Mycobacterium tuberculosis survives within host macrophages by actively inhibiting phagosome fusion with lysosomes. Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular mycobacteria. The following studies were undertaken to characterize the effector pathway(s) involved. Macrophages were obtained from p47phox and inducible NO synthase gene-disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals, respectively) and P2X7 gene-disrupted mice. RAW murine macrophages transfected with either the natural resistance-associated macrophage protein gene 1 (Nramp1)-resistant or Nramp1-susceptible gene were also used. The cells were infected with bacille Calmette-Guérin (BCG), and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy. P2X7 receptor activation was essential for effective ATP-induced mycobacterial killing, as its bactericidal activity was radically diminished in P2X7−/− macrophages. ATP-mediated killing of BCG within p47phox−/−, inducible NO synthase−/−, and Nramps cells was unaffected, demonstrating that none of these mechanisms have a role in the ATP/P2X7 effector pathway. Following ATP stimulation, BCG-containing phagosomes rapidly coalesce and fuse with lysosomes. Blocking of macrophage phospholipase D activity with butan-1-ol blocked BCG killing, but not macrophage death. ATP stimulates phagosome-lysosome fusion with concomitant mycobacterial death via P2X7 receptor activation. Macrophage death and mycobacterial killing induced by the ATP/P2X7 signaling pathway can be uncoupled, and diverge proximal to phospholipase D activation.

Macrophage apoptosis occurs within the granuloma, which is essential for successful immunity to tuberculosis. In vitro macrophage apoptosis is associated with the killing of intracellular Mycobacterium tuberculosis. A greater understanding of these observations will lead to new immunotherapies and improved vaccine design. The relevant apoptotic stimuli, the anti-mycobacterial mechanisms that they stimulate and their physiological relevance are reviewed in this paper.

Introduction: Mycobucterium tuberculosis survives within host macrophages by actively inhibiting phagosome fusion with lysosomes. Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular mycobacteria. The following studies were undertaken to characterize the effector pathway(s) involved. Methods: Macrophages were obtained from p47phox and iNOS gene disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals respectively) and P2x7 gene disrupted mice. RAW murine macrophages transfected with either the Nrampl resistant or Nrampl susceptible gene were also utilized. The cells were infected with BCG and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy. Results: E X 7 receptor activation was essential for effective ATP-induced mycobacterial killing as its bactericidal activity was radically diminished in P2X7-I-macrophages. ATPmediated killing of BCG within p47phox-/-, iNOS-/-and Nramps cells was unaffected, demonstrating that none of these mechanisms have a role in the ATPIP2X7 effector pathway. Following ATP stimulation, BCG-containing phagosomes rapidly coalesce and fuse with lysosomes. Blocking of macrophage PLD activity with hutan-1-01 blocked BCG killing but not macrophage death. Conclusion: ATP stimulates phagosome-lysosome fusion with concomitant mycobacterial death via P2X7 receptor activation.