Inhibition of calcium signaling affects phagosomal maturation and intracellular survival of M. tuberculosis.

摘要

Complement receptor (CR)-mediated phagocytosis of Mycobacterium tuberculosis (Mtb) by mononuclear phagocytes (MP) results in intracellular survival, suggesting that Mtb interferes with MP microbicidal mechanisms. Since Ca2+ is required for many phagocyte antimicrobial responses, we hypothesized that CR-mediated phagocytosis of Mtb is accompanied by alterations in Ca2+ signaling. In contrast to other complement (C)-opsonized particles, incubation of MDMs with live, virulent, complement (C)-opsonized Mtb was not accompanied by a change in cytosolic Ca2+ levels, despite phagocytosis of the bacilli. Inhibition of macrophage Ca2+ signaling required viable bacilli since complement-opsonized, gamma-irradiated Mtb induced changes in [Ca2+]c. The rise in cytosolic calcium levels in response to gamma-irradiated Mtb was not mediated by increased IP3 levels but, rather, relied on sphingosine 1-phosphate (S1-P) production. This sphingolipid metabolite was not produced, however, during incubation of MPs with live, C-opsonized Mtb. This lack of calcium signaling is an important determinant in the inhibition of phagosome-lysosome fusion in Mtb infection of human MPs. Ionophore-mediated elevation of [Ca 2+]c induced the maturation of phagosomes containing live bacilli, as evidenced by increased acidification and staining with lysosomal protein markers. Conversely, chelation of [Ca2+]c inhibited the fusion of vesicles containing gamma-irradiated bacilli with lysosomes. The calcium sensitivity of Mtb phagosome trafficking to lysosomal compartments was dependent on the activation of calmodulin-dependent protein kinase II (CaMKII). Pharmacological inhibition of CaMKII prevented the maturation of phagosomes containing gamma-irradiated Mtb and, also, the trafficking of live Mtb phagosomes to lysosomes in ionophore-treated cells. Furthermore, the activation (autophosphorylation) of CaMKII on phagosomes was dependent on the viability of the bacilli and, consequently, a rise in cytosolic calcium levels. These studies demonstrate that live Mtb does not stimulate CR-dependent calcium signaling and indicate that this alteration of macrophage activation contributes to inhibition of phagosome-lysosome fusion.