TransLeish: Fitness phenotyping of Leishmania transporter mutants

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.

Membrane transporters are fundamental to cellular function, facilitating uptake of nutrients and building blocks, waste disposal, shuttling of molecules between cellular compartments and maintaining physiological pH and osmotic pressure. Leishmania spp. are protists with a parasitic life-style, cycling between an insect vector and mammalian host where they infect macrophages and cause disease through interference with normal immune functions. The specific environments the parasite inhabits determine which transporters are required to meet the basic needs for parasite proliferation and to enable it to adapt and respond to environmental changes. Detailed studies on a few individual transporters underscored the fundamental roles played by Leishmania transporters in meeting basic nutritional needs, intracellular survival and drug resistance. There are however around 300 predicted transporters encoded in the genome, most of which have not been assessed. Here we propose to investigate the L. mexicana transportome to determine which transporters are important on the two major life cycle stages and in the effect of anti-leishmanial drugs. We will exploit a mutant library which we have generated that consists of >200 transporter null mutants and propose to generate additional mutants: around 100 heterozygous mutants for transporters that could not be deleted, and 18 deletions of transporter arrays. Each of these mutants carries a unique barcode that enables "bar-seq" fitness screens, where we will test the relative fitness of each mutant under three types of conditions: (i) growth of promastigote forms under nutrient limitation and pH stress conditions, (ii) growth of amastigotes in macrophages and (iii) survival under drug pressure. These data will identify non-dispensable transporters, and provide the necessary context for targeted screens of novel anti-leishmanial compounds.

The primary objective of this research is to understand how the Leishmania parasite is equipped with the right set of membrane transporters to maintain cellular physiology and scavenge from the different environments it encounters during its life cycle, and which of these play a role in the action of anti-leishmanial drugs.
The beneficiaries of this research outside the academic research community are the 350 million people worldwide estimated to be at risk of infection with the Leishmania parasite. Leishmaniasis belongs to a group of neglected tropical diseases (NTD) that poses a huge health burden on some of the poorest communities in the world with a detrimental impact on economic development in endemic areas. These include many of the least developed countries on the DAC list, from Afghanistan to Yemen.
Within the research community, this work benefits academics with an interest in basic cellular processes as well as researchers seeking to exploit biological discoveries for new therapies. This fundamental research employs advanced molecular biology methods to use data from genome sequences and provide new biological information through forward genetic screens. It builds on the successful generation of a library of over 200 null mutants for L. mexicana membrane transporters and channels and the development of a powerful new method for fitness phenotyping of mutants by barcode sequencing. This work effectively harnesses genomic data, built up by the scientific community over the last 20 years, and generates new biological information through systematic standardised assays to assess the importance of individual genes in different contexts. The project is designed to gain efficiently a global overview of transporter importance. This will generate new hypotheses with relevance to studies on drug development: It will allow deprioritisation of non-essential targets and enable focused follow-up studies on transporters that may provide new targets and those that play a role in drug action. Links with industry have already been established; this discovery research is intended in time to provide tangible health benefits for the affected communities.
Researchers in training, such as UK undergraduate and graduate students and postdocs will also benefit. This project will train researchers in powerful new gene editing methods for discovery of mutant phenotypes and gene function. Tools and protocols for genetic modification of Leishmania and bar-seq screens resulting from this work will find many applications in other areas of Leishmaniasis research, and the same principles can be applied to other biological systems. This work will also benefit students from countries on the DAC list, as it offers opportunities for training in gene editing methods and collaborative projects, particularly on drug resistance research in disease endemic countries such as Brazil or India.
The project will generate new knowledge about a parasite's strategy for survival in the human body and pinpoint parasite genes important for virulence. These outputs will be publicised to a wider audience through articles and science exhibitions so that the public can engage with basic discovery science and understand the impact of scientific research on health, and to inspire the next generation of school children to become researchers themselves.