In vitro reconstitution of Type III secretion system - basic and applied research -

SptP是沙门氏菌的毒力效应蛋白，其参与细菌侵入宿主细胞。为了有效分泌，SptP通过其伴侣结合结构域，残基35-139与SptP特异性伴侣SicP形成复合物。我们通过体外重构实验表明SptP的106-136位残基对于与SicP形成复合物是重要的。SptP/SicP复合物的解离需要InvC的ATP酶活性。因此，我们纯化了InvC，然后表征其ATP酶活性，并测试InvC ATP酶是否解离复合物。InvC ATP酶活性显示正协同性，表明InvC亚基形成同源寡聚体。此外，ATP酶活性在脂质体的存在下被刺激，这表明InvC与质膜相互作用，并且活性受到相互作用的调节。在脂质体和SptP/SicP复合物的存在下，InvC ATP酶活性没有进一步增加，也没有解离SptP/SicP复合物。

SptP is a virulence effector protein of Salmonella that is involved in bacterial invasion into a host cell. For effective secretion, SptP forms a complex with SptP-specific chaperone SicP through its chaperone-binding domain, residues 35-139. We suggested that residues 106-136 of SptP are important for complex formation with SicP by in vitro reconstitution experiments. The dissociation of the SptP/SicP complex requires ATPase activity of InvC. So, we purified InvC, and then characterized its ATPase activity and tested whether InvC ATPase dissociates the complex. InvC ATPase activity showed positive cooperativity, suggesting that InvC subunits form a homo-oligomer. Furthermore the ATPase activity was stimulated in the presence of liposomes, suggesting that InvC interacts with plasma membrane and the activity is regulated by the interaction. In the presence of liposomes and SptP/SicP complexes, InvC ATPase activity was not increased further and did not dissociate SptP/SicP complexes.