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MOLECULAR AND CELLULAR PATHOGENESIS

Tripeptidyl Peptidase II Promotes Maturation of Caspase-1 in Shigella flexneri-Induced Macrophage Apoptosis

Hubert Hilbi, Robyn J. Puro, Arturo Zychlinsky
Hubert Hilbi
The Skirball Institute, Department of Microbiology and Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016
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Robyn J. Puro
The Skirball Institute, Department of Microbiology and Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016
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Arturo Zychlinsky
The Skirball Institute, Department of Microbiology and Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016
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DOI: 10.1128/IAI.68.10.5502-5508.2000
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    Fig. 1.

    TPPII is involved in Shigella-induced macrophage apoptosis. (A) Mouse J774 macrophages were treated with the TPPII inhibitor AAF-cmk (●), the proteasome-TPPII inhibitorclasto-Lactacystin β-lactone (lactacystin) (■), or the proteasome inhibitors LLnL (▴), LLM (□), LLL-vs (○), or with zFA-fmk (▵) and then infected with S. flexneri wild-type strain M90T or the nonvirulent strain BS176 (⧫) at an MOI of 10. The cytotoxicity was assayed by measuring the LDH release after 2 h. (B) J774 macrophages were treated with AAF-cmk (5 μM), LLM (100 μM), or the caspase-1 inhibitor YVAD (50 μM) and washed (AAF/w, LLM/w, and YVAD/w) prior to M90T infection. (C) Peritoneal macrophages were preincubated with a 10 μM concentration of the proteasome-TPPII inhibitors indicated or with YVAD-cmk (100 μM) and infected with M90T. (D) J774 macrophages were infected with M90T grown in the presence of 1 μM AAF-cmk (M90T/AAF) or 5 μM lactacystin (M90T/Lacta). In parallel, untreated macrophages (none) or macrophages treated with 1 μM AAF-cmk (AAF) or 5 μM lactacystin (Lacta) were infected with mock-treated M90T or BS176. Means and standard deviations of experiments done in triplicate are shown. Similar results were obtained in at least three (A and D) or two (B and C) independent experiments.

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    Fig. 2.

    TPPII is required for the maturation of caspase-1 inShigella-infected macrophages. (A) Peritoneal macrophages (None) and macrophages treated with AAF-cmk (AAF, 10 μM), lactacystin (Lacta, 30 μM), or YVAD-cmk (YVAD, 100 μM) were infected with virulent (M90T, 0.5 to 4 h) or nonvirulent (BS, 4 h)Shigella strains. Maturation of caspase-1 was analyzed by Western blotting in lysates of infected macrophages. (B) Differentiated, LPS-stimulated THP.1 cells (None) and cells treated with AAF-cmk (AAF, 10 μM), lactacystin (Lacta, 135 μM), or YVAD-cmk (YVAD, 0.5 mM) were infected with virulent (M90T, 0.5 to 4 h) or nonvirulent (BS, 4 h) Shigella strains. Maturation of IL-1β was analyzed by Western blotting in lysates of infected and naive (−LPS) macrophages. (C) The specific activity of purified caspase-1 against the fluorogenic peptide substrate YVAD-amc was determined in the absence or in presence of AAF-cmk, lactacystin, or YVAD-cmk at the concentrations indicated. The specific activity is defined as the number of micromoles of amc released per minute per milligram of protein. The experiments were done at least twice, and results similar to those shown were obtained each time.

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    Fig. 3.

    Shigella is protected within macrophages from gentamicin by AAF-cmk or lactacystin. J774 macrophages were treated with the TPPII inhibitors AAF-cmk (AAF, 1 μM) or lactacystin (Lacta, 5 μM), with the caspase inhibitors YVAD-cmk or zVAD-fmk (YVAD or zVAD, 100 μM), or with cytochalasin B (CytoB, 2.5 μg/ml), an inhibitor of actin polymerization. After infection with either wild-type (M90T) or avirulent (BS176) S. flexneri and gentamicin treatment, the CFU were determined. Means and standard deviations of experiments done in quadruplicate are shown. Similar results were obtained in three independent experiments.

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    Fig. 4.

    TPPII is involved in apoptosis induced by staurosporine and ATP. J774 macrophages were incubated with staurosporine (1 μM, 12 h) (A) or LPS-activated mouse peritoneal macrophages were treated with ATP (5 mM, 30 min) (B) in the absence of in the presence of AAF-cmk (●), lactacystin (■), or LLnL (▴) at the concentrations indicated. The cytotoxicity was determined by measuring the LDH release and is corrected for cell death caused by lactacystin alone. Means and standard deviations of experiments done in triplicate are shown. Similar results were obtained in three (A) or two (B) independent experiments.

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Tripeptidyl Peptidase II Promotes Maturation of Caspase-1 in Shigella flexneri-Induced Macrophage Apoptosis
Hubert Hilbi, Robyn J. Puro, Arturo Zychlinsky
Infection and Immunity Oct 2000, 68 (10) 5502-5508; DOI: 10.1128/IAI.68.10.5502-5508.2000

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Tripeptidyl Peptidase II Promotes Maturation of Caspase-1 in Shigella flexneri-Induced Macrophage Apoptosis
Hubert Hilbi, Robyn J. Puro, Arturo Zychlinsky
Infection and Immunity Oct 2000, 68 (10) 5502-5508; DOI: 10.1128/IAI.68.10.5502-5508.2000
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KEYWORDS

apoptosis
Caspase 1
macrophages
Serine Endopeptidases
Shigella flexneri

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