TransLeish: Fitness phenotyping of Leishmania transporter mutants

寄生虫从宿主身上偷东西，却没有给宿主任何有用的东西，从而削弱了宿主。一些较大的寄生虫，如蜱虫和钩虫，生活在皮肤上或肠道里，吸血。有些寄生虫很小，生活在我们身体的细胞里。利什曼原虫只有一个单细胞，能够引起一种被忽视的热带病——利什曼病。感染利什曼原虫的人可能出现轻微症状，伴有局部皮肤损伤，或危及生命的全身感染。目前全世界约有1200万人感染；每年大约有3万人死于这种疾病，而那些幸存下来的人往往会留下毁容的伤疤。利什曼原虫生命周期的一部分生活在吸血沙蝇的肠道内。当它们咬人时，寄生虫就会游进伤口，在那里被白细胞捕获。这些免疫细胞通常会杀死微生物，但利什曼原虫寄生虫已经进化到生活在一种叫做巨噬细胞的细胞内。在这里，利什曼原虫可以接触到许多有用的分子，如糖、脂类、DNA和蛋白质的组成部分，以及寄生虫生存和复制所需的其他营养物质。利什曼原虫需要选择有用的物质并将其运输穿过作为屏障的细胞膜。膜上布满了蛋白质，即所谓的膜转运蛋白或通道，它们起到了决定哪些物质可以进入的看门人的作用。拥有正确的转运体可以确保寄生虫有效地利用宿主细胞。因此，转运蛋白对寄生虫的生存至关重要。然而，它们也是一个潜在的弱点，因为一些旨在杀死利什曼原虫的药物通过其自身的转运体进入寄生虫细胞。对利什曼原虫基因组中所有基因的分析表明，它可以制造大约300种不同的转运体。其中一些已经被详细研究过，例如葡萄糖转运体和铁转运体的三种转运体；其他的还没有被研究过，但由于它们与在其他细胞中研究过的转运蛋白非常相似，我们可以很好地猜测它们可能运输的是什么，以及寄生虫为什么需要它们。然而，仍有大量的转运蛋白，其功能和作用我们尚未了解。我们已经开发出一种快速而简单的方法，通过一种叫做CRISPR的方法从基因组中去除基因，一个接一个地去除这些转运蛋白。通过这种方法，我们产生了200多个利什曼原虫突变系。在这个项目中，我们想在实验室研究这些寄生虫，发现哪些转运体对生存特别重要。我们将测试在营养缺乏的情况下，哪些突变寄生虫还能生长。它们还能感染巨噬细胞吗？他们对抗利什曼病药物治疗的反应如何？如果转运体对药物的摄取很重要，那么转运体的缺失可能会导致耐药性。相反，失去一种帮助将药物泵出细胞的转运蛋白可能会使寄生虫更容易受到药物的影响。在这个项目中收集的数据将精确定位对寄生虫在正常环境和药物压力下生存至关重要的转运蛋白。了解这一点是有用的：它将帮助我们更好地了解利什曼原虫是如何在人类细胞内作为寄生虫生存的，它们是如何逃脱目前使用的药物的杀死，并确定潜在的新药靶点。

Parasites steal from their host without giving anything useful back, and weakening the host. Some of the bigger parasites like ticks and hookworms live on the skin or in the gut and suck blood. Some parasites are microscopically small and live inside cells of our body. The parasite Leishmania is only a single cell, able to cause a neglected tropical disease called leishmaniasis. A person infected with Leishmania can develop mild symptoms, with localized skin lesions, or a life-threatening infection of the whole body. About 12 million people across the world are currently infected; about 30,000 people die every year from the disease and those that survive are often left with disfiguring scars. For part of their life cycle Leishmania live inside the gut of blood-feeding sand flies. When these bite a human, the parasite swims into the wound where it is captured by white blood cells. These immune cells normally kill microbes but Leishmania parasites have evolved to live inside a type of cell called the macrophage. Here the Leishmania have access to many useful molecules such as sugars, lipids, building blocks for DNA and proteins, and other nutrients, which the parasite needs for survival and replication. Leishmania need to select what is useful and transport it across its cell membrane, which acts as a barrier. The membrane is studded with proteins, so-called membrane transporters or channels, that act as gatekeepers to determine which substances can enter. Possessing the correct set of transporters ensures the parasite can exploit its host cell effectively. Transporters are therefore critical for parasite survival. They are however also a potential weakness, as some drugs designed to kill Leishmania enter the parasite cell through its own transporters. The analysis of all genes in the Leishmania genome showed that it can make about 300 different transporters. Some of these have already been studied in detail, for example three transporters for glucose or transporters for iron; others have not been studied but since they closely resemble transporters that have been studied in other cells we can make a good guess what they may be transporting, and why the parasite needs them. There are however still a large number of transporters whose function and role we do not yet understand. We have developed a rapid and simple method to remove each of these transporters one-by-one, by removing the gene from the genome with a method called CRISPR. By this method we produced over 200 mutant Leishmania lines. In this project we want to study these parasites in the laboratory, to discover which transporters are particularly important for survival. We will test which of these mutant parasites can still grow if nutrients are scarce. Can they still infect macrophages? How will they respond to treatment with anti-leishmanial drugs? If the transporter it important for the uptake of a drug, the loss of the transporter might cause drug-resistance. Conversely, loss of a transporter that helps to pump drugs out of the cell may render the parasite more susceptible to the drug. The data gathered in this project will pinpoint transporters that are vital for parasite survival in their normal environments and under drug pressure. Knowing this is useful: It will help us to understand better how Leishmania are equipped to live as parasites inside human cells, how they can escape killing by currently used drugs and pinpoint potential new drug targets.