TransLeish: Fitness phenotyping of Leishmania transporter mutants

TransLeish：利什曼原虫转运蛋白突变体的适应性表型

• 批准号: MR/V000446/1
• 负责人: Richard McCulloch
• 金额: $ 55.46万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FV000446%2F1
• 关键词: TransLeish; Fitness; phenotyping; Leishmania; transporter

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.

寄生虫从宿主那里偷东西，却不回馈任何有用的东西，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。有些寄生虫在显微镜下很小，生活在我们身体的细胞内。利什曼原虫只有一个单细胞，能够引起一种被忽视的热带疾病，称为利什曼病。感染利什曼原虫的人可以出现轻微的症状，局部皮肤病变，或危及生命的全身感染。目前全世界约有1 200万人受到感染;每年约有30 000人死于这种疾病，幸存者往往留下毁容的疤痕。利什曼原虫的部分生命周期生活在吸血沙蝇的肠道内。当这些寄生虫叮咬人类时，寄生虫游进伤口，在那里被白色血细胞捕获。这些免疫细胞通常会杀死微生物，但利什曼原虫已经进化到生活在一种称为巨噬细胞的细胞内。在这里，利什曼原虫可以获得许多有用的分子，如糖，脂质，DNA和蛋白质的构建模块，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什么是有用的，并将其运输通过其细胞膜，这是一个障碍。细胞膜上布满了蛋白质，即所谓的膜转运蛋白或通道，它们充当守门人，决定哪些物质可以进入。拥有正确的转运蛋白可以确保寄生虫能够有效地利用其宿主细胞。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蛋白进入寄生虫细胞。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种不同的转运蛋白。其中一些已经被详细研究过，例如三种葡萄糖转运蛋白或铁转运蛋白;其他的还没有被研究过，但由于它们与其他细胞中已经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在转运什么，以及寄生虫为什么需要它们。然而，仍然有大量的转运蛋白的功能和作用，我们还不了解。我们已经开发出一种快速简单的方法，通过一种称为CRISPR的方法从基因组中移除基因，逐个移除这些转运蛋白。通过这种方法，我们产生了200多个突变的利什曼原虫株系。在这个项目中，我们希望在实验室中研究这些寄生虫，以发现哪些转运蛋白对生存特别重要。我们将测试哪些突变寄生虫在营养缺乏的情况下仍然可以生长。它们还能感染巨噬细胞吗？他们对抗利什曼病药物治疗有何反应？如果转运蛋白对药物的摄取起重要作用，则转运蛋白的丢失可能导致耐药性。相反，帮助将药物泵出细胞的转运蛋白的丢失可能使寄生虫更容易受到药物的影响。该项目收集的数据将确定在正常环境和药物压力下对寄生虫生存至关重要的转运蛋白。知道这一点很有用：它将帮助我们更好地了解利什曼原虫如何在人体细胞内作为寄生虫生活，它们如何逃脱目前使用的药物的杀伤，并确定潜在的新药靶点。