Leishmania parasites are the causative agents of a broad spectrum of diseases. The parasites migrate between sand-fly vectors and mammalian hosts, adapting to changing environments by driving a regulated program of gene expression, with translation regulation playing a key role. The leishmanias encode six different paralogs of eIF4E, the cap-binding translation initiation factor. Since these vary in function, expression profile, and assemblage, it is assumed that each is assigned a specific role throughout the life cycle. Using the CRISPR-Cas9 system for Leishmania, we generated a null mutant of LeishIF4E1, eliminating both alleles. Although the mutant cells were viable, their morphology was altered and their ability to synthesize the flagellum was impaired. Elimination of LeishIF4E1 affected their protein expression profile and decreased their ability to infect cultured macrophages. Restoring LeishIF4E1 expression restored the affected features. This study highlights the importance of LeishIF4E1 in diverse cellular events during the life cycle of Leishmania.

The genomes of Leishmania and trypanosomes encode six paralogs of the eIF4E cap-binding protein, known in other eukaryotes to anchor the translation initiation complex. In line with the heteroxenous nature of these parasites, the different LeishIF4E paralogs vary in their biophysical features and their biological behavior. We therefore hypothesize that each has a specialized function, not limited to protein synthesis. Of the six paralogs, LeishIF4E-3 has a weak cap-binding activity. It participates in the assembly of granules that store inactive transcripts and ribosomal proteins during nutritional stress that is experienced in the sand fly. We investigated the role of LeishIF4E-3 in Leishmania mexicana promastigotes using the CRISPR-Cas9 system. We deleted one of the two LeishIF4E-3 alleles, generating a heterologous deletion mutant with reduced LeishIF4E-3 expression. The mutant showed a decline in de novo protein synthesis and growth kinetics, altered morphology, and impaired infectivity. The mutant cells were rounded and failed to transform into the nectomonad-like form, in response to purine starvation. Furthermore, the infectivity of macrophage cells by the LeishIF4E-3(+/−) mutant was severely reduced. These phenotypic features were not observed in the addback cells, in which expression of LeishIF4E-3 was restored. The observed phenotypic changes correlated with the profile of transcripts associated with LeishIF4E-3. These were enriched for cytoskeleton- and flagellum-encoding genes, along with genes for RNA binding proteins. Our data illustrate the importance of LeishIF4E-3 in translation and in the parasite virulence. IMPORTANCE Leishmania species are the causative agents of a spectrum of diseases. Available drug treatment is toxic and expensive, with drug resistance a growing concern. Leishmania parasites migrate between transmitting sand flies and mammalian hosts, experiencing unfavorable extreme conditions. The parasites therefore developed unique mechanisms for promoting a stage-specific program for gene expression, with translation playing a central role. There are six paralogs of the cap-binding protein eIF4E, which vary in their function, expression profiles, and assemblages. Using the CRISPR-Cas9 system for Leishmania, we deleted one of the two LeishIF4E-3 alleles. Expression of LeishIF4E-3 in the deletion mutant was low, leading to reduction in global translation and growth of the mutant cells. Cell morphology also changed, affecting flagellum growth, cell shape, and infectivity. The importance of this study is in highlighting that LeishIF4E-3 is essential for completion of the parasite life cycle. Our study gives new insight into how parasite virulence is determined.

Leishmania parasites lack pathways for de novo purine biosynthesis. The depletion of purines induces differentiation into virulent metacyclic forms. In vitro, the parasites can survive prolonged periods of purine withdrawal changing their morphology to long and slender cells with an extended flagellum, and decreasing their translation rates. Reduced translation leads to the appearance of discrete granules that contain LeishIF4E-3, one of the six eIF4E paralogs encoded by the Leishmania genome. We hypothesize that each is responsible for a different function during the life cycle. LeishIF4E-3 is a weak cap-binding protein paralog, but its involvement in translation under normal conditions cannot be excluded. However, in response to nutritional stress, LeishIF4E-3 concentrates in specific cytoplasmic granules. LeishIF4E-3 granulation can be induced by the independent elimination of purines, amino acids and glucose. As these granules contain mature mRNAs, we propose that these bodies store inactive transcripts until recovery from stress occurs. In attempt to examine the content of the nutritional stress-induced granules, they were concentrated over sucrose gradients and further pulled-down by targeting in vivo tagged LeishIF4E-3. Proteomic analysis highlighted granule enrichment with multiple ribosomal proteins, suggesting that ribosome particles are abundant in these foci, as expected in case of translation inhibition. RNA-binding proteins, RNA helicases and metabolic enzymes were also enriched in the granules, whereas no degradation enzymes or P-body markers were detected. The starvation-induced LeishIF4E-3-containing granules, therefore, appear to store stalled ribosomes and ribosomal subunits, along with their associated mRNAs. Following nutritional stress, LeishIF4E-3 becomes phosphorylated at position S75, located in its less-conserved N-terminal extension. The ability of the S75A mutant to form granules was reduced, indicating that cellular signaling regulates LeishIF4E-3 function.

Leishmania parasites cycle between sand fly vectors and mammalian hosts, transforming from extracellular promastigotes that reside in the vectors’ alimentary canal to obligatory intracellular non-motile amastigotes that are harbored by macrophages of the mammalian hosts. The transition between vector and host exposes them to a broad range of environmental conditions that induces a developmental program of gene expression, with translation regulation playing a key role. The Leishmania genome encodes six paralogs of the cap-binding protein eIF4E. All six isoforms show a relatively low degree of conservation with eIF4Es of other eukaryotes, as well as among themselves. This variability could suggest that they have been assigned discrete roles that could contribute to their survival under the changing environmental conditions. Here, we describe LeishIF4E-5, a LeishIF4E paralog. Despite the low sequence conservation observed between LeishIF4E-5 and other LeishIF4Es, the three aromatic residues in its cap-binding pocket are conserved, in accordance with its cap-binding activity. However, the cap-binding activity of LeishIF4E-5 is restricted to the promastigote life form and not observed in amastigotes. The overexpression of LeishIF4E-5 shows a decline in cell proliferation and an overall reduction in global translation. Immuno-cytochemical analysis shows that LeishIF4E-5 is localized in the cytoplasm, with a non-uniform distribution. Mass spectrometry analysis of proteins that co-purify with LeishIF4E-5 highlighted proteins involved in RNA metabolism, along with two LeishIF4G paralogs, LeishIF4G-1 and LeishIF4G-2. These vary in their conserved eIF4E binding motif, possibly suggesting that they can form different complexes.

Translation of most cellular mRNAs in eukaryotes proceeds through a cap-dependent pathway, whereby the cap-binding complex, eIF4F, anchors the preinitiation complex at the 5′ end of mRNAs and regulates translation initiation. The requirement of Leishmania to survive in changing environments can explain why they encode multiple eIF4E (LeishIF4Es) and eIF4G (LeishIF4Gs) paralogs, as each could be assigned a discrete role during their life cycle. Here we show that the expression and activity of different LeishIF4Es change during the growth of cultured promastigotes, urging a search for regulatory proteins. We describe a novel LeishIF4E-interacting protein, Leish4E-IP2, which contains a conserved Y(X)4LΦ IF4E-binding-motif. Despite its capacity to bind several LeishIF4Es, Leish4E-IP2 was not detected in m7GTP-eluted cap-binding complexes, suggesting that it could inhibit the cap-binding activity of LeishIF4Es. Using a functional assay, we show that a recombinant form of Leish4E-IP2 inhibits the cap-binding activity of LeishIF4E-1 and LeishIF4E-3. Furthermore, we show that transgenic parasites expressing a tagged version of Leish4E-IP2 also display reduced cap-binding activities of tested LeishIF4Es, and decreased global translation. Given its ability to bind more than a single LeishIF4E, we suggest that Leish4E-IP2 could serve as a broad-range repressor of Leishmania protein synthesis.