Molecular mechanisms of bacterial type III protein translocation across membranes

细菌III型蛋白跨膜易位的分子机制

• 批准号: RGPIN-2018-05999
• 负责人: Coombes, Brian
• 金额: $ 4.23万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=648040
• 关键词: Molecular; mechanisms; bacterial; type; III

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.

III型分泌系统（T3 SS）将细菌蛋白质递送到宿主细胞中，从而实现多种生活方式，例如互利共生、寄生和共生。T3 SS促进了E.大肠杆菌转化为温血哺乳动物，最终在反刍动物宿主中进化出亲缘关系。许多E。大肠杆菌菌株现在广泛分布在商业牲畜中，它们作为特定的掺杂物和环境污染物危及食品和水的安全。T3 SS抑制剂在新兴的畜牧业实践中具有潜在的应用，其目标是从源头上使动物去殖民化。这将有利于农业和食品部门，以减轻因E。大肠杆菌污染。然而，这样的应用需要通过对T3 SS功能的细致理解来了解，这是我们的重点。该提案研究了神秘的“分泌层次”过程，该过程描述了T3 SS货物如何被选择并以逐步的方式优先释放。我们的重点一直是T3 SS相关的伴侣蛋白，通过未知的机制对货物选择施加监管控制。我们的短期目标有两个方面。我们将首先确定E.使用多货物分子伴侣CesT和调节T3 SS货物选择的效应子Tir，我们解决了Tir-CesT复合物的晶体结构，揭示了Tir中以前未知的CesT结合结构域。根据我们的结构，我们将使用Tir突变体来探测Tir-CesT复合物如何与T3 SS看门蛋白相互作用，以及这种相互作用是否是中晚期效应物释放之间的调节步骤。在第二个目标中，我们试图建立可逆的翻译后修饰分子伴侣如何调节其功能的货物选择。我们的初步数据表明，可逆磷酸化的CesT伴侣控制分泌层次通过相互作用与mRNA调节CsrA。我们已经确定了磷酸酶的参与，并将表征何时以及如何作用于磷酸化的CesT底物。我们设计了一个遗传筛选，利用CsrA拮抗作用的磷酸化CesT，以确定CesT激酶，并将确定CesT-CsrA相互作用的晶体学的结构基础。这项研究计划将提供一个新的分子理解如何T3 SS货物选择监管，与农业和食品部门的影响，其中含T3 SS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，寄生和互利关系，以便我们可能利用其作为细菌世界中的殖民因子的广泛作用。