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Inhibition of Ca(2+) signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages.

Malik ZA, Denning GM, Kusner DJ - J. Exp. Med. (2000)

Bottom Line: Increased ¿Ca(2+)(c) induced by Ab-opsonized bacilli was associated with a 76% reduction in intracellular survival, compared with C-opsonized M. tuberculosis.Similarly, reversible elevation of macrophage ¿Ca(2+)(c) with the ionophore A23187 reduced intracellular viability by 50%.Ionophore-mediated elevation of ¿Ca(2+)(c) promoted the maturation of phagosomes containing live C-opsonized bacilli, as evidenced by acidification and accumulation of lysosomal protein markers.

View Article: PubMed Central - PubMed

Affiliation: Inflammation Program, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA.

ABSTRACT
Complement receptor (CR)-mediated phagocytosis of Mycobacterium tuberculosis by macrophages results in intracellular survival, suggesting that M. tuberculosis interferes with macrophage microbicidal mechanisms. As increases in cytosolic Ca(2+) concentration (¿Ca(2+)(c)) promote phagocyte antimicrobial responses, we hypothesized that CR phagocytosis of M. tuberculosis is accompanied by altered Ca(2+) signaling. Whereas the control complement (C)-opsonized particle zymosan (COZ) induced a 4.6-fold increase in ¿Ca(2+)(c) in human macrophages, no change in ¿Ca(2+)(c) occurred upon addition of live, C-opsonized virulent M. tuberculosis. Viability of M. tuberculosis and ingestion via CRs was required for infection of macrophages in the absence of increased ¿Ca(2+)(c), as killed M. tuberculosis or antibody (Ab)-opsonized, live M. tuberculosis induced elevations in ¿Ca(2+)(c) similar to COZ. Increased ¿Ca(2+)(c) induced by Ab-opsonized bacilli was associated with a 76% reduction in intracellular survival, compared with C-opsonized M. tuberculosis. Similarly, reversible elevation of macrophage ¿Ca(2+)(c) with the ionophore A23187 reduced intracellular viability by 50%. Ionophore-mediated elevation of ¿Ca(2+)(c) promoted the maturation of phagosomes containing live C-opsonized bacilli, as evidenced by acidification and accumulation of lysosomal protein markers. These data demonstrate that M. tuberculosis inhibits CR-mediated Ca(2+) signaling and indicate that this alteration of macrophage activation contributes to inhibition of phagosome-lysosome fusion and promotion of intracellular mycobacterial survival.

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Elevation of macrophage cytosolic Ca2+ during infection with live M. tuberculosis results in increased phagosomal maturation. (A) MDMs were preincubated with a 1:10,000 dilution of LysoTracker Red for 2 h, after which they were infected with live, C-op M. tuberculosis (H37Rv strain) at a 1:1 ratio for 20 min in CHBSS. Cells were then washed and repleted with RH, 1% serum, 20 μg/ml phosphatidylcholine vesicles, and LysoTracker (left column). 24 h after infection, MDMs were fixed with paraformaldehyde and prepared for confocal microscopy as described in Materials and Methods. Where indicated, infection occurred in the presence of 1 μM A23187 (center column) or A23187 in Ca2+-free EGTA buffer (right column). 24 h after infection, cells were fixed and prepared for confocal microscopy. Samples were stained with LysoTracker Red (top row, red) and auramine to detect M. tuberculosis (center row, green). Acquisition of both fluorescence emission maxima demonstrates colocalization of LysoTracker Red and live M. tuberculosis in MDMs incubated in A23187 (center column, yellow) but not in control Ca2+ buffer (left column) or EGTA buffer (right column). Crossover fluorescence did not contribute to the colocalization of signals, as cells labeled with either LysoTracker Red or auramine alone did not emit a detectable fluorescence signal when observed through the narrow bandpass filter appropriate for the other fluorochrome. (B) Summary of the percentage of phagosomes containing C-op, live M. tuberculosis that colocalize with cathepsin D, LAMP-1, CD63, or LysoTracker Red in each of the three buffer conditions. Each value is the mean percentage (± SEM) from at least three experiments.
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Figure 7: Elevation of macrophage cytosolic Ca2+ during infection with live M. tuberculosis results in increased phagosomal maturation. (A) MDMs were preincubated with a 1:10,000 dilution of LysoTracker Red for 2 h, after which they were infected with live, C-op M. tuberculosis (H37Rv strain) at a 1:1 ratio for 20 min in CHBSS. Cells were then washed and repleted with RH, 1% serum, 20 μg/ml phosphatidylcholine vesicles, and LysoTracker (left column). 24 h after infection, MDMs were fixed with paraformaldehyde and prepared for confocal microscopy as described in Materials and Methods. Where indicated, infection occurred in the presence of 1 μM A23187 (center column) or A23187 in Ca2+-free EGTA buffer (right column). 24 h after infection, cells were fixed and prepared for confocal microscopy. Samples were stained with LysoTracker Red (top row, red) and auramine to detect M. tuberculosis (center row, green). Acquisition of both fluorescence emission maxima demonstrates colocalization of LysoTracker Red and live M. tuberculosis in MDMs incubated in A23187 (center column, yellow) but not in control Ca2+ buffer (left column) or EGTA buffer (right column). Crossover fluorescence did not contribute to the colocalization of signals, as cells labeled with either LysoTracker Red or auramine alone did not emit a detectable fluorescence signal when observed through the narrow bandpass filter appropriate for the other fluorochrome. (B) Summary of the percentage of phagosomes containing C-op, live M. tuberculosis that colocalize with cathepsin D, LAMP-1, CD63, or LysoTracker Red in each of the three buffer conditions. Each value is the mean percentage (± SEM) from at least three experiments.

Mentions: 24 h after infection of human MDMs, live, C-op M. tuberculosis was located in immature phagosomes that exhibited low amounts of the lysosomal protein markers. The percentage of phagosomes positive for cathepsin D, LAMP-1, and CD63 were 32, 37, and 25%, respectively (Fig. 7). Additionally, only 41% of phagosomes containing live M. tuberculosis colocalized with LysoTracker Red. These results are in agreement with previous characterizations of the maturational state of M. tuberculosis–containing phagosomes in macrophages, as determined by epifluorescence, confocal immunofluorescence, and cryoimmunoelectron microscopy 10111223245455.


Inhibition of Ca(2+) signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages.

Malik ZA, Denning GM, Kusner DJ - J. Exp. Med. (2000)

Elevation of macrophage cytosolic Ca2+ during infection with live M. tuberculosis results in increased phagosomal maturation. (A) MDMs were preincubated with a 1:10,000 dilution of LysoTracker Red for 2 h, after which they were infected with live, C-op M. tuberculosis (H37Rv strain) at a 1:1 ratio for 20 min in CHBSS. Cells were then washed and repleted with RH, 1% serum, 20 μg/ml phosphatidylcholine vesicles, and LysoTracker (left column). 24 h after infection, MDMs were fixed with paraformaldehyde and prepared for confocal microscopy as described in Materials and Methods. Where indicated, infection occurred in the presence of 1 μM A23187 (center column) or A23187 in Ca2+-free EGTA buffer (right column). 24 h after infection, cells were fixed and prepared for confocal microscopy. Samples were stained with LysoTracker Red (top row, red) and auramine to detect M. tuberculosis (center row, green). Acquisition of both fluorescence emission maxima demonstrates colocalization of LysoTracker Red and live M. tuberculosis in MDMs incubated in A23187 (center column, yellow) but not in control Ca2+ buffer (left column) or EGTA buffer (right column). Crossover fluorescence did not contribute to the colocalization of signals, as cells labeled with either LysoTracker Red or auramine alone did not emit a detectable fluorescence signal when observed through the narrow bandpass filter appropriate for the other fluorochrome. (B) Summary of the percentage of phagosomes containing C-op, live M. tuberculosis that colocalize with cathepsin D, LAMP-1, CD63, or LysoTracker Red in each of the three buffer conditions. Each value is the mean percentage (± SEM) from at least three experiments.
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Figure 7: Elevation of macrophage cytosolic Ca2+ during infection with live M. tuberculosis results in increased phagosomal maturation. (A) MDMs were preincubated with a 1:10,000 dilution of LysoTracker Red for 2 h, after which they were infected with live, C-op M. tuberculosis (H37Rv strain) at a 1:1 ratio for 20 min in CHBSS. Cells were then washed and repleted with RH, 1% serum, 20 μg/ml phosphatidylcholine vesicles, and LysoTracker (left column). 24 h after infection, MDMs were fixed with paraformaldehyde and prepared for confocal microscopy as described in Materials and Methods. Where indicated, infection occurred in the presence of 1 μM A23187 (center column) or A23187 in Ca2+-free EGTA buffer (right column). 24 h after infection, cells were fixed and prepared for confocal microscopy. Samples were stained with LysoTracker Red (top row, red) and auramine to detect M. tuberculosis (center row, green). Acquisition of both fluorescence emission maxima demonstrates colocalization of LysoTracker Red and live M. tuberculosis in MDMs incubated in A23187 (center column, yellow) but not in control Ca2+ buffer (left column) or EGTA buffer (right column). Crossover fluorescence did not contribute to the colocalization of signals, as cells labeled with either LysoTracker Red or auramine alone did not emit a detectable fluorescence signal when observed through the narrow bandpass filter appropriate for the other fluorochrome. (B) Summary of the percentage of phagosomes containing C-op, live M. tuberculosis that colocalize with cathepsin D, LAMP-1, CD63, or LysoTracker Red in each of the three buffer conditions. Each value is the mean percentage (± SEM) from at least three experiments.
Mentions: 24 h after infection of human MDMs, live, C-op M. tuberculosis was located in immature phagosomes that exhibited low amounts of the lysosomal protein markers. The percentage of phagosomes positive for cathepsin D, LAMP-1, and CD63 were 32, 37, and 25%, respectively (Fig. 7). Additionally, only 41% of phagosomes containing live M. tuberculosis colocalized with LysoTracker Red. These results are in agreement with previous characterizations of the maturational state of M. tuberculosis–containing phagosomes in macrophages, as determined by epifluorescence, confocal immunofluorescence, and cryoimmunoelectron microscopy 10111223245455.

Bottom Line: Increased ¿Ca(2+)(c) induced by Ab-opsonized bacilli was associated with a 76% reduction in intracellular survival, compared with C-opsonized M. tuberculosis.Similarly, reversible elevation of macrophage ¿Ca(2+)(c) with the ionophore A23187 reduced intracellular viability by 50%.Ionophore-mediated elevation of ¿Ca(2+)(c) promoted the maturation of phagosomes containing live C-opsonized bacilli, as evidenced by acidification and accumulation of lysosomal protein markers.

View Article: PubMed Central - PubMed

Affiliation: Inflammation Program, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA.

ABSTRACT
Complement receptor (CR)-mediated phagocytosis of Mycobacterium tuberculosis by macrophages results in intracellular survival, suggesting that M. tuberculosis interferes with macrophage microbicidal mechanisms. As increases in cytosolic Ca(2+) concentration (¿Ca(2+)(c)) promote phagocyte antimicrobial responses, we hypothesized that CR phagocytosis of M. tuberculosis is accompanied by altered Ca(2+) signaling. Whereas the control complement (C)-opsonized particle zymosan (COZ) induced a 4.6-fold increase in ¿Ca(2+)(c) in human macrophages, no change in ¿Ca(2+)(c) occurred upon addition of live, C-opsonized virulent M. tuberculosis. Viability of M. tuberculosis and ingestion via CRs was required for infection of macrophages in the absence of increased ¿Ca(2+)(c), as killed M. tuberculosis or antibody (Ab)-opsonized, live M. tuberculosis induced elevations in ¿Ca(2+)(c) similar to COZ. Increased ¿Ca(2+)(c) induced by Ab-opsonized bacilli was associated with a 76% reduction in intracellular survival, compared with C-opsonized M. tuberculosis. Similarly, reversible elevation of macrophage ¿Ca(2+)(c) with the ionophore A23187 reduced intracellular viability by 50%. Ionophore-mediated elevation of ¿Ca(2+)(c) promoted the maturation of phagosomes containing live C-opsonized bacilli, as evidenced by acidification and accumulation of lysosomal protein markers. These data demonstrate that M. tuberculosis inhibits CR-mediated Ca(2+) signaling and indicate that this alteration of macrophage activation contributes to inhibition of phagosome-lysosome fusion and promotion of intracellular mycobacterial survival.

Show MeSH
Related in: MedlinePlus