TransLeish: Fitness phenotyping of Leishmania transporter mutants

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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.