Peracino Barbara, Wagner Carina, Balest Alessandra, Balbo Alessandra, Pergolizzi Barbara, Noegel Angelika A, Steinert Michael, Bozzaro Salvatore
Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy.
Traffic. 2006 Jan;7(1):22-38. doi: 10.1111/j.1600-0854.2005.00356.x.
Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H(+)ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.
盘基网柄菌变形虫是专业的吞噬细胞，以摄取细菌作为主要食物来源。我们已经克隆了人类天然抗性相关膜蛋白1（Nramp1）[溶质载体家族11成员1（Slc11a1）]的盘基网柄菌同源物，Nramp1是一种内溶酶体膜蛋白，可赋予巨噬细胞对多种细胞内细菌和原生动物感染的抗性。盘基网柄菌Nramp1基因编码一个53 kDa的蛋白质，具有11个推定的跨膜结构域。Nramp1基因在生长阶段转录，饥饿时下调至几乎检测不到的水平。为了深入了解它们在细胞内的定位，我们将Nramp1或V-H(+)ATP酶的vatB亚基与绿色荧光蛋白融合并在细胞中表达。绿色荧光蛋白-vatB插入到所有酸性区室和收缩液泡网络的膜中，并装饰大胞饮体和吞噬体。绿色荧光蛋白-Nramp1除了装饰内体SNARE蛋白Vti1或空泡蛋白（外排途径的标志物）呈阳性的细胞内囊泡区室外，还装饰大胞饮体和吞噬体。Nramp1缺失产生的突变体对嗜肺军团菌和鸟分枝杆菌的细胞内生长比野生型细胞更具包容性。Nramp1过表达保护细胞免受嗜肺军团菌感染。有证据表明，Nramp1以ATP依赖的过程将金属阳离子转运出吞噬溶酶体，并且嗜肺军团菌和鸟分枝杆菌使用不同机制来中和Nramp1活性。
