| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
(1) Shigella, the causative agent of bacillary dysentery, are capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. We investigated the role of Cdc42 in actin-based movement of Shigella. The several experiments using microinjection, in vitro motility assay in Xenopus egg extracts, and pyrene actin assay revealed that Cdc42 activity is involved in initiating actin nucleation mediated by VirG-N-WASP-actin-related protein (Arp) 2/3 complex formed on intracellular Shigella.
(2) We showed that profilin I is essential to the rapid movement of Shigella in infected cells. Two mutants of profilin which failed to bind G-actin, or to bind with poly-L-proline, did not support fast moving of Shigella. The results indicate that profilin I associated with N-WASP is an essential host factor for sustaining efficient intra- and intercellular spreading of Shigella.
(3) Since all of the WASP family proteins including N-WASP induce actin polymerization by recruiting Arp2/3 complex, we investigated their involvement in Shigella motility. We showed that VirG binds to N-WASP but not the other WASP family proteins. We observed that in hemtopoietic cells such as macrophages, polymorphonuclear leukocytes (PMNs) and platelets, WASP was predominantly expressed, while the expression of N-WASP was greatly suppressed. Indeed, unlike Listeria, Shigella was unable to move in macrophages at all, though the movement was restored as N-WASP was ectopically expressed. Thus, our findings demonstrate that N-WASP is a specific ligand of VirG, which determines the host cell type allowing actin-based spreading of Shigella.
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