Heterologous deletion of LeishIF4E-2, a cap-binding protein in Leishmania

Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to alternating environments. Stage-differentiation involves changes in the proteome profile. Translation regulation plays a central role in driving the differential program of gene expression, since gene regulation is controlled mostly by post transcriptional mechanisms. The Leishmania genome encodes six eIF4E candidates, each assumed to be responsible for specific functions, although overlaps are expected. Some are bound by eIF4G candidates, and others have no such binding partners. LeishIF4E-2 does not bind with any known eIF4G paralog. It was also shown to migrate in the polysomal fractions of sucrose gradients, unlike other initiation factors that usually migrate co-migrate with lighter fractions. Using the CRISPR-Cas9 methodology we deleted one of the two LeishIF4E2 gene copies. The deletion caused severe alterations in the morphology of mutant cells, causing them to be round and a-flagellated. Our lab used the CRISPR-Cas9 methodology to silence other cap-binding proteins, and in all cases such deletions impaired their infectivity. Unlike with other LeishIF4Es, silencing of LeishIF4E-2 had an opposite effect, as it enhanced the ability of the mutant cells to infect cultured macrophages, as compared to the infectivity of control parasites. The proteomic profile of the mutant cells and is currently analyzed, along with identification of associated transcripts. The combination of these two analyses should shed light on its potential functions. Our current results further support the possibility that the different LeishIF4Es are assigned unique functions.