Periodic Reporting for period 1 - transLEISHion (A targeted knockout screen for identification of Leishmania membrane transporters required for infection of macrophages)
Période du rapport: 2018-09-01 au 2020-08-31
Leishmania parasites cause a disease in humans known as Leishmaniasis. Widespread in 98 countries, leishmaniasis constitutes a risk for 1 billion people worldwide with an incidence of over 1 million cases per year. Leishmaniasis clinical manifestations range from cutaneous lesions to fatal visceral forms estimated to cause 20,000-40,000 deaths/year. Clinical manifestations and course of the infection depend on factors such as parasite species and the immune system of the infected individual.
When a female sandfly bites a mammalian host and feeds on its blood, it injects infective forms of Leishmania parasites called metacyclic promastigotes, which are rapidly taken up by white blood cells, called macrophages, then it differentiates into aflagellated forms called amastigotes.
Uniquely among organisms of the same family, Leishmania parasites are adapted to survival inside macrophages, a niche in which only few pathogens thrive. This poses specific challenges to the parasite, and it mandates the activation of mechanisms for parasite survival such as acquisition of nutrients and tolerance to acidic conditions.
As there are no human vaccines available, there is a critical need for safe, non-toxic and cost-effective new drugs to treat leishmaniasis.
Some transporter proteins have been implicated in parasite survival, defence from host immune attack as well as uptake of drug formulations. However significant gaps exist with regards to the importance of transporter proteins in the biology of Leishmania parasites.
The Gluenz lab recently discovered several uncharacterized transporter proteins, among a number of transcripts upregulated in amastigote forms when compared to promastigote forms. These discoveries called for follow-up studies on the biological functions and relative importance of these amastigote-upregulated gene products.
Due to unique genomic features, the functional study of different genes of Leishmania parasites lagged in comparison to other protozoan parasites. Taking advantage of the revolution in genome editing brought about by the discovery of the CRISPR-Cas9 system and its adaptation for biotechnology, the Gluenz lab developed a novel high-throughput genome editing method for Leishmania parasites, which allows the rapid generation of gene knockout mutants enabling for the first-time large-scale loss-of-function screens to discover genes essential for Leishmania survival.
Taking advantage of these resources the main aim of transLEISHion, was (1) to generate a library of Leishmania mexicana knockout mutants for 48 genes, whose transcripts in amastigotes were observed upregulated; (2) identify membrane transporters required for the viability of vector stage promastigotes and (3) identify membrane transporters required for the viability of amastigotes.
When a female sandfly bites a mammalian host and feeds on its blood, it injects infective forms of Leishmania parasites called metacyclic promastigotes, which are rapidly taken up by white blood cells, called macrophages, then it differentiates into aflagellated forms called amastigotes.
Uniquely among organisms of the same family, Leishmania parasites are adapted to survival inside macrophages, a niche in which only few pathogens thrive. This poses specific challenges to the parasite, and it mandates the activation of mechanisms for parasite survival such as acquisition of nutrients and tolerance to acidic conditions.
As there are no human vaccines available, there is a critical need for safe, non-toxic and cost-effective new drugs to treat leishmaniasis.
Some transporter proteins have been implicated in parasite survival, defence from host immune attack as well as uptake of drug formulations. However significant gaps exist with regards to the importance of transporter proteins in the biology of Leishmania parasites.
The Gluenz lab recently discovered several uncharacterized transporter proteins, among a number of transcripts upregulated in amastigote forms when compared to promastigote forms. These discoveries called for follow-up studies on the biological functions and relative importance of these amastigote-upregulated gene products.
Due to unique genomic features, the functional study of different genes of Leishmania parasites lagged in comparison to other protozoan parasites. Taking advantage of the revolution in genome editing brought about by the discovery of the CRISPR-Cas9 system and its adaptation for biotechnology, the Gluenz lab developed a novel high-throughput genome editing method for Leishmania parasites, which allows the rapid generation of gene knockout mutants enabling for the first-time large-scale loss-of-function screens to discover genes essential for Leishmania survival.
Taking advantage of these resources the main aim of transLEISHion, was (1) to generate a library of Leishmania mexicana knockout mutants for 48 genes, whose transcripts in amastigotes were observed upregulated; (2) identify membrane transporters required for the viability of vector stage promastigotes and (3) identify membrane transporters required for the viability of amastigotes.
The initial objective was exceeded by expanding the knockout screen to all 314 putative transporter proteins encoded in the genome of Leishmania mexicana parasites. Of the 314 targeted genes, 257 homozygous and 46 heterozygous knock-out cell lines, lacking one or more copies of the target gene, were successfully isolated. This generated a library of barcoded knockout cell lines which enables in-depth studies of transporter functions in L. mexicana. For the ten remaining genes, whose attempt of deletion systematically failed, heterozygous mutants utilising single drug selection as opposed to double-drug selection, were isolated.
These results, suggest that about 18% of the transportome of L. mexicana parasites is important for parasite survival or fitness. To successfully verify this, future follow-up experiments will include conditional knock-out strategies to show definitively whether this sub-set of protein encoding genes is essential.
Due to the COVID-19 pandemic and mandatory lab closure between March and June 2020, the determination of the relative fitness of these mutants in the amastigote stage, was not completed during the time-frame of this project, however this is currently being pursued with funding secured by the line manager for a wider-ranging study on Leishmania transporter proteins that builds on the initial results from this project.
Despite the pandemic restrictions, the work carried out during this action was able to provide valuable resources which will allow to fill in multiple gaps in Leishmania cell biology. In addition, by identifying a sub-set of genes potentially essential for the survival of Leishmania promastigote parasites, this project has partially contributed to aid in the discovery of urgently needed new drug targets.
Due to the COVID-19 pandemic and mandatory lab closure between March and June 2020, the determination of the relative fitness of these mutants in the amastigote stage, was not completed during the time-frame of this project, however this is currently being pursued with funding secured by the line manager for a wider-ranging study on Leishmania transporter proteins that builds on the initial results from this project.
Despite the pandemic restrictions, the work carried out during this action was able to provide valuable resources which will allow to fill in multiple gaps in Leishmania cell biology. In addition, by identifying a sub-set of genes potentially essential for the survival of Leishmania promastigote parasites, this project has partially contributed to aid in the discovery of urgently needed new drug targets.