ATP stimulates human macrophages to kill intracellular virulent Mycobacterium tuberculosis via calcium-dependent phagosome-lysosome fusion.
Advances in therapy for tuberculosis will require greater understanding of the molecular mechanisms of pathogenesis and the human immune response in this disease. Exposure of Mycobacterium tuberculosis-infected human macrophages to extracellular ATP (ATP(e)) results in bacterial killing, but the molecular mechanisms remain incompletely characterized. In this study, we demonstrate that ATP(e)-induced bactericidal activity toward virulent M. tuberculosis requires an increase in cytosolic Ca(2+) in infected macrophages. Based on our previous work with primary infection of human macrophages, we hypothesized that the Ca(2+) dependence of ATP-induced killing of intracellular M. tuberculosis was linked to promotion of phagosome-lysosome fusion. Using confocal laser-scanning microscopy, we demonstrate that ATP(e) induces fusion of the M. tuberculosis-containing phagosome with lysosomes, defined by accumulation of three lysosomal proteins and an acidophilic dye. Stimulation of phagosome-lysosome fusion by ATP(e) exhibited distinct requirements for both Ca(2+) and phospholipase D and was highly correlated with killing of intracellular bacilli. Thus, key signal transduction pathways are conserved between two distinct models of human macrophage antituberculous activity: primary infection of naive macrophages and physiologic stimulation of macrophages stably infected with M. tuberculosis.
Kusner
D J
DJ
Department of Medicine, Inflammation Program, and Graduate Program in Immunology, University of Iowa and Veterans Affairs Medical Center, Iowa City, IA 52242, USA. kusner@uiowa.edu
Barton
J A
JA
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000328:Adult; D001433:Bacteriolysis / Q000187:drug effects*; D015415:Biomarkers; D002118:Calcium / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D006801:Humans; D008247:Lysosomes / Q000187:drug effects* / Q000502:physiology; D008264:Macrophages / Q000187:drug effects* / Q000382:microbiology / Q000502:physiology; D008561:Membrane Fusion / Q000187:drug effects* / Q000502:physiology; D018613:Microscopy, Confocal; D009169:Mycobacterium tuberculosis / Q000472:pathogenicity; D010588:Phagosomes / Q000187:drug effects* / Q000502:physiology; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000502:physiology; D014774:Virulence
D015415:Biomarkers; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D000431:Ethanol; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; C058180:5,5'-dimethyl-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; D010739:Phospholipase D; D002118:Calcium