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

Abstract

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.

Sphingosine 1–Phosphate Induces Antimicrobial Activity Both In Vitro and In Vivo

“…S1P and acidification of M. tuberculosis-containing phagosomes. Because macrophage PLD is involved in phagolysosome biogenesis [20] and M. tuberculosis is known to reside in nonacidified vacuoles sequestered from late endosomal compartments, the acidification of phagosomes that contain M. tuberculosis in S1P-stimulated cells was investigated by use of confocal fluorescent microscopy ( figure 5A). In the absence of S1P, phagocytosed M. tuberculosis bacilli appeared green, which indicated their presence in nonacidic vesicles separate from the red-stained acidic vacuoles.…”

“…Macrophage PLD-mediated S1P-induced antimicrobial activity. Because human macrophage PLDs have been reported to be involved in the molecular pathway leading to the activation of antimicrobial mechanisms [19] and in adenosine triphosphate (ATP)-induced intracellular mycobacterial killing [20], the activity of PLD after M. tuberculosis infection and stimulation with S1P was tested by use of TLC. Figure 4A shows the formation of [ 3 H]PetOH, as an index of PLD activity, 15, 90, and 180 min after stimulation with S1P.…”

“…Exogenous S1P has been reported to induce PLD activity that leads to the formation of intracellular PA [11], which is involved in the generation of a number of macrophage antimicrobial activities, such as phagocytosis [19], the production of reactive oxygen intermediates (ROIs) [24], the intracellular trafficking of endocytosed immunocomplexes to lysosomes [25], and phagolysosome maturation during the course of ATPinduced mycobacterial killing [20]. On these grounds, the role of PLD in S1P-induced antimicrobial activity was hypothesized.…”

Biochemical Characterization of a Pseudomonas aeruginosa Phospholipase D

“…Roles for human PLD (hPLD) in a variety of signaling processes include cytoskeleton rearrangement, vesicular trafficking, endocytosis, and cell survival. − The involvement of hPLD in bacterial infections has long been recognized, but details on its function are lacking. PLD expressed in human leukocytes has been proposed to function in antimicrobial mechanism such as phagocytosis, degranulation, respiratory burst, and chemotaxis. − However, host PLD can be manipulated to promote internalization and intracellular survival of bacterial pathogens also. , Several intracellular pathogens are known to secrete one or more phospholipase D enzymes to promote cell internalization, intracellular survival, or in vivo infectivity. − …”

Gene Dosage Determines the Negative Effects of Polymorphic Alleles of the P2X7Receptor on Adenosine Triphosphate–Mediated Killing of Mycobacteria by Human Macrophages

“…Activation of P2X 7 in macrophages stimulates a number of signalling pathways, including the caspase cascade, with resultant apoptosis, and activation of phospholipase D (PLD) [23][24][25]. PLD promotes phagolysosomal fusion, thereby exposing mycobacteria to destructive lysosomal enzymes [26,27].…”