| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
In this study, we focused on the analysis of the mechanism of autophagy induced by invasive pathogens, and planned to analyze the mechanism of autophagy induced by Shigella and another pathogens. Since, in antuohagy induced by Shigella, Atg5 play as a sensor for intracellular Shigella, we, at first, tried to find novel Atg5 interacting host proteins. By yeast-two-hybrid screening, we obtained novel protein, named Afp, as a candidate of Atg5-interacting protein. By using mammalian cells expressing GFP-LC3 and Afp-Myc, we found that Afp is localized on the surface of autophagosomes containing Shigella and group A Sreptococcus. In knock-down experiment, the reduction of the Afp expression level leads to the decrease of the autophagic activity induced by rapamycin treatment. By the result from domain analysis of Afp for the binding to Atg5, we succeeded in producing one-amino acid altered Afp mutant protein that is deficient in Atg5 binding. Next, we analyzed the autophagy inducd by Listeria monocytogenes infection in epithelial cells. L. monocytogenes is known as a intracellular motile pathogen like Shigella. As a result from screening using L. moncytogenes mutant strains, we found ActA has a pivotal role in escaping from autophagy. The recognition mechanism of autophagy for L. moncytogenesi looks different from that for Shigella and GAS. Our data suggests that L. moncytogenes is recognized as protein aggregate by host cells.
Based on our data, we may conclude that Afp is a novel protein that plays a pivotal role in autophagy induced by pathogens and mTOR inhibition, and intracellular motile pathogens such as Shigella and Listeria have a common strategy to escape from autophagy in cytoplasm of host cells.
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