title: ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X(7)-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 p47(phox) and inducible NO synthase gene-disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals, respectively) and P2X(7) 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. P2X(7) receptor activation was essential for effective ATP-induced mycobacterial killing, as its bactericidal activity was radically diminished in P2X(7)(-/-) macrophages. ATP-mediated killing of BCG within p47(phox-/-), inducible NO synthase(-/-), and Nramp(s) cells was unaffected, demonstrating that none of these mechanisms have a role in the ATP/P2X(7) 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 P2X(7) receptor activation. Macrophage death and mycobacterial killing induced by the ATP/P2X(7) signaling pathway can be uncoupled, and diverge proximal to phospholipase D activation.
authors.affiliation: Medical Research Council Centre for Immune Regulation, Birmingham Medical School, Birmingham University, Edgbaston, Birmingham, United Kingdom. i.p.fairbairn@bham.ac.uk
authors.forename: I P
authors.initials: IP
authors.lastname: Fairbairn
authors.forename: C B
authors.initials: CB
authors.lastname: Stober
authors.forename: D S
authors.initials: DS
authors.lastname: Kumararatne
authors.forename: D A
authors.initials: DA
authors.lastname: Lammas
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001433:Bacteriolysis / Q000187:drug effects* / Q000502:physiology; D000440:Butanols / Q000494:pharmacology; D027682:Cation Transport Proteins / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000502:physiology*; D008264:Macrophages / Q000187:drug effects* / Q000382:microbiology / Q000502:physiology; D008561:Membrane Fusion / Q000187:drug effects*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000382:microbiology / Q000502:physiology; D009163:Mycobacterium bovis; D019255:NADPH Oxidases; D019001:Nitric Oxide Synthase / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D052247:Nitric Oxide Synthase Type II; D010588:Phagosomes / Q000502:physiology*; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000502:physiology; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D014617:Vacuoles / Q000382:microbiology; D000099173:Natural Resistance-associated Macrophage Protein 1
chemical_list: D000255:Adenosine Triphosphate; D000440:Butanols; D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D019255:NADPH Oxidases; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D010739:Phospholipase D; D010750:Phosphoproteins; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000099173:Natural Resistance-associated Macrophage Protein 1; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; C496319:NOS2 protein, human; C496317:Nos2 protein, mouse; C060505:neutrophil cytosolic factor 1