| Co-Investigator(Kenkyū-buntansha) |
TATEDA Kazuhiro Toho University, School of Medicine, Assistant Professor, 医学部, 講師 (20236558)
ISHII Yoshikazu Toho University, School of Medicine, Research Associate, 医学部, 助手2 (90246695)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2006: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
1. Development of quorum-sensing inhibitors
We synthesized the quorum-sensing autoinducer analogs. Their analogs are based on the information of the three-dimensional structure of LasR-autoinducer complex provided for the homology modeling with TraR-autoinducer complex. The efficient screening assay of Pseudomonas LasR-dependent lasI promoter activity in 96-well format was established using derivative strain from E.coli MC4100. The assay was found to be quite sensitive to find active principles because ED_<50> for the agonistic activity of natural ligand,3-oxo-C_<12>-homoserine lactone (3-oxo-C_<12>-HSL), was as low as 0.4 nM. Some of the reported antagonists conversely showed agonistic activity in this system and some others showed toxic effect on the reporter E.coli strain used. We concluded that they are not true antagonists in our system. We found two true antagonistic compounds of homoserine lactone analogs, although their potencies were moderate. In the presence of these two subst
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ances at 10 μM, biofilm formation was enhanced significantly in the system using P.aeruginosa PAO1,probably perturbing quorum sensing signaling cascade.
2. Mechanistic studies of the function of quorum-sensing autoinducer against the host cells
3-oxo-C_<12>-HSL has an immunomodulatory activity, such as induction of IL-8 and prostaglandin E_1. Recently, we reported that 3-oxo-C_<12>-HSL induces apoptosis in eukaryotic cells. Interestingly, induction of apoptosis was observed in macrophages and neutrophils, but not fibroblast or epithelial cells, although molecular mechanisms of this phenomenon are largely unknown. We synthesized a series of acyl-HSL analogs to characterize crucial structures required for induction of apoptosis in macrophages. The results of structure-activity relationship revealed the structural characteristics of the acyl-HSL analogs necessary for the apoptosis-inducing activity in macrophages, suggesting the presence of a putative receptor in eukaryotic cells. Further investigation to discover the molecular target of 3-oxo-C_<12>-HSL is ongoing not only for induction of apoptosis in macrophages, but also for IL-8 production in epithelial cells.
Furthermore, we examined potential of bactericidal activity of the tetramic acids, which was derived from the autoiducers, against Clostridium difficile, an anerobic gram-positive bacterium. The structure-activity correlation and the mechanisms of action were examined in several ways, such as time-kill assay and observation of changes of morphology by electron microscopy. The cell surface was suggested to be one of the targets for bactericidal activity of tetramic acid, because the disturbance of cell membrane was demonstrated by electron microscopy after exposure to tetramic acid. Less
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