| Shigella, the etiological agent of dysentery, kills macrophages by inducing apoptosis. Deletion mutants in the invasion plasmid antigen B (ipaB) of Shigella flexneri are not cytotoxic. Here, we localized IpaB to the cytoplasm of macrophages infected with S. flexneri. Purified IpaB induced apoptosis when microinjected into macrophages, indicating that IpaB is sufficient to induce apoptosis. Using a GST-IpaB fusion protein as a ligand in affinity purification we isolated four IpaB-binding proteins from macrophages which were identified as the precursor and the mature polypeptides of Interleukin-1{dollar}beta{dollar} Converting Enzyme (ICE) or a highly homologous protease in the caspase family.; To elucidate which caspase binds to IpaB, ligand blot assays using {dollar}sp{lcub}32{rcub}{dollar}P labeled GST-IpaB as a probe were performed to test the direct binding between IpaB and ICE (caspase-1), Cpp32 (caspase-3) or Nedd-2 (caspase-2), representatives of the three known groups of the caspase family. IpaB was detected to bind to His-ICE fusion protein but not His-Cpp32 or His-Nedd-2. Furthermore, ICE is activated during S. flexneri infection, and specific inhibitors of ICE prevented Shigella induced apoptosis. The data suggest that ICE is the executioner of the apoptosis induced by IpaB.; The mechanism of ICE activation by IpaB was investigated. During Shigella infection, mature ICE accumulated with an increasing cleavage of IL-1{dollar}beta{dollar}. By ligand blot assay, IpaB was shown to bind to ICE precursor, but not to mature ICE P10 or P20 suggesting that ICE activation is due to ICE maturation. However, IpaB could not activate ICE maturation in vitro. Probably, intact cell structure is critical for IpaB to function as a ICE activator. |
