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Cellular Microbiology: Pathogen-Host Cell Molecular Interactions

Listeria monocytogenes Desensitizes Immune Cells to Subsequent Ca2+ Signaling via Listeriolysin O-Induced Depletion of Intracellular Ca2+ Stores

Nelson O. Gekara, Lothar Groebe, Nuno Viegas, Siegfried Weiss
Nelson O. Gekara
1Department of Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
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  • For correspondence: nelson.gekara@helmholtz-hzi.de
Lothar Groebe
2Department of Mucosal Immunity, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
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Nuno Viegas
1Department of Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
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Siegfried Weiss
1Department of Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
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DOI: 10.1128/IAI.00622-07
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  • FIG. 1.
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    FIG. 1.

    L. monocytogenes (L.m) and LLO renders BMMCs inert to antigen-induced Ca2+ mobilization. (A) BMMCs were exposed to L. monocytogenes and loaded with Indo 1-AM and then incubated on ice with an IgE antibody specific for DNP. After the unbound antibody was washed off, cells were stored on ice. When ready for calcium measurements, cells were warmed up to 37°C before DNP-bovine serum albumin (BSA) was added to cross-link the FcεRI. Stimulation was done in a Ca2+-containing medium. (B) BMMCs were left untreated or pretreated with LLO (0.25 μg/ml/ml) or thapsigargin (Thaps) (1 μM) for 4 h, loaded with Indo 1-AM, and then stimulated via FcεRI as described for panel A. Each trace represents the average of intracellular Ca2+ levels in the cells during the time of acquisition. A 30-s baseline was recorded each time before stimulation. The arrows indicate the time points of stimulation. The experiment was repeated at least three times with similar results.

  • FIG. 2.
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    FIG. 2.

    Preincubation with L. monocytogenes (L.m) renders cells resistant to LLO-induced calcium signals. After incubation with (or without) L. monocytogenes or the LLO-deficient L. monocytogenes Δhly mutant (L.mΔhly) (multiplicity of infection, 100) for 3 h, BMMCs were washed and then loaded with Indo 1-AM in penicillin-streptomycin-supplemented medium for 45 min (to kill all the bacteria). Cells were again washed and then suspended in Ca2+-containing medium and analyzed by flow cytometry for intracellular Ca2+ mobilization following stimulation with LLO (0.25 μg/ml). The arrow indicates the time point of stimulation. The experiment was repeated at least three times with similar results.

  • FIG. 3.
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    FIG. 3.

    LLO-pretreated cells exhibit diminished intracellular Ca2+ release but respond normally to ionophore-mediated Ca2+ influx. (A) Pretreated cells are refractory to LLO but not to ionomycin-mediated Ca2+ mobilization. BMMCs were pretreated (or not) with LLO (0.25 μg/ml) for 3 h, loaded with Indo 1-AM, and resuspended in Ca2+-containing medium. LLO was added to such cells at the time point indicated by the arrow marked “LLO,” and intracellular Ca2+ was measured. At the time point indicated by the other arrow, 1 μM ionomycin was added. (B) LLO-induced Ca2+ responses in pretreated cells under Ca2+-free conditions. BMMCs were incubated with (or without) LLO (0.25 μg/ml) for 4 h and then loaded with Indo 1-AM. To evaluate only the Ca2+ release from the intracellular stores, cells were thoroughly washed in Ca2+-free medium and then resuspended in Ca2+-free medium before stimulation with LLO (0.25 μg/ml) at the time point indicated by the arrow.

  • FIG. 4.
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    FIG. 4.

    LLO depletes intracellular Ca2+ stores. LLO (0.25 μg/ml) was added to Indo 1-AM-loaded BMMCs, and intracellular Ca2+ was measured. Then, thapsigargin (Thaps; 1 μM) was added to evaluate the remaining Ca2+ release from intracellular stores (purple trace). Vice versa, thapsigargin was first added to cells to achieve maximum Ca2+ release from intracellular stores before the addition of 0.25 μg/ml LLO (green trace). Note that unlike in untreated cells, hardly any Ca2+ was released from intracellular stores by thapsigargin after LLO pretreatment. Ca2+ mobilization induced by LLO was also greatly diminished in thapsigargin-pretreated cells. Since the experiment was done in Ca2+-containing medium, note that the minor peak elicited by LLO in thapsigargin-pretreated cells is most likely due to a Ca2+ influx via the toxin pores. (B) Depletion of intracellular Ca2+ stores by L. monocytogenes (L.m). BMMCs were incubated for 3 h with L. monocytogenes or the Δhly mutant (L.mΔhly) (multiplicity of infection, 100) and then loaded with Indo 1-AM and treated with thapsigargin (1 μM) to evaluate the maximum Ca2+ release from intracellular stores. Arrows indicate the time points of additions of LLO and thapsigargin.

  • FIG. 5.
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    FIG. 5.

    Refractory Ca2+ signaling wanes with longer periods of toxin exposure. Primary T cells were pretreated (or untreated) with LLO (0.25 μg/ml) for 4 h or 18 h. To evaluate the intracellular Ca2+ stored in such cells, cells were labeled with Indo 1-AM and washed thoroughly as described in Materials and Methods and then resuspended in Ca2+-free medium before they were restimulated with 0.25 μg/ml LLO (the arrow indicates the point of stimulation). It is, however, worth pointing out that with our culture conditions, the indicated T cells showed no appreciable proliferation. Thus, with or without cellular growth, cells do recover from the indicated toxin effects.

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Listeria monocytogenes Desensitizes Immune Cells to Subsequent Ca2+ Signaling via Listeriolysin O-Induced Depletion of Intracellular Ca2+ Stores
Nelson O. Gekara, Lothar Groebe, Nuno Viegas, Siegfried Weiss
Infection and Immunity Jan 2008, 76 (2) 857-862; DOI: 10.1128/IAI.00622-07

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Listeria monocytogenes Desensitizes Immune Cells to Subsequent Ca2+ Signaling via Listeriolysin O-Induced Depletion of Intracellular Ca2+ Stores
Nelson O. Gekara, Lothar Groebe, Nuno Viegas, Siegfried Weiss
Infection and Immunity Jan 2008, 76 (2) 857-862; DOI: 10.1128/IAI.00622-07
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KEYWORDS

bacterial toxins
calcium
Calcium Signaling
Heat-Shock Proteins
Hemolysin Proteins
Listeria monocytogenes
mast cells

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