Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated hemizygous deletion

Leishmania parasites cycle between sand-fly vectors and mammalian hosts adapting to alternating environments by stage-differentiation accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E candidates, some of which bind a dedicated eIF4G candidate, and each eIF4E is assumed to have specific functions with perhaps some overlap. However, LeishIF4E2 does not bind any known eIF4G ortholog and was previously shown to comigrate with the polysomal fractions of sucrose gradients in contrast to the other initiation factors that usually comigrate with pre-initiation and initiation complexes. Here we deleted one of the two LeishIF4E2 gene copies using the CRISPR-Cas9 methodology. The deletion caused severe alterations in the morphology of the mutant cells that became round, small, and equipped with a very short flagellum that did not protrude from its pocket. Reduced expression of LeishIF4E2 had no global effect on translation and growth, unlike other LeishIF4Es; however, there was a change in the proteome profile of the LeishIF4E2(+/-) cells. Upregulated proteins were related mainly to general metabolic processes including enzymes involved in fatty acid metabolism, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included flagellar rod and cytoskeletal proteins, as well as surface antigens involved in virulence. Moreover, the LeishIF4E2(+/-) cells were impaired in their ability to infect cultured macrophages. Overall, LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Our results also suggest that the individual LeishIF4Es perform unique functions. Author summary Leishmania parasites cause a broad spectrum of diseases with different pathological symptoms. During their life cycle the parasites shuffle between sand-fly vectors and mammalian hosts adapting to the changing environments via a stage specific program of gene expression that promotes their survival. Translation initiation plays a key role in control of gene expression and in Leishmania this is exemplified by the presence of multiple cap-binding complexes that interact with mRNAs. The parasites encode multiple paralogs of the cap-binding translation initiation factor eIF4E and of its corresponding binding partner eIF4G forming complexes with different potential functions. The role of LeishIF4E2 remains elusive: it does not bind any of the LeishIF4G candidate subunits and associates with polysomes, a feature less common for canonical translation factors. Here we generated a hemizygous Leishmania mutant of the least studied cap-binding paralog, LeishIF4E2, by eliminating one of the two alleles using the CRISPR-Cas9 methodology. The mutant showed morphological defects with short and rounded cells, and a significant reduction in their flagellar length. Moreover, the LeishIF4E2(+/-) cells were impaired in their ability to infect cultured macrophages. The mutants showed differences in their proteome: upregulated proteins were related mainly to general metabolic processes including enzymes involved in fatty acid metabolism, DNA repair and replication, signaling, and cellular motor activity. Downregulated proteins included flagellar rod and cytoskeletal proteins, as well as surface antigens involved in virulence. Overall, LeishIF4E2 does not behave like a general translation factor and its function remains elusive. It could affect translation of a particular set of transcripts, causing direct or downstream effects that do not affect global translation. Our results suggest that individual LeishIF4Es perform specific functions.

Automated summary

Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to changing environments through stage-specific gene expression regulation. Translation initiation is controlled post-transcriptionally in Leishmania, with LeishIF4E2 showing unique characteristics and functions that differ from other translation factors. Deletion of one LeishIF4E2 gene copy led to altered morphology, changes in proteome profiles, and impaired infectivity in macrophages, suggesting specific functions for individual LeishIF4Es.