Molecular mechanisms of bacterial type III protein translocation across membranes

III型分泌系统（T3SS）将细菌蛋白质输送到宿主细胞中，从而实现了共生、寄生和共生等多种生活方式。T3SS促进了大肠杆菌在温血哺乳动物中的扩张，并最终在反刍动物宿主中进化出共生关系。许多这些大肠杆菌菌株现在广泛分布在商业牲畜中，它们作为特定的掺假物和环境污染物危及食品和水的安全。T3SS抑制剂在新兴的畜牧业实践中有潜在的应用，其目标是在源头上去殖民化动物。这将有利于农业和食品部门，作为减轻大肠杆菌污染造成的数十亿美元损失的一种方式。然而，这样的应用程序需要通过对T3SS功能的细致理解来了解，这是我们关注的重点。该提案研究了神秘的“分泌层次”过程，该过程描述了如何选择T3SS货物并以逐步的方式优先释放。我们的重点是通过未知机制对货物选择施加调节控制的t3ss相关伴侣蛋白。我们的短期目标是双重的。我们将首先使用多货物伴侣CesT和调节T3SS货物选择的效应物Tir确定大肠杆菌分泌层次的分子要求。我们解决了Tir-CesT配合物的晶体结构，揭示了Tir中未知的CesT结合域。在我们的结构指导下，我们将使用Tir突变体来探索Tir- cest复合物如何与T3SS看门人蛋白相互作用，以及这种相互作用是否是中后期效应释放之间的调节步骤。在第二个目标中，我们试图确定伴侣蛋白的可逆翻译后修饰如何调节其在货物选择中的功能。我们的初步数据表明，CesT伴侣的可逆磷酸化通过与mRNA调节因子CsrA相互作用来控制分泌层次。我们已经确定了所涉及的磷酸酶，并将描述它何时以及如何作用于磷酸化的CesT底物。我们设计了一个基因筛选，利用磷酸化CesT对CsrA的拮抗作用来鉴定CesT激酶，并将通过晶体学确定CesT-CsrA相互作用的结构基础。该研究项目将提供对T3SS货物选择如何受到监管的新分子理解，对农业和食品部门具有重要意义，其中含有T3SS的细菌是常见的污染物。我们的长期目标是了解细菌如何利用III型分泌物与宿主进行寄生、共生和互惠关系，以便我们可能利用它作为细菌世界中的定植因子的广泛作用。

Type III secretion systems (T3SS) deliver bacterial proteins into host cells, enabling diverse lifestyles such as mutualism, parasitism, and commensalism. T3SS facilitated the expansion of E. coli into warm-blooded mammals where they eventually evolved commensal relationships in ruminant hosts. Many of these E. coli strains are now widely distributed in commercial livestock where they imperil food and water safety as specific adulterants and environmental contaminants. T3SS inhibitors have potential applications in emerging husbandry practices where the goal is to de-colonize animals at the source. This would benefit the agricultural and food sectors as a way to mitigate the billion-dollar losses suffered due to E. coli contamination. However, such applications need to be informed by a meticulous understanding of T3SS function and this provides our focus. This proposal examines the enigmatic process of ‘secretion hierarchy', which describes how T3SS cargo is selected and prioritized for release in a stepwise fashion. Our focus has been on T3SS-associated chaperone proteins that impose regulatory control on cargo selection through unknown mechanisms. Our short-term objectives are two-fold. We will first determine the molecular requirements for secretion hierarchy in E. coli using the multi-cargo chaperone CesT and the effector Tir that regulates T3SS cargo selection. We solved the crystal structure of a Tir-CesT complex that revealed a previously unknown CesT binding domain in Tir. Guided by our structure, we will use Tir mutants to probe how the Tir-CesT complex interacts with the T3SS gatekeeper protein and whether this interaction is the regulatory step between middle to late effector release. In the second objective, we seek to establish how reversible post-translational modifications in chaperones regulate their functions in cargo selection. Our preliminary data shows that reversible phosphorylation of the CesT chaperone controls secretion hierarchy through interaction with the mRNA regulator CsrA. We have identified the phosphatase involved and will characterize when and how it acts on phosphorylated CesT substrate. We designed a genetic screen that takes advantage of CsrA antagonism by phosphorylated CesT to identify the CesT kinase and will determine the structural basis of CesT-CsrA interaction by crystallography. This research program will deliver a new molecular understanding of how T3SS cargo selection is regulated, with implications for agriculture and food sectors where T3SS-containing bacteria are common contaminants. Our long-term objective is to understand how bacteria use type III secretion for parasitic, commensal and mutualistic associations with hosts so that we may perhaps harness its widespread role as a colonization factor in the bacterial world.