Purine depletion in Leishmania inhibits translation and changes cell morphology, while targeting inactive ribosomal particles and mRNAs to LeishIF4E3-containing storage granules

Leishmania are ancient unicellular eukaryotes that cycle between sand-fly vectors and mammalian hosts, adapting to alternating environments. Stage-differentiation involves changes in the proteome profile, which are subject to translation regulation. The Leishmania genome encodes six paralogs of the cap-binding protein eIF4E, each assumed to be responsible for specific functions. One of them, LeishIF4E3, concentrates in dynamic stress granules that were shown to store inactive ribosomes and mRNAs during the nutritional stress. Leishmania parasites lack pathways for de novo purine biosynthesis and the depletion of purines that occurs in the sand fly, induces differentiation into the virulent metacyclic forms. In response to purine depletion, LeishIF4E3 is phosphorylated at its N-terminus, and concentrates in specific cytoplasmic granules. The purified granules contain mainly ribosomal particles and mature mRNAs, along with RNA-binding proteins, RNA-helicases and metabolic enzymes. However, no degradation enzymes or P-body markers were detected in these granules, excluding their participation in RNA degradation. Using the CRISPR-Cas9 methodology we deleted one of the two LeishIF4E3 gene copies. Expression of LeishIF4E3 in these cells reduced, affecting parasite morphology and infectivity. The mutant LeishIF4E3(+/-) cells generated the storage bodies already under normal conditions, suggesting that the cell senses changes in the level of LeishIF4E3. Our data indicate that Leishmania recruited one of their LeishIF4E paralogs to overcome temporary starvation periods that they experience during their life cycle, by assembling temporary storage organelles in the cells. ­­­