About LeishGEM

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. 

Deletion bar-seq

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

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.