Shigellae are the etiological agents of bacillary dysentery, a severe form of diarrhea that is often fatal in infants. Shigellosis is an acute inflammatory disease. Here we propose to investigate the role of apoptosis in the initiation of inflammation.
We have demonstrated that Shigella induces apoptosis in macrophages in vitro and in vivo. Shigella first invades cells and then escapes from the phagocytic vacuole into the cytoplasm. In the cytoplasm, Shigella secretes the plasmid-encoded virulence factor Invasion Plasmid Antigen (Ipa) B which is necessary to induce cell death. IpaB binds to caspase (Casp)-1, a host cysteine protease that is required for Shigella induced apoptosis. Apoptosis mediated by Casp-1 appears to be pro- inflammatory in Shigella infections, since Casp-1 proteolytically activates the cytokines pro-Interleukin (IL)-1beta and pro-IL-18. Macrophages infected with Shigella release mature IL-1beta and IL-18. Furthermore, casp-1 knock-out mice do not mount an acute inflammation in response to Shigella infection. In vivo, some apoptotic cells are localized to regions of the lymphoid follicle where Shigella is not detectable. This difference in distribution suggested that Shigella possesses a second cytotoxic molecule, not IpaB, that can diffuse within infected tissue.
We identified the novel diffusible cytotoxic activity in Shigella culture supernatants as Bacterial Lipoproteins (BLP). We also demonstrated that BLP activates both apoptosis and the host cell transcription factor Nuclear Factor - kappa B (NF-kappaB) through the Toll Like Receptor (TLR)2. In this application we propose to further understand the significance of apoptosis in Shigella infections. More specifically we will determine: (1) the role of Casp-1 activated cytokines in acute inflammation and whether apoptosis is required for the release of mature IL-1beta and IL-18 and (2) the signal transduction pathway activated by TLR2 after treatment with BLP and the role of BLP and TLR2 in vivo.