The cellular mechanisms underpinning the host restriction of Salmonella Typhi

细菌病原体伤寒沙门氏菌会引起人类严重的全身性疾病，称为伤寒，在全球具有重要意义，每年导致超过2700万例病例和20万人死亡。伤寒沙门氏菌是一种适应宿主的病原体，仅限于人类，但控制这种宿主特异性的疾病机制尚不清楚。伤寒沙门氏菌通过将毒力蛋白注射到人类宿主细胞中，以指导在称为含沙门氏菌空泡（SCV）的细胞内膜结合区室中的摄取和复制来引发伤寒。伤寒沙门氏菌不能在小鼠细胞中建立感染，在小鼠细胞中哺乳动物Rab32 GTB定位于SCV并指导病原体降解。当Rab32被基因工程从细胞中消除时，伤寒沙门氏菌在小鼠体内存活。这表明Rab32对于病原体的严格宿主特异性至关重要。Rab GTdR家族（约60个成员）通过将专门的“效应”蛋白募集到膜结合区室来控制细胞通讯途径。小鼠Rab32如何破坏伤寒沙门氏菌，以及通过哪些效应器，是未知的。引人注目的是，人Rab32定位于感染的人细胞中的SCV，病原体在其中存活并建立感染。这证明了小鼠和人Rab32途径的关键差异。伤寒沙门氏菌如何在人Rab32及其同源效应物的作用下存活尚不清楚。Rab功能的基础是它们所锚定的膜，但研究这一点提出了艰巨的挑战。我建议建立一个新的实验重建方法，通过专注于Rabs和它们的膜之间的关键关系来解决这个非常重要的问题。我将设计展示宿主特异性Rab32的膜结合颗粒，这些颗粒将模拟小鼠和人类无细胞提取物中的SCV。通过这种方式，我将捕获和识别神秘的Rab32效应器，并了解它们在生理膜环境中的基本运作方式。SCV蛋白质组学和基于感染的筛选将形成识别关键Rab32效应子的互补方法。Rab32效应子的作用和调节及其与细胞内伤寒沙门氏菌的相互作用将在小鼠和人细胞感染期间确定。解决伤寒沙门氏菌宿主限制的细胞机制将揭示伤寒的关键细胞生物学，并有可能加快我们抗感染药物库的发展，扩大治疗干预的范围。此外，强大的重建系统将适用于任何Rab GTdR组合，使其成为未来解决人类疾病的多种研究途径的有效工具。

The bacterial pathogen Salmonella Typhi causes a severe systemic disease in humans called typhoid fever, which is of major global importance and results in over 27 million cases of disease and 200,000 deaths each year. S.Typhi is a host-adapted pathogen that is exclusively restricted to humans but the disease mechanisms governing this host specificity are unknown.S.Typhi initiates typhoid fever by injecting virulence proteins into human host cells to direct uptake and replication within an intracellular membrane-bound compartment called the Salmonella-containing vacuole (SCV). S.Typhi is incapable of establishing infection in mouse cells where the mammalian Rab32 GTPase localises to the SCV and directs the pathogen for degradation. When Rab32 is eliminated from cells using genetic engineering S.Typhi survives within a mouse. This shows that Rab32 is critical for the pathogen's strict host-specificity. The Rab GTPase family (~60 members) control cellular communication pathways by recruiting specialised 'effector' proteins to membrane-bound compartments. How mouse Rab32 destroys S.Typhi, and through which effectors, is unknown. Strikingly, human Rab32 localises to SCVs in infected human cells where the pathogen survives and establishes infection. This demonstrates a critical difference in the mouse and human Rab32 pathways. How S.Typhi survives the action of human Rab32 and its cognate effectors is not understood.Fundamental to Rab function is the membrane to which they are anchored but studying this presents formidable challenges. I propose to build a new experimental reconstitution approach that tackles this very important problem by focusing on the key relationship between Rabs and their membrane. I will engineer membrane-bound particles displaying host-specific Rab32 that will mimic SCVs in mouse and human cell-free extracts. In this way, I will capture and identify the mystery Rab32 effectors, and understand how they operate fundamentally in the physiological membrane environment. SCV proteomics and infection-based screens will form complementary approaches for identifying the pivotal Rab32 effectors. The role and regulation of the Rab32-effectors and their interaction with intracellular S.Typhi will be determined during infection of mouse and human cells. Resolving the cellular mechanisms of S.Typhi's host restriction will reveal pivotal cell biology underlying typhoid, and has the potential to speed the development of our anti-infectives arsenal and broaden the scope for therapeutic intervention. Furthermore, the powerful reconstitution system would be applicable to any Rab GTPase combination making it an effective tool for multiple research avenues in the future addressing human diseases.