Mycobacterium tuberculosis Phagosomes Exhibit Altered Calmodulin-Dependent Signal Transduction: Contribution to Inhibition of Phagosome-Lysosome Fusion and Intracellular Survival in Human Macrophages

Abstract

Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca2+ ([Ca2+]c), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca2+]c that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca2+-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca2+]c resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca2+ ionophore-induced phagosomal maturation and enhanced the bacilli’s intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca2+-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.

ATP Stimulates Human Macrophages to Kill Intracellular Virulent Mycobacterium tuberculosis Via Calcium-Dependent Phagosome-Lysosome Fusion

“…Because the confocal measurements were acquired in a blinded fashion, inherent limitations of the technique (e.g., the lack of a single, fully specific marker of M lysosomes) should be distributed equally among the experimental groups. The use of this technique by our laboratory (9,22) and others (23)(24)(25) has resulted in strong interobserver correlations, as well as excellent agreement with the results of immunoelectron microscopy (25)(26)(27).…”

“…Our previous work (9,22) and the results presented herein demonstrate that [Ca 2ϩ ] c regulates the intracellular viability of M. tuberculosis in two different experimental models designed to represent distinct stages in the host-pathogen interaction. In the first case, primary infection of naive human M , the intracellular survival of M. tuberculosis requires inhibition of M Ca 2ϩ signaling, which closely correlates with inhibition of P-L fusion (9,22).…”

“…In the first case, primary infection of naive human M , the intracellular survival of M. tuberculosis requires inhibition of M Ca 2ϩ signaling, which closely correlates with inhibition of P-L fusion (9,22). Transient (20 min) pharmacologic elevation of [Ca 2ϩ ] c at the time of infection results in P-L fusion and decreased viability of intracellular bacilli.…”

“…The degree of maturation of phagosomes containing live or killed M. tuberculosis was assessed by colocalization of the bacilli with the acidophilic dye LysoTracker Red (Molecular Probes, Eugene, OR) and the lysosomal protein markers cathepsin D, CD63, and lysosome-associated membrane protein (LAMP)-1, as described previously (9,22). LysoTracker Red was incubated at a 1/10,000 dilution with M monolayers in RPMI 1640, 20 mM HEPES, and 2.5% autologous serum for 2 h at 37°C.…”

“…To characterize the biochemical determinants of ATP e -stimulated killing of intracellular M. tuberculosis, we used an in vitro model system in which blood monocytes are differentiated to a M phenotype by cultivation for 5 days, followed by purification via adherence to tissue culture plates (6,17,22). M were infected with the virulent H37Rv strain of M. tuberculosis, at a MOI of 1:1 for 24 h, followed by incubation with 3 mM ATP.…”