Mammalian metal transporters & Salmonella infection

描述(申请人提供)：沙门氏菌可引起人类的急性胃肠炎或全身发热性疾病伤寒。这两种症状都是世界范围内的重大公共卫生问题。导致沙门氏菌入侵和细胞内生长的沙门氏菌与哺乳动物细胞之间的相互作用取决于细菌和宿主因素。铁、镁、锰等金属离子在这些相互作用中起着重要的作用。参与这些金属结合或运输的细菌蛋白突变对沙门氏菌的毒力有重大影响。相应地，哺乳动物二价金属转运蛋白Nramp1的突变导致对沙门氏菌感染的易感性增加。基于这些观察，我推测除了Nramp1之外，哺乳动物的金属转运蛋白也可能影响沙门氏菌与宿主细胞的相互作用。为了支持这一观点，我提出了初步的证据表明，铁外流蛋白铁蛋白1(FPN1)是包括Nramp1在内的溶质载体(SLC)家族的成员，当在HeLa细胞中表达时，显著抑制沙门氏菌的细胞内生长。这项建议中的实验将把这一研究范围扩展到：(A)通过研究fpn1的亚细胞定位、其铁运输功能在抑菌中的参与以及它对铁进入或流出吞噬小体的影响来阐明fpn1的抑菌作用的机制。(B)利用siRNA技术下调蛋白的表达，阐明FPN1在巨噬细胞内沙门氏菌生长中的作用；巨噬细胞是在全身疾病中长期感染该细菌的一种细胞类型；(C)通过对小鼠基因组中所有此类转运体进行基于siRNA的功能筛选，确定SLC家族中可能影响沙门氏菌细胞内生长的其他金属离子转运体。这些研究将确定沙门氏菌-宿主细胞相互作用的新方面，阐明哺乳动物金属转运蛋白在沙门氏菌感染中的作用，并提出治疗沙门氏菌病的新方法。

DESCRIPTION (provided by applicant): Salmonellae cause either acute gastroenteritis or the systemic febrile illness typhoid in humans. Both syndromes are significant public health problems worldwide. The interactions between Salmonella and mammalian cells that result in invasion and intracellular growth of the bacteria depend on both bacterial and host factors. Metal ions such as iron, magnesium and manganese play important roles in these interactions. Mutations in bacterial proteins that are involved in the binding or transport of these metals have major effects on Salmonella virulence. Correspondingly, mutations in the mammalian divalent metal transporter Nrampl result in increased susceptibility to Salmonella infection. Based on these observations, I hypothesized that mammalian metal transporters other than Nramp1 might also influence Salmonella-host cell interactions. In support of this idea, I present preliminary evidence showing that the iron efflux protein ferroportin 1 (FPN1), a member of the solute carrier (SLC) family of transporters that includes Nramp1, significantly inhibits the intracellular growth of Salmonella when expressed in HeLa cells. The experiments in this proposal will extend this line of investigation to: (a) Elucidate the mechanism of the bacteriostatic effect of FPN1, by examining its sub-cellular localization, involvement of its iron transport function in bacteriostasis, and its effects on movement of iron into or out of the phagosome. (b) Clarify the role of FPN1 in the growth of Salmonella inside macrophages, the cell type that is chronically infected by this organism in vivo in systemic disease, using siRNA technology to knock-down expression of the protein, (c) Identify other metal ion transporters of the SLC family that might influence the intracellular growth of Salmonella by using an siRNA-based functional screen of all such transporters in the mouse genome. These studies will identify new aspects of the Salmonella-host cell interaction, shed light on the role of mammalian metal transporters in Salmonella infection, and suggest novel approaches to treating salmonellosis.