Authors

S Alqarni¹; JI Asseri¹;  ¹ University of Glasgow , UK

Discussion

Leishmaniasis is a potentially fatal disease caused by the insect vector-borne parasite Leishmania, prevalent in over 80 countries worldwide. Current treatments for leishmaniasis are inadequate to address the growing public health crisis, necessitating the development of novel, more effective therapeutics. During its digenetic life cycle, Leishmaniaalternates between two major developmental stages. The flagellated extracellular promastigote develops in the digestive tract of the sand fly vector and is injected together with the saliva into a mammalian host during blood feeding. Once within the vertebrate host, promastigotes are internalized by macrophages and differentiate into intracellular non-motile amastigotes that survive and replicate. This initial interaction between the host cell and Leishmania promastigotes is dependent on surface proteins of both organisms. Membrane proteins are fundamental molecules involved in a variety of tasks, including recognition, penetration in the host cell and transport of nutrients. The analysis of the membrane proteome profile of Leishmania promastigotes may permit a more detailed understanding of the host- parasite interaction and aspects of Leishmania biology. Hence, this project aims to glean insight into membrane proteins by first identifying the functional roles of two putative amino acid transporters encoded in L. mexicana, then, specifying membrane proteins that interact with our proteins of interest. These genes are annotated as LmxM.30.1820 and LmxM.30.1800.

CRISPR-Cas9 knockout strategy was used to generate two mutant cell lines for the selected genes in L. mexicana. A complete knockout was successfully generated for LmxM.30.1820, while one allele of LmxM.30.1800 was retained despite the deletion of 2 alleles. Subsequent, various assays were used to evaluate these mutants, LmxM.30.1820^(-/-)and LmxM.30.1800^(-/-/+).

The uptake of radiolabelled amino acids, including tryptophan, lysine, glycine, and threonine, was performed in mutant promastigotes and wild type to investigate the involvement of these genes in specific amino acid transport. While bioinformatic analysis initially suggested both genes might function as tryptophan transporters, the uptake assay shows that neither LmxM.30.1820 nor LmxM.30.1800 significantly contribute to tryptophan transport in the promastigote stage, whereas the uptake of glycine in both mutants was reduced by 90% compared with the control cell line.

Furthermore, gene expression in the absence of glycine was examined, and an up-regulation in the mRNA level encoding both genes was observed in the mutants compared to the control. Metabolic changes were then assessed in the mutant cell line LmxM.30.1820^(-/-) using untargeted LC-MS metabolomics. Significant metabolic changes occurred in glycerophospholipid and fatty acid metabolism, which were significantly decreased in LmxM.30.1820^(-/-).

In order to identify potential partners that interact with our proteins of interest, tagged cell lines have been generated, and Co-IP have been performed to pull down LmxM.30.1800 & LmxM.30.1820 and their protein complexes. A different pull-down approach, TurboID, has also been established to compare outcomes.

The next step is to disrupt specific proteins in these complexes and investigate the repercussions in terms of their functions and parasite biology. Assessing phenotypes such as cell growth and drug susceptibility. Examining the hypothesis of these being a potential focus for therapeutic intervention against this parasite pathogenicity.