Aerolysin Induces G-protein Activation and Ca2+Release from Intracellular Stores in Human Granulocytes

Abstract

Aerolysin is a pore-forming toxin that plays a key role in the pathogenesis of Aeromonas hydrophila infections. In this study, we have analyzed the effect of aerolysin on human granulocytes (HL-60 cells). Proaerolysin could bind to these cells, was processed into active aerolysin, and led to membrane depolarization, indicating that granulocytes are potential targets for this toxin. Fura-2 measurements were used to analyze the effect of aerolysin on cytosolic [Ca 2؉] homeostasis. As expected for a pore-forming toxin, aerolysin addition led to Ca 2؉ influx across the plasma membrane. In addition, the toxin triggered Ca 2؉ release from agonist and thapsigargin-sensitive intracellular Ca 2؉ stores. This Ca 2؉ release was independent of the aerolysin-induced Ca 2؉ influx and occurred in two kinetically distinct phases: an initial rapid and transient phase and a second, more sustained, phase. The first, but not the second phase was sensitive to pertussis toxin. Activation of pertussis toxin-sensitive G-proteins appeared to be a consequence of pore formation, rather than receptor activation through aerolysinbinding, as it: (i) was not observed with a binding competent, insertion-incompetent aerolysin mutant, (ii) had a marked lag time, and (iii) was also observed in response to other bacterial pore-forming toxins (staphylococcal ␣-toxin, streptolysin O) which are thought to bind to different receptors. G-protein activation through pore-forming toxins stimulated cellular functions, as evidenced by pertussis toxin-sensitive chemotaxis. Our results demonstrate that granulocytes are potential target cells for aerolysin and that in these cells, Ca 2؉ signaling in response to a pore-forming toxin involves G-proteindependent cell activation and Ca 2؉ release from intracellular stores.

Channels formed by subnanomolar concentrations of the toxin aerolysin trigger apoptosis of T lymphomas

“…At least two other channel-forming toxins, Staphylococcus aureus alpha toxin and Actinobacillus actinomycetemcomitans leukotoxin (Chen and Zychlinsky, 1994), also cause apoptosis at low concentrations, and it has been shown recently that, like aerolysin, the outer membrane porin PorB of Neisseria gonorrhoeae causes rapid calcium in¯ux into epithelial cells, which may induce apoptosis by activating cysteine proteases (Muller et al, 1999). It seems reasonable to suppose that the vacuolation of BHK endoplasmic reticulum by aerolysin, which was reported by Abrami et al (1998b), was also a consequence of apoptosis, as was the G-protein activation the same group reported in granulocytes (Krause et al, 1998). Our results are not consistent with their claim that intracellular stores of calcium are released by aerolysin.…”

“…5). This rules out the possibility that aerolysin somehow causes the release of calcium from intracellular stores, as suggested by Krause et al (1998) for the action of aerolysin on granulocytes.…”

Xenorhabdus nematophila (Enterobacteriacea) Secretes a Cation-selective Calcium-independent Porin Which Causes Vacuolation of the Rough Endoplasmic Reticulum and Cell Lysis

“…Another case of ER vacuolation by bacterial toxin was reported by Abrami et al (26) for aerolysin on baby hamster kidney cells. For aerolysin (37)(38)(39), as for ␣X (this study), the first target of the toxins is the plasma membrane, where they form channels selective for small cations before they trigger ER vacuolation. However, a fundamental difference between aerolysin and ␣X is their respective effects on intracellular Ca 2ϩ concentration.…”

Ras and Its Signals Diffuse through the Cell on Randomly Moving Nanoparticles

“…To further study the rasosomes and determine whether they function as platforms for singling, it was necessary to develop ways to isolate them physically and characterize them biochemically. We first used COS cells expressing GFP-H-Ras and treated them with 0.1% digitonin or with streptolysin-O, both of which form small pores in the cell membrane (36). We then used time-lapse imaging under epifluorescence to track GFP-H-Ras particles at 0 time and during digitonization.…”