Diarrhea is one of the main problems of public health in the world. Shigellae are the etiological agents of dysentery, a very severe form of diarrhea that is often fatal in infants. We have recently described that Shigella flexneri induces host macrophages to die by apoptosis. The bacterial protein IpaB (Invasion plasmid antigen) is required for the induction of apoptosis. IpaB is localized in aggregates in the macrophage cytoplasm where it probably binds to specific proteins. The interaction between the bacterial pathogenic proteins and the host cell program for death is not understood. It is our long term goal to determine how shigella induces programmed cell death and the relevance of this phenomenon during in vivo infections.
Specific aims : (1) To determine the function of IpaB in the eukaryotic host. We will express IpaB in macrophages to determine whether this protein alone is sufficient to induce apoptosis. If IpaB is not sufficient to provoke cell suicide we plan to determine whether other bacterial factors are required. We will determine what is the final destination of IpaB within the host macrophage. We will establish whether the macrophage IpaB-binding proteins, identified through affinity purification, are biologically relevant and if they are, we will identify them. (2) To establish the cell specificity and the role of the host cell in shigella induced apoptosis. We will test cells of the myeloid lineage as well as other cell lines to investigate whether shigella sensitivity is developed during differentiation. Macrophage-resistant cell hybrids will be infected to determine whether macrophage sensitivity or cell resistance is a dominant trait. We will also identify the putative host cell factors that confer either sensitivity or resistance. In addition, we will examine the relationship between IpaB induced apoptosis and cellular genes and enzymes known to interfere or promote programmed cell death. (3) To determine whether macrophage apoptosis occurs in in vivo infections. We will compare in animals experimentally infected with either wild type or non-pathogenic strains of S. flexneri, the induction of apoptosis in intestinal macrophages and other cell types.
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