ATP-Mediated Killing of Intracellular Mycobacteria by Macrophages Is a P2X7-Dependent Process Inducing Bacterial Death by Phagosome-Lysosome Fusion

Abstract

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.

Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation

“…Classical activation of macrophages is intimately linked to induction of microbicidal activity and several studies have shown that P2X 7 R play a role in killing of Mycobacterium tuberculosis via activation of phospholipase D [8,9,35]. The down-regulation of surface P2X 4 R in classically activated alveolar macrophages makes it unlikely that P2X 4 R are involved in the killing of bacteria.…”

A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

“…Confocal microscopy shows P2X7 in the phagosome membrane surrounding engulfed particles or bacteria [29,30] and recent reports suggest that P2X7 together with pannexin-1 may facilitate the delivery of bacterial products from phagosome lumen into the cytosol, where they can activate inflammasome assembly [31,32]. Moreover P2X7 has a role in facilitating the fusion of phagosome with lysosome by a process involving stimulation of phospholipase D activity presumably on the phagolysosomal membrane [33,34].…”

Role of macrophage phospholipase D in natural and CpG-induced antimycobacterial activity

“…Phospholipase D has been recently involved in ATPinduced killing of intracellular mycobacteria by human macrophages (Fairbairn et al ., 2001). Because macrophages show a different ability to control intracellular replication of pathogenic and non-pathogenic mycobacteria such as MTB and Msm, we tested the hypothesis that such differences in intracellular growth control could reflect differences in host PLD activity.…”

“…As the involvement of PLD activation has recently been shown in ATP-induced killing of intracellular mycobacteria (Fairbairn et al ., 2001) and we have shown that it is crucial in the control of Msm viability (Fig. 3), we investigated the enzyme role in CpG-induced MTB killing.…”

“…Phagocyte activation in response to extracellular agonist is mediated by different classes of enzymes, including phospholipases (Liscovitch et al ., 2000). Moreover, phospholipase D (PLD) has been recently reported to be actively associated with ATPinduced killing of intracellular mycobacteria by human macrophages (Fairbairn et al ., 2001). Phospholipase D catalyses hydrolysis of the terminal diester bond of phosphatidylcholine as the favourite substrate, to liberate phosphatidic acid (PA) and choline (Liscovithc et al ., 2000;Exton, 2002).…”