The role of macrophage sphingosine kinase 1 in phagocytosis, phagosome maturation, and mycobacterial infection.

摘要

Mycobacterium tuberculosis (M.tb) is a leading cause of global infectious mortality. The pathogenesis of tuberculosis involves inhibition of phagosome maturation, leading to survival of M.tb within human macrophages. A key determinant of this is M.tb-induced inhibition of macrophage sphingosine kinase (SK) activity, which normally induces Ca2+-signaling and phagosome maturation. Our first objective was to determine the localization of SK during phagocytosis and during its inhibition by M.tb. Stimulation of SK activity by killed M.tb, live Staphylococcus aureus, or latex beads, but not by live M.tb induced translocation of cytosolic SK1 to the phagosome. The magnitude and kinetics of translocation of catalytically-inactive (CI)- and phosphorylation-defective (PD)-SK1 to latex bead phagosomes were indistinguishable from wild type (WT)-SK1, indicating that novel determinants regulate the association of SK1 with nascent phagosomes.; The translocation of SK1 from the cytosol to the phagosome membrane contributes to the spatial restriction of Ca2+-signaling. Thus, our second objective was to further characterize the determinants of SK1 localization to specific compartments: the phagosome membrane and the plasma membrane. Resting RAW 264.7 macrophages exhibited constitutive association of WT-SK1 with cortical actin filaments which was increased by stimulation with PMA, especially in plasma membrane ruffles. In contrast, cells transfected with CI-SK1 or with PD-SK1 exhibited significantly less membrane translocation and colocalization. Surprisingly, WT-, CI- and PD-SK1 each colocalized with actin filaments at the membrane of nascent phagosomes containing model particles. Biochemical analysis confirmed the constitutive- and stimulus-enhanced association of SK1 with the F-actin-containing detergent-insoluble fraction. Inhibition of actin cytoskeletal dynamics with latrunculin B or jasplakinolide disrupted the localization of SK1 and modulated its enzymatic activity. Conversely, inhibition of SK activity resulted in dysregulation of actin filaments.; Taken together, these studies suggest that M.tb inhibits both the activation and phagosomal translocation of SK1 to block the localized Ca2+ transients required for phagosome maturation. Furthermore, it may accomplish this by augmenting actin polymerization since subcellular localization and activity of SK1 is tightly coupled to actin cytoskeletal dynamics.