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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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Viability of M. tuberculosis is required for inhibition of macrophage Ca2+ signaling. (A) An aliquot of Erdman M. tuberculosis was heated to 100°C for 10 min, and efficiency of killing was demonstrated by absence of growth on 7H11 agar. After opsonization in 50% autologous serum, heat-killed bacilli (Hk-MTb) were incubated with Fura2-loaded macrophages at a bacteria/MDM ratio of 10:1. Levels of macrophage [Ca2+]c were determined from the ratio of fluorescence of Fura2, as noted in Materials and Methods. (B) MDMs were incubated with F(ab′)2 fragments of α-CD18 mAb for 30 min at 15°C, followed by repeated washing to remove unbound Ab. MDMs were warmed to 37°C and incubated with heat-killed M. tuberculosis. At the indicated time, 100 nM PAF was added to evaluate CD18-independent Ca2+ signaling. (C) After loading with Fura2-AM, macrophages were incubated in EHBSS for 5 min before addition of Hk-MTb and determination of [Ca2+]c. Thapsigargin (1 μM) was added at the indicated time. (D) MDMs were incubated in EHBSS with 25 μM MAPTAM for 30 min at 37°C, followed by addition of Hk-MTb (arrow). (E) Gamma-irradiated H37Rv, preopsonized in 50% autologous serum, was incubated with MDMs in CHBSS, and [Ca2+]c levels were determined via fluorescence of Fura2. Results in A are representative of data from 16 identical experiments; at least 4 separate experiments were conducted for each of the other conditions.
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Figure 4: Viability of M. tuberculosis is required for inhibition of macrophage Ca2+ signaling. (A) An aliquot of Erdman M. tuberculosis was heated to 100°C for 10 min, and efficiency of killing was demonstrated by absence of growth on 7H11 agar. After opsonization in 50% autologous serum, heat-killed bacilli (Hk-MTb) were incubated with Fura2-loaded macrophages at a bacteria/MDM ratio of 10:1. Levels of macrophage [Ca2+]c were determined from the ratio of fluorescence of Fura2, as noted in Materials and Methods. (B) MDMs were incubated with F(ab′)2 fragments of α-CD18 mAb for 30 min at 15°C, followed by repeated washing to remove unbound Ab. MDMs were warmed to 37°C and incubated with heat-killed M. tuberculosis. At the indicated time, 100 nM PAF was added to evaluate CD18-independent Ca2+ signaling. (C) After loading with Fura2-AM, macrophages were incubated in EHBSS for 5 min before addition of Hk-MTb and determination of [Ca2+]c. Thapsigargin (1 μM) was added at the indicated time. (D) MDMs were incubated in EHBSS with 25 μM MAPTAM for 30 min at 37°C, followed by addition of Hk-MTb (arrow). (E) Gamma-irradiated H37Rv, preopsonized in 50% autologous serum, was incubated with MDMs in CHBSS, and [Ca2+]c levels were determined via fluorescence of Fura2. Results in A are representative of data from 16 identical experiments; at least 4 separate experiments were conducted for each of the other conditions.

Mentions: The specific virulence determinants that enable M. tuberculosis to survive within the phagosomes of human macrophages are unknown. In addition, there are no avirulent strains of M. tuberculosis that may be used to define the molecular mechanisms that regulate essential pathogenic interactions between tubercle bacilli and mononuclear phagocytes. Despite these limitations, considerable evidence indicates that the failure of M. tuberculosis–containing phagosomes to mature into acidic microbicidal phagolysosomes is an important component of tuberculous pathogenesis 245455. Clemens and Horwitz have demonstrated that this inhibition of phagosomal maturation is dependent on the viability of M. tuberculosis, as phagosomes containing heat-killed M. tuberculosis develop into mature phagolysosomes 2324. We tested the hypothesis that the M. tuberculosis–induced inhibition of macrophage Ca2+ signaling would demonstrate a similar requirement for bacterial viability. Erdman M. tuberculosis was killed by heating to 100°C for 10 min, followed by opsonization in autologous, nonimmune serum as described above for live bacilli 2324. Particular care was taken to ensure that the preparation of heat-killed M. tuberculosis consisted of >95% single bacilli, as noted in Materials and Methods. The loss of viability of heat-killed M. tuberculosis was verified by absence of growth on 7H11 agar. Heat-killed Erdman M. tuberculosis induced a rapid and significant rise in macrophage [Ca2+]c (Fig. 4 A; fold-increase in [Ca2+]c = 3.8; range, 2.1–6.5-fold; n = 16), which closely resembled that induced by COZ. Utilization of an alternate protocol for heat killing (80°C, 60 min; reference 5) resulted in similar stimulation of increased [Ca2+]c by dead, C-op M. tuberculosis (data not shown). The increase in levels of macrophage [Ca2+]c induced by heat-killed M. tuberculosis was completely inhibited by preincubation of these cells with F(ab′)2 fragments of α-CD18 mAb (Fig. 4 B), indicating a major role for CR3 and/or CR4 in the initiation of this response. Studies with Ca2+-free media (Fig. 4 C) and intracellular Ca2+ buffering (Fig. 4 D) indicated that the increase in [Ca2+]c stimulated by heat-killed M. tuberculosis resulted from both release of Ca2+ from intracellular stores as well as influx of extracellular Ca2+.


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)

Viability of M. tuberculosis is required for inhibition of macrophage Ca2+ signaling. (A) An aliquot of Erdman M. tuberculosis was heated to 100°C for 10 min, and efficiency of killing was demonstrated by absence of growth on 7H11 agar. After opsonization in 50% autologous serum, heat-killed bacilli (Hk-MTb) were incubated with Fura2-loaded macrophages at a bacteria/MDM ratio of 10:1. Levels of macrophage [Ca2+]c were determined from the ratio of fluorescence of Fura2, as noted in Materials and Methods. (B) MDMs were incubated with F(ab′)2 fragments of α-CD18 mAb for 30 min at 15°C, followed by repeated washing to remove unbound Ab. MDMs were warmed to 37°C and incubated with heat-killed M. tuberculosis. At the indicated time, 100 nM PAF was added to evaluate CD18-independent Ca2+ signaling. (C) After loading with Fura2-AM, macrophages were incubated in EHBSS for 5 min before addition of Hk-MTb and determination of [Ca2+]c. Thapsigargin (1 μM) was added at the indicated time. (D) MDMs were incubated in EHBSS with 25 μM MAPTAM for 30 min at 37°C, followed by addition of Hk-MTb (arrow). (E) Gamma-irradiated H37Rv, preopsonized in 50% autologous serum, was incubated with MDMs in CHBSS, and [Ca2+]c levels were determined via fluorescence of Fura2. Results in A are representative of data from 16 identical experiments; at least 4 separate experiments were conducted for each of the other conditions.
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Related In: Results  -  Collection

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Figure 4: Viability of M. tuberculosis is required for inhibition of macrophage Ca2+ signaling. (A) An aliquot of Erdman M. tuberculosis was heated to 100°C for 10 min, and efficiency of killing was demonstrated by absence of growth on 7H11 agar. After opsonization in 50% autologous serum, heat-killed bacilli (Hk-MTb) were incubated with Fura2-loaded macrophages at a bacteria/MDM ratio of 10:1. Levels of macrophage [Ca2+]c were determined from the ratio of fluorescence of Fura2, as noted in Materials and Methods. (B) MDMs were incubated with F(ab′)2 fragments of α-CD18 mAb for 30 min at 15°C, followed by repeated washing to remove unbound Ab. MDMs were warmed to 37°C and incubated with heat-killed M. tuberculosis. At the indicated time, 100 nM PAF was added to evaluate CD18-independent Ca2+ signaling. (C) After loading with Fura2-AM, macrophages were incubated in EHBSS for 5 min before addition of Hk-MTb and determination of [Ca2+]c. Thapsigargin (1 μM) was added at the indicated time. (D) MDMs were incubated in EHBSS with 25 μM MAPTAM for 30 min at 37°C, followed by addition of Hk-MTb (arrow). (E) Gamma-irradiated H37Rv, preopsonized in 50% autologous serum, was incubated with MDMs in CHBSS, and [Ca2+]c levels were determined via fluorescence of Fura2. Results in A are representative of data from 16 identical experiments; at least 4 separate experiments were conducted for each of the other conditions.
Mentions: The specific virulence determinants that enable M. tuberculosis to survive within the phagosomes of human macrophages are unknown. In addition, there are no avirulent strains of M. tuberculosis that may be used to define the molecular mechanisms that regulate essential pathogenic interactions between tubercle bacilli and mononuclear phagocytes. Despite these limitations, considerable evidence indicates that the failure of M. tuberculosis–containing phagosomes to mature into acidic microbicidal phagolysosomes is an important component of tuberculous pathogenesis 245455. Clemens and Horwitz have demonstrated that this inhibition of phagosomal maturation is dependent on the viability of M. tuberculosis, as phagosomes containing heat-killed M. tuberculosis develop into mature phagolysosomes 2324. We tested the hypothesis that the M. tuberculosis–induced inhibition of macrophage Ca2+ signaling would demonstrate a similar requirement for bacterial viability. Erdman M. tuberculosis was killed by heating to 100°C for 10 min, followed by opsonization in autologous, nonimmune serum as described above for live bacilli 2324. Particular care was taken to ensure that the preparation of heat-killed M. tuberculosis consisted of >95% single bacilli, as noted in Materials and Methods. The loss of viability of heat-killed M. tuberculosis was verified by absence of growth on 7H11 agar. Heat-killed Erdman M. tuberculosis induced a rapid and significant rise in macrophage [Ca2+]c (Fig. 4 A; fold-increase in [Ca2+]c = 3.8; range, 2.1–6.5-fold; n = 16), which closely resembled that induced by COZ. Utilization of an alternate protocol for heat killing (80°C, 60 min; reference 5) resulted in similar stimulation of increased [Ca2+]c by dead, C-op M. tuberculosis (data not shown). The increase in levels of macrophage [Ca2+]c induced by heat-killed M. tuberculosis was completely inhibited by preincubation of these cells with F(ab′)2 fragments of α-CD18 mAb (Fig. 4 B), indicating a major role for CR3 and/or CR4 in the initiation of this response. Studies with Ca2+-free media (Fig. 4 C) and intracellular Ca2+ buffering (Fig. 4 D) indicated that the increase in [Ca2+]c stimulated by heat-killed M. tuberculosis resulted from both release of Ca2+ from intracellular stores as well as influx of extracellular Ca2+.

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