|Budget Amount *help
¥14,100,000 (Direct Cost : ¥14,100,000)
Fiscal Year 1998 : ¥6,700,000 (Direct Cost : ¥6,700,000)
Fiscal Year 1997 : ¥7,400,000 (Direct Cost : ¥7,400,000)
The project was aimed to investigate the molecular mechanisms of shigellae invasion and the subsequent spreading process within and among cells that is a critical step leading to shigellosis at early stage of infection of colonic epithelial cells by Shigella. In the study, the following findings are obtained.
Shigellae invasion : Ipa proteins secreted from shigellae play a crucial role in invasion of epithelial cells, since the Ipa proteins can interact with a5b1 integrin which promote bacterial invasion. The interaction was found to be involved in the activation of focal adhesion plaques, an even downstream of RhoA-regulated cellular signal, since C3 transferase can almost completely shut off the bacterial entry, while injection of Val l4RhoA, a constitutively activated form of RhoA protein, can promote bacterial internalization. The addition of Ipa proteins to the serum-starved fibroblast or epithelial cells can induce rapid rearrangement of actin cytoskeletons and condensation of maj
or components of focal adhesion plaques. For the efficient shigellae invasion of epithelial cells, we noted another set of proteins including VirA secreted through Type-III system is crucial. Indeed, the expression of VirA or injection of GST-VirA fusions in mammalian cells can induce membrane ruffling. These results strongly indicate that shigellae invasion, a process resembling macropinocytosis, could be conducted by a cellular signal regulated by RhoA together with the induction of an additional signal possibly directed by the subset of shigellae proteins injected by the Type III system.
Shigellae spreading : Shigellae spreading within and among epithelial cells has been shown to be depended on the actin polymerization at one pole of bacterium in infected cells, that is mediated by shigellae VirG protein. In this study, we found that VirG can directly interact with two host factors, one is vinculin and the other is N-WASP, and the binding of the proteins under in vitro and in vivo conditions are mediated by the glycine-rich repeats domain of VirG.We also demonstrated that the bingeing of VirG to the two host proteins are functionally involved in the formation of actin tail form intracellular shigellae. Less