Substrate-translocase interactions during two-partner secretion in Gram-negative bacteria

在致病性革兰氏阴性菌中发现的双伴侣分泌（TPS-）途径专门用于分泌毒力因子（称为TPS-蛋白）。tpssa蛋白通过Sec易位子转运到外周质，再通过外膜上同源的TpsB蛋白形成的β桶状孔出口。一个相关的运输系统是自体转运体，其中分泌的蛋白质在c端融合到一个介导跨外膜易位的β桶结构域。最近，我们开发了一种新的体外系统，用于研究TpsA蛋白在其tpsb孔中的易位。我们现在想用这个系统来鉴定参与陪伴和靶向TpsA蛋白到tpsb孔的质周蛋白。我们将使用位点特异性光交联结合TpsB孔中停滞的TpsB蛋白的易位中间体的产生，来确定TpsA和TpsB之间的相互作用位点，跨TpsB的实际易位路径，以及TpsA插入TpsB的方向。通过使用失活突变的tpsa蛋白，我们想要检查它的易位后折叠是否提供了运输的驱动力和单向性。我们将进一步利用无细胞系统来研究相关的自转运体通路。

The two-partner secretion (TPS-) pathway found in pathogenic Gram-negative bacteria is dedicated to the secretion of virulence factors (termed TpsA-proteins). TpsA-proteins are translocated via the Sec translocon to the periplasm, from where they are exported through a β-barrel pore that is formed by the cognate TpsB protein in the outer membrane. A related transport system is that of autotransporters, in which the secreted protein is C-terminally fused to a β-barrel domain that mediates translocation across the outer membrane. Recently we have developed a novel in vitro system for the study of translocation of a TpsA protein across its TpsB-pore. We now want to use this system to identify periplasmic proteins involved in chaperoning and targeting a TpsA protein to the TpsB-pore. We will use site-specific photo-crosslinking in combination with the generation of translocation intermediates of a TpsA-protein stalled in the TpsB-pore, to determine the interacting sites between TpsA and TpsB, the actual translocation path across TpsB, and the orientation by which TpsA is threaded into TpsB. By use of inactivating mutations of the TpsA-protein we want to examine if its post-translocational folding provides the driving force and unidirectionality of transport. We will further exploit the cell-free system to study the related autotransporter pathway.