytosis) [76][77][78]. The same mechanism of Ca 2+ -dependent microvesicle shedding was reported for the CDC listeriolysin O (LLO), perfringolysin O (PFO), intermedilysin (ILY), and PLY [79][80][81]. Another possibility to prevent cell lysis is the Ca 2+ -dependent endocytic removal and internal degradation of the plasmalemmal lesion, which has also been described for SLO [52], as well as for other pore-forming toxins [82,83].…”

Interaction of Macrophages and Cholesterol-Dependent Cytolysins: The Impact on Immune Response and Cellular Survival

“…In contrast, reduction or lack of calcium did not increase cell death from LLO to the same extent that it increased PLY-dependent killing [269,270]. While reducing calcium from 2 mM to~1 mM or 0 mM increased PLY killing of glial cells up to 10-fold, it improved LLO-mediated killing only 2.5-fold [269,270]. This was attributed to a decrease in the extent of vesicle shedding triggered by LLO compared to PLY, though shedding was observed for both CDCs [270].…”

“…While small pore-forming toxins such as aerolysin did not show calcium dependence [78], CDCs such as SLO, PFO, or PLY generally triggered calcium-dependent repair mechanisms [35,47,55,56,268]. In contrast, reduction or lack of calcium did not increase cell death from LLO to the same extent that it increased PLY-dependent killing [269,270]. While reducing calcium from 2 mM to~1 mM or 0 mM increased PLY killing of glial cells up to 10-fold, it improved LLO-mediated killing only 2.5-fold [269,270].…”

“…While reducing calcium from 2 mM to~1 mM or 0 mM increased PLY killing of glial cells up to 10-fold, it improved LLO-mediated killing only 2.5-fold [269,270]. This was attributed to a decrease in the extent of vesicle shedding triggered by LLO compared to PLY, though shedding was observed for both CDCs [270]. However, these findings could be cell-type specific because HeLa cells did not show an increase cell death between 2 mM or 0.4 mM calcium when challenged with PFO or SLO [55].…”

Phobalysin: Fisheye View of Membrane Perforation, Repair, Chemotaxis and Adhesion

“…As observations are usually made at a very early time point after addition of toxin, it is tacitly assumed that cellular channel forming proteins or ruptures do not contribute to the influx of dye. At any rate, important insight into the consequences of membrane damage, e.g., [67,68], and various mechanism of CIDRE [72,73,74,75,76,77,78,79,80], has been gained by studies on CDC. In essence, these studies showed that removal of CDC pores from the plasma membrane is important for recovery of target cells and that this may involve uptake or shedding of membrane lesions.…”