Arginine sensing in Leishmania

Abstract Protozoa of the genus Leishmania are the causative agents of leishmaniasis in humans. These parasites cycle between promastigotes in the sand fly mid-gut and amastigotes in phagolysosome of mammalian macrophages. During infection, they up-regulate host nitric oxide synthase and arginase expression, both of which use arginine as a substrate. These elevated activities deplete macrophage arginine pools, a situation that invading Leishmania must overcome since it is an essential amino acid. Leishmania donovani imports exogenous arginine via a mono-specific amino acid transporter (AAP3) and utilizes it primarily through the polyamine pathway to provide precursors for trypanothione biosynthesis. Here we report the discovery of a pathway whereby promastigote and amastigote forms of the Leishmania sense the lack of environmental arginine and respond with rapid up-regulation in AAP3 expression and activity, as well as several other transporters. Significantly, this arginine deprivation response is also activated in parasites during macrophage infection. Phosphoproteomic analyses of L. donovani promastigotes have implicated a mitogen activated protein kinase 2 (MPK2)-mediated signaling cascade in this response and L. mexicana mutants lacking MPK2 are unable to respond to arginine deprivation. In addition, these mutants cannot differentiate into amastigotes in axenic culture or in peritoneal macrophages, and fail to establish an infection in mice. We propose that sensing arginine levels plays a critical role in Leishmania virulence by activating a rapid metabolic reaction for salvaging this amino acid in response to the lower arginine concentration in the macrophage phagolysosome.摘要 利什曼原虫属（Leishmania）的原生动物是人类利什曼病（leishmaniasis）的致病病原体。这类寄生虫在白蛉中肠的前鞭毛体（promastigotes）与哺乳动物巨噬细胞吞噬溶酶体（phagolysosome）内的无鞭毛体（amastigotes）之间完成生活史循环。感染宿主过程中，它们会上调宿主一氧化氮合酶（nitric oxide synthase）与精氨酸酶（arginase）的表达，这两类酶均以精氨酸作为催化底物。上述酶活性的升高会耗竭巨噬细胞内的精氨酸储备池，而入侵的利什曼原虫必须克服这一困境，因为精氨酸是其必需氨基酸。 杜氏利什曼原虫（Leishmania donovani）可通过单特异性氨基酸转运蛋白（AAP3，mono-specific amino acid transporter）摄取外源性精氨酸，并主要通过多胺通路（polyamine pathway）利用精氨酸，为锥虫硫酮（trypanothione）的生物合成提供前体。 本研究报道了一条全新通路的发现：利什曼原虫的前鞭毛体与无鞭毛体能够感知环境中精氨酸的缺乏，并通过快速上调AAP3的表达与活性，以及其他多种转运蛋白的表达来做出响应。值得注意的是，在巨噬细胞感染过程中，寄生虫体内的这种精氨酸剥夺响应同样会被激活。 对杜氏利什曼原虫前鞭毛体的磷酸蛋白质组学（phosphoproteomic）分析显示，丝裂原活化蛋白激酶2（MPK2，mitogen activated protein kinase 2）介导的信号级联反应参与了该响应；而缺失MPK2的墨西哥利什曼原虫（L. mexicana）突变体无法对精氨酸剥夺做出响应。 此外，这类突变体既无法在无菌培养（axenic culture）或腹腔巨噬细胞中分化为无鞭毛体，也无法在小鼠体内建立感染。我们据此提出：感知精氨酸水平在利什曼原虫的毒力（virulence）形成中发挥关键作用——其通过激活快速代谢反应来回收该氨基酸，以应对巨噬细胞吞噬溶酶体内较低的精氨酸浓度。