The cellular mechanisms underpinning the host restriction of Salmonella Typhi

细菌病原体伤寒沙门氏菌在人类中引起一种严重的系统性疾病，称为伤寒，这是一种具有重大全球重要性的疾病，每年导致2700多万例疾病和20万人死亡。伤寒沙门氏菌是一种宿主适应的病原体，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，直接在细胞内膜结合的空泡(SCV)内摄取和复制，从而引发伤寒。伤寒沙门氏菌不能在小鼠细胞中建立感染，而哺乳动物Rab32 GTP酶定位于SCV并指导病原体降解。当使用基因工程从细胞中消除Rab32时，伤寒沙门氏菌在小鼠体内存活下来。这表明Rab32对于病原菌的严格宿主特异性是至关重要的。Rab GTPase家族(约60个成员)通过招募特殊的“效应器”蛋白到膜结合的间隔来控制细胞通讯途径。老鼠Rab32如何摧毁伤寒沙门氏菌，以及通过哪些效应器，尚不清楚。引人注目的是，人类Rab32定位于受感染的人类细胞中的SCV，在那里病原体存活并建立感染。这证明了小鼠和人类Rab32通路的关键差异。伤寒沙门氏菌如何在人类Rab32及其同源效应器的作用下存活尚不清楚。Rab功能的基础是它们所锚定的膜，但研究这一点面临着巨大的挑战。我建议建立一种新的实验性重建方法，通过关注RAB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，这种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，我将捕捉和鉴定神秘的Rab32效应器，并从根本上了解它们在生理膜环境中是如何运作的。SCV蛋白质组学和基于感染的筛查将形成识别关键Rab32效应器的互补方法。在小鼠和人类细胞感染过程中，将确定Rab32-效应器的作用和调节，以及它们与伤寒沙门氏菌的相互作用。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，并有可能加快我们的抗感染药物武器库的发展，并扩大治疗干预的范围。此外，强大的重组系统将适用于任何Rab GTP酶组合，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.