Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization

Abstract

Summary The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.

Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and α-Hemolysin from Escherichia coli

“…Standard curves with increasing concentrations of HlyA identified the EC 50 of the toxin. The EC 50 for LtxA was found at a concentration of 0.122 g ml Ϫ1 , which is slightly more potent in this purification than what was previously published (13). The EC 50 for HlyA varies and has to be determined every experimental week because of the faster degradation of protein activity.…”

“…The EC 50 for LtxA was found at a concentration of 0.122 g ml Ϫ1 , which is slightly more potent in this purification than what was previously published (13). The EC 50 for HlyA varies and has to be determined every experimental week because of the faster degradation of protein activity. The amount of active protein which corresponded to EC 50 in our preparation has been determined to be 25 ng ml Ϫ1 (4 Statistics.…”

“…Washed THP-1 monocytes were centrifuged at 280 ϫ g, suspended in HBS (0.5 ϫ 10 6 cells ml Ϫ1 ), and incubated with a propidium iodide (PI) cell permeabilization kit (5 M, 5 min, 37°C; Invitrogen). Cells were incubated in blockers of interest in Eppendorf tubes for 5 min before addition of either HlyA (EC 50 ) or LtxA (EC 50 ). The cells were incubated in the dark with toxins in the presence of compounds or vehicles (150 rpm, 60 min, 37°C).…”

Bacterial RTX Toxins Allow Acute ATP Release from Human Erythrocytes Directly through the Toxin Pore

“…When considering this option, one has to acknowledge that true pore formation has been questioned for both toxins, in particular LtxA (36,43). Nevertheless, it is well established that both HlyA and LtxA are membrane-damaging toxins (44 -49) but conclusive details regarding their membrane interaction are unknown for both proteins as structural models are currently lacking.…”

“…Patch clamp studies on HL-60 cells supported this by showing a large conductance and collapse of the membrane potential when LtxA was present (64). Recent data on T lymphocytes and artificial membranes argues against direct pore formation and suggest that the cytotoxicity of LtxA originates from membrane destabilization without the formation of transmembrane pores (36,43). The latter studies showed membrane interaction with artificial membranes, and the conclusion from these studies was that LtxA binds via two steps: (i) membrane association and (ii) membrane destabilization, which occurs through a modification of the bilayer structure demonstrated by leakage of dye (calcein or 8-aminonaphtalene-1,3,6-trisulfonic acid/p-xylene-bis(N-pyridium bromide) (ANTS/DPX)).…”