Atomic insight into virulence factor translocation by Gram-negative bacterial type-II secretion systems

许多革兰氏阴性细菌病原体使用ii型分泌系统（T2SS），一种类似注射器的机制，将蛋白质底物转运到周围环境中。这些输出的底物具有多种作用，包括形成/建立生物膜，颠覆宿主对感染的反应，促进细胞内宿主的入侵/生存。底物首先被运送到外质中，在那里它们折叠成它们的原始状态。在P. aeruginosa中，底物与其LspC等同物（跨膜螺旋（TM）和同源区（HR）之间的非结构化连接物）的TMHR相互作用2,3，导致其T2SS内部的构象变化和进入。据认为，在ATP水解作用的驱动下，货物随后由假柄通过孔运输1,3。对属于不同细菌的几种底物的突变分析表明，分泌所必需的残基位于很远的地方，并在折叠时聚集在一起形成构象结构。然而，基于突变的方法很难解开这些事件，并且由于T2SS靶向基序是3D基序，因此高分辨率结构策略是必不可少的。我的小组目前正专注于嗜肺军团菌T2SS5-8，它非常适合进行结构研究。我们已经证明T2SS的几个底物，如在铜绿假单胞菌中，也能识别TMHR连接体中的残基。LspC和LspD之间的相互作用连接了T2SSs9的内外膜成分，我们观察到TMHR也提供了与LspD的相互作用。因此，LspC对底物的识别可能与LspC- lspd复合物的位移相耦合，从而允许进入系统。此外，与底物识别位点相邻的LspC-TMHR序列与LspL/LspM距离很近，可能与这两种蛋白相互作用。因此，LspC的底物识别也可能触发LspL/LspM的构象变化，这可能为信号转导提供途径，导致活性底物输出。该项目的具体目标将是：a)使用x射线晶体学和/或NMRb确定一个或多个嗜肺乳杆菌T2SS底物与LspC TMHR复合物的原子结构)使用x射线晶体学和/或NMRc研究LspC TMHR残基与LspL/LspM之间的相互作用)确定(a)和(b)中鉴定的相互作用是嗜肺乳杆菌所特有的还是也观察到其他细菌(铜绿假单胞菌，大肠杆菌和D. dadantii)通过分析体外蛋白质：蛋白质相互作用（如NMR，化学交联，ITC, BiaCore）

Many Gram-negative bacterial pathogens use a type-II secretion system (T2SS), a syringe-like mechanism, to transport protein substrates into their surroundings. These exported substrates have diverse roles including the formation/establishment of biofilms, subversion of host responses to infection and promoting intracellular host invasion/survival. Substrates are first transported into the periplasm where they fold into their native states. In P. aeruginosa, substrates interact with the TMHR of its LspC equivalent (an unstructured linker between the transmembrane helix (TM) and homologous region (HR))2,3, causing conformational changes within and entry to its T2SS. It is thought that cargo is then transported through the pore by the pseudopilus, driven by ATP hydrolysis1,3. Mutational analyses of several substrates belonging to different bacteria indicate that residues essential for secretion are distantly located and come together to form a conformational structure upon folding4. However, with mutation-based approaches it is difficult to uncouple these events and as the T2SS targeting motif is a 3D one, a high-resolution structural strategy is essential. My group is currently focussing on the Legionella pneumophila T2SS5-8, which is highly amenable to structural studies. We have shown that several substrates of this T2SS, as in P. aeruginosa, also recognize residues within the TMHR linker. Interactions between LspC and LspD connect the inner and outer membrane components of T2SSs9, and we observe that the TMHR also provides interactions with LspD.Therefore, substrate recognition by LspC may be coupled to displacement of the LspC-LspD complex, allowing entry into the system. Moreover, the LspC-TMHR sequence adjacent to the substrate recognition site is in close proximity with LspL/LspM and likely interacts with these two proteins. Therefore substrate recognition of LspC may also trigger a conformational change in LspL/LspM that could provide a pathway for signal transduction resulting in active substrate export. The specific aims of this project will be to:a) Determine the atomic structures of one or more L. pneumophila T2SS substrates in complex with the TMHR of LspC using X-ray crystallography and/or NMRb) Study interactions between LspC TMHR residues and LspL/LspM using X-ray crystallography and/or NMRc) Determine whether the interactions identified in (a) and (b) are unique to L. pneumophila or are also observed other bacteria (P. aeruginosa, E. coli and D. dadantii) by analysing in vitro protein:protein interactions (e.g. NMR, chemical cross-linking, ITC, BiaCore)