| 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 |
This study was aimed to identify bacterial genes that are responsible for the "host-dependency". The phenomena "host-dependency" includes findings that bacteria express some virulence genes, or secrete some virulence factors, in response to a direct contact of bacterium with host cells. For this purpose, we focused on enteropathogenic. Escherichia coli, because our study has indicated that the bacteria secrete certain virulence factors termed effectors only in a host-dependent manner. We tried to construct a system using a Cre recombinase as a reporter, and a loxP-sacB-tet-loxP gene cassette to monitors the reporter activity. Initial attempts to insert the gene cassette into bacterial chromosome were failed, probably because of structure of the constructed plasmid. We are now using rpsL, a streptomycin resistant marker gene, instead of sacB, which gave us good results.
We also have established a relatively simple system monitoring "host-dependency" in vivo and in vitro. In vivo system uses cultured cells, in which effector translocation into host cytoplasm can be detected by a simple colorirnetric assay. In vitro system uses a recombinant Tir protein, which consists of GST and intimin-binding region of Tir. This protein (TirIBD) binds to the bacterial surface intimin, so that an artificial Tir-intimin binding can be reproduced in a cell-free (in vitro) system.
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