Identification and Functional Characterisation of Putative Amino Acid Transporters in Leishmania mexicana

Abstract

The genus Leishmania comprises haemoflagellate kinetoplastids responsible for leishmaniasis, a vector-borne disease endemic in over 90 countries. Current treatments are inadequate, necessitating the development of novel therapeutics. Leishmania spp. rely on scavenging essential amino acids (AAs) via amino acid transporter (AAT) proteins, making these systems potential therapeutic targets. However, most AATs in Leishmania remain uncharacterized.

This study aimed to investigate the roles of two putative AATs, LmxM.30.1820 and LmxM.30.1800, in L. mexicana. Bioinformatics identified 27 putative AAT genes, including homologues of known AATs, and predicted LmxM.30.1820 and LmxM.30.1800 as potential tryptophan transporters. CRISPR-Cas9 was used to generate mutants for both genes, revealing LmxM.30.1820 (-/-) lacked a growth phenotype, while LmxM.30.1800 (-/-/+) exhibited modest amastigote growth defects. Radiolabelled uptake assays indicated neither protein transported tryptophan but implicated LmxM.30.1820 (-/-) in glycine transport and LmxM.30.1800 (-/-/+) in glycine and threonine uptake. Inhibition assays suggested a shared glycine, serine, threonine, and alanine transport system, with high threonine affinity. In the absence of glycine, mRNA levels of both genes were upregulated in mutants, indicating compensatory mechanisms.

Metabolomics analysis of LmxM.30.1820 (-/-) revealed significant reductions in glycerophospholipid and fatty acid metabolism, highlighting glycine's role as a precursor in these pathways.

This thesis identifies LmxM.30.1820 as a glycine transporter affecting lipid metabolism and LmxM.30.1800 as a glycine and threonine transporter influencing amastigote growth. These findings expand our understanding of AAT function in L. mexicana and may inform future therapeutic strategies targeting leishmaniasis.