Leishmania exosomal tRNA-derived small non-coding RNA fragments modulate host THP-1 derived macrophage proteins: A...

Protozoan parasites of the genus Leishmania, the causative agents of human leishmaniasis, have evolved mechanisms to manipulate host cell functions to their advantage. Recently, small non-coding RNAs have been identified as contributors to the pathogenesis of Leishmania infection. An increasing number of studies have demonstrated that Leishmania tRNA-derived small non-coding RNAs, also known as Leishmania tRNA-derived fragments (Ld-tRFs), can be delivered to host cells via exosomes, thereby influencing host cell function. Here, we investigated the potential effects of Leishmania exosomal Ld-tRF-Asp and Ld-tRF-Leu in THP-1-derived host macrophages. SILAC (Stable Isotope Labelling by Amino acids in Cell culture) based quantitative proteomics was used to investigate the effects of synthetic Ld-tRF-Asp and Ld-tRF-Leu by analyzing how these Ld-tRFs affect the macrophage proteome. Ld-tRF-Asp modulated 20 proteins, whereas Ld-tRF-Leu affected 18 proteins in macrophages. Interestingly, 7 of the Ld-tRF-Asp-modulated proteins and 7 of the Ld-tRF-Leu-modulated proteins showed potential pro-Leishmania effects. Biochemical isolation of Argonaute (Ago) protein complexes by "Ago proteins Affinity Purification by Peptide," followed by identification of interacting small non-coding RNAs, revealed the selective presence of both tRFs in Leishmania-infected macrophages. This striking finding suggested that Ld-tRFs engage macrophage Ago proteins for their effects. In addition, an in vivo biotin-RNA pull-down assay showed that Ld-tRF-Leu selectively interacted with Ago 1, suggesting that Ago 1 is the preferred Ago guiding Ld-tRFs to target host genes. Taken together, this study shows, for the first time, that Leishmania exosomal tRFs significantly alter the host macrophage proteome in favour of Leishmania survival. This may offer new insights into the mechanisms of leishmaniasis and may provide future therapeutic interventions targeting these regulatory molecules.