Mammalian metal transporters & Salmonella infection

描述（由申请方提供）：沙门氏菌引起人类急性胃肠炎或全身性发热性疾病伤寒。这两种综合征都是世界范围内严重的公共卫生问题。沙门氏菌和哺乳动物细胞之间的相互作用导致细菌的侵入和细胞内生长，这取决于细菌和宿主因素。金属离子如铁、镁和锰在这些相互作用中起重要作用。参与这些金属的结合或运输的细菌蛋白质的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Nrampl中的突变导致对沙门氏菌感染的易感性增加。基于这些观察，我假设哺乳动物金属转运蛋白Nramp 1以外的其他可能也影响沙门氏菌宿主细胞的相互作用。为了支持这一想法，我提出的初步证据表明，铁外排蛋白ferroportin 1（FPN 1），溶质载体（SLC）家族的成员，包括Nramp 1，显着抑制沙门氏菌的细胞内生长时，在HeLa细胞中表达。本提案中的实验将扩展这一研究方向：（a）通过检查FPN 1的亚细胞定位、其铁转运功能在抑菌中的参与及其对铁进入或离开吞噬体的运动的影响，阐明FPN 1的抑菌作用机制。(b)阐明FPN 1在巨噬细胞内沙门氏菌生长中的作用，巨噬细胞是在全身性疾病中被这种生物体体内慢性感染的细胞类型，使用siRNA技术敲低蛋白质的表达，（c）通过使用小鼠基因组中所有此类转运蛋白的基于siRNA的功能筛选，鉴定可能影响沙门氏菌细胞内生长的SLC家族的其他金属离子转运蛋白。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.