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的注射体利用保守的III型分泌系统（T3 SS）用于输出底物蛋白。T3 SS由嵌入基体中的出口门和可溶性ATP酶复合物（在鞭毛的情况下为FliH、FliI和FliJ）组成。T3 SS的一个独特特征是其能够将底物选择性从早期型切换到晚期型底物，这有助于适当的鞭毛组装。ATP酶复合物可能有助于靶向分子伴侣-底物复合物和底物蛋白的解折叠，然而，T3 SS在ATP酶缺陷突变体中也保留了一定程度的功能。此外，它最近已被证明，第二信使环二GMP（cdG）结合到ATP酶FliI，这表明第二信使在鞭毛组装过程中可能的新作用。因此，在T3 SS蛋白分泌过程中，cdG结合和ATP水解通过ATP酶FliI的生理作用仍然habitu.Based上的初步结果，我们假设，cdG结合调节寡聚化状态，因此ATP酶活性的FliI，这反过来又调节鞭毛T3 SS从早期型到晚期型底物的底物特异性开关。在这个项目中，我们的目标是澄清在蛋白质分泌过程中ATP水解和cdG结合到ATP酶复合物的作用。具体而言，我们想研究：（i）cdG结合如何调节FliI的ATP酶活性，从而调节底物特异性;（ii）FliI是否以及如何有助于分泌底物解折叠和伴侣底物释放;（iii）ATP酶的功能和CdG结合如何调节亚磷酸酶的活性。FliI的细胞定位和寡聚化。通过分析蛋白质分泌，使用遗传学和生物化学方法的组合以及单分子/超分辨率显微镜技术，对野生型FliI和缺乏ATP水解和/或推定的cdG结合的突变体进行ATP酶寡聚化和亚细胞定位。我们期望这些实验将建立ATP水解和假定的cdG-结合FliI的功能，并研究几种可能的机制，如何cdG结合可能调节蛋白输出通过T3 SS。

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.