ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X(7)-dependent process inducing bacterial death by phagosome-lysosome fusion

Mycobacterium tuberculosis survives within host macrophages by actively inh ibiting phagosome fusion with lysosomes. Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular myc obacteria. The following studies were undertaken to characterize the effect or pathway(s) involved. Macrophages were obtained from p47(phox) and induci ble NO synthase gene-disrupted mice (which are unable to produce reactive o xygen and nitrogen radicals, respectively) and P2X(7) gene-disrupted mice. RAW murine macrophages transfected with either the natural resistance-assoc iated macrophage protein gene 1 (Nramp1)-resistant or Nramp1-susceptible ge ne were also used. The cells were infected with bacille Calmette-Guerin (BC G), and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy. P2X(7) receptor activation was essential for effe ctive ATP-induced mycobacterial killing, as its bactericidal activity was r adically diminished in P2X(7)(-/-) macrophages. ATP-mediated killing of BCG within p47(phox-/-), inducible NO synthase(-/-), and Nramp(s) cells was un affected, demonstrating that none of these mechanisms have a role in the AT P/P2X(7) effector pathway. Following ATP stimulation, BCG-containing phagos omes rapidly coalesce and fuse with lysosomes. Blocking of macrophage phosp holipase D activity with butan-1-ol blocked BCG killing, but not macrophage death. ATP stimulates phagosome-lysosome fusion with concomitant mycobacte rial death via P2X(7) receptor activation. Macrophage death and mycobacteri al killing induced by the ATP/P2X(7) signaling pathway can be uncoupled, an d diverge proximal to phospholipase D activation.