Function of the type-III secretion system ATPase complex in protein export of the bacterial flagellum

细菌鞭毛和许多革兰氏阴性病原体的进化相关注射剂利用保守的III型分泌系统(T3SS)输出底物蛋白。T3SS由嵌入在基体中的出口门和可溶性的ATPase复合体(鞭毛中的FliH、FliI和FliJ)组成。T3SS的一个独特功能是它能够将底物选择性从早型底物切换到晚型底物，这对正确的鞭毛组装是有利的。ATPase复合体可能有助于伴侣-底物复合体的靶向和底物蛋白质的打开，然而，T3SS在ATPase缺陷突变体中也保留了一定程度的功能。此外，最近的研究表明，第二信使环二核苷酸(CDG)与ATPase FliI结合，提示第二信使在鞭毛组装过程中可能起到新的作用。因此，CDG结合和ATPase FliI在T3SS蛋白分泌过程中的生理作用尚不清楚。根据初步结果，我们假设CDG结合调节FliI的寡聚化状态，从而调节FliI的ATPase活性，进而调节鞭毛T3SS从早型底物到晚期底物的底物特异性开关。在这个项目中，我们的目标是阐明ATP水解和CDG与ATPase复合体结合在蛋白质分泌过程中的作用。具体地说，我们想要研究：(I)CDG结合如何调节FliI的ATPase活性，从而调节底物特异性；(Ii)FliI是否以及如何促进分泌底物的展开和伴侣-底物的释放；(Iii)ATPase功能和CDG结合如何调控FliI的亚细胞定位和寡聚化。这些问题将通过分析野生型FliI蛋白分泌、ATPase寡聚和亚细胞定位，以及结合遗传学和生化方法的野生型FliI和/或假定的CDG结合突变体，以及单分子/超分辨显微镜技术，以比较的方法解决各种问题。总之，我们预计这些实验将建立ATP水解和可能的CDG结合与FliI功能的生理相关性，并研究CDG结合如何通过T3SS调节蛋白质输出。

The bacterial flagellum and the evolutionarily related injectisome of many Gram-negative pathogens utilize a conserved type-III secretion system (T3SS) for the export of substrate proteins. The T3SS consists of the export gate embedded in the basal body and a soluble ATPase complex (FliH, FliI and FliJ in case of the flagellum). An unique feature of T3SS is its ability to switch substrate selectivity from early-type to late-type substrates that is instrumental for proper flagellum assembly. The ATPase complex presumably contributes to targeting of chaperone-substrate complexes and unfolding of substrate proteins, yet, the T3SS retains some degree of function also in ATPase-deficient mutants. Further, it recently has been shown that the second messenger cyclic di-GMP (cdG) binds to the ATPase FliI, suggesting a possible novel role of the second messenger during flagellar assembly. Accordingly, the physiological roles of cdG binding and ATP hydrolysis via the ATPase FliI during the T3SS protein secretion process remain elusive.Based on preliminary results, we hypothesize that cdG binding modulates the oligomerization state and therefore ATPase activity of FliI, which in turn regulates the substrate specificity switch of the flagellar T3SS from early-type to late-type substrates. In this project, we aim to clarify the role of ATP hydrolysis and cdG binding to the ATPase complex during the protein secretion process. Specifically, we want to investigate: (i) how cdG binding modulates the ATPase activity of FliI and thereby regulates the substrate specificity; (ii) if and how FliI contributes to secretion substrate unfolding and chaperone-substrate release and; (iii) how the ATPase function and cdG-binding modulates the sub-cellular localization and oligomerization of FliI.The various questions will be addressed in a comparative approach by analyzing protein secretion, ATPase oligomerization and sub-cellular localization of wildtype FliI and mutants deficient in ATP hydrolysis and/or putative cdG binding using a combination of genetics and biochemical approaches, as well as single-molecule/superresolution microscopy techniques.In summary, we expect that these experiments will establish the physiological relevance of ATP hydrolysis and the putative cdG-binding for the function of FliI and investigate several potential mechanisms how cdG binding might regulate protein export via T3SS.