LeishGEM (the Leishmania genetic modification project) will use high throughput genetic tools to understand how Leishmania parasites are such successful pathogens.
This collaborative project will have two genome-wide reverse genetics screens as a foundation, followed up with cutting edge molecular biology, cell biology and data science.
LeishGEM will determine which proteins are needed for Leishmania parasites to progress through their life cycle and survive in their hosts, which stages they're important for and where they are found in the cell.
This approach will not just find important proteins, it will find the most important proteins. We can then start assembling this knowledge to find the most important pathways for pathogenicity.
The two genome-wide screens which are the LeishGEM foundations will also be community resources: Deletion bar-seq and Leishtag. These will transform our understanding Leishmania molecular cell biology.
This sub-project will will generate deletion mutants for every Leishmania protein-coding gene, with each mutant carrying a unique genetic barcode. We will put pools of these mutants through series of assays in host organisms, host cells or in vitro imitations of host environments, then sequence the barcodes in the population (bar-seq) to determine which genes are required under each condition. It builds on the success of our previous, smaller, bar-seq experiments to understand Leishmania molecular cell biology (Beneke et al. 2019, Baker et al. 2021).
The deletion bar-seq fitness data will be made available as it is generated. This will become a genome-wide database of quantitative parasite fitness defects across multiple in vitro and in vivo conditions caused by deletion of a gene. The approximately 170 pooled populations of 48 barcoded deletion mutants, together covering the entire genome, will be cryopreserved as a resource for the future.
LeishTag will determine the subcellular localisation of Leishmania proteins, prioritising proteins which appear to be recent evolutionary adaptations in Leishmania or identified as important for pathogenicity by deletion bar-seq. This will be supplemented with LOPIT-DC evidence. It builds on to the success of TrypTag, carried out in the related parasite Trypanosoma brucei and prioritises genes not conserved in T. brucei.
The LeishTag data will be made available as it is generated on LeishTag.org. This will be a database of microscope images of the promastigote and amastigote life cycle stages and annotations of the protein subcellular localisation.
LeishGEM is a collaborative project being carried out primarily in the UK. There are three key sites.
Gluenz lab (Wellcome Centre for Integrative Parasitology, University of Glasgow)
Eva Gluenz's group will have a main focus on the cell biology of Leishmania mutants in vitro. Bar-seq deletion mutant generation and in vitro phenotyping will be carried out here.
Mottram lab (in the York Biomedical Research Institute, University of York)
Work in Jeremy Mottram's group will focus on understanding the interaction of the Leishmania parasite with its host. Bar-seq deletion in vitro and in vivo phenotyping will be carried out here, as will the LOPIT-DC contribution to LeishTag.
The Oxford-based research groups will focus on subcellular protein localisation. Jack Sunter's group will focus on microscopy of Leishmania and will host the LeishTag sub-project. Richard Wheeler's group will focus on computational analysis and integration of the different data types from the different LeishGEM sites.
Collaborators will include Petr Volf (Charles University, Prague) for work in the sandfly vector and Samuel Dean (University of Warwick) for his expertise from TrypTag.
LeishGEM is an enormous project, possible only through our devoted integrated team of post-docs and research assistants.
Eden Ramalho de Araujo Ferreira
Ana Raquel Pereira
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LeishGEM is funded by a Wellcome Trust Collaborative Award.