Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of Leishmania

Abstract

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.

LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity

“…While LeishIF4E1 is nonessential for parasite viability and both alleles could be eliminated, similar attempts to delete the two LeishIF4E3 alleles were unsuccessful. However, eliminating even a single gene copy of LeishIF4E3 resulted in parasites with impaired growth and infectivity, along with altered morphology and restricted growth of their flagellum (43). The involvement of both LeishIF4Es in the regulation of cell morphology and flagellar growth suggests that different LeishIF4Es may have overlapping cellular functions, as deletion of both cap-binding paralogs leads to comparable phenotypes.…”

eIF4E and Interactors from Unicellular Eukaryotes

“…They are more abundant and carry an aminoterminal extension not observed for other eIF4Es (see Figure 4B). Leishmania eIF4E4 is essential for growth of the parasite [84], deletion of a single eIF4E3 allele (out of two) results in altered morphology and impaired infectivity of the parasite [85]. While Leishmania eIF4E3 interacts with eIF4G4, eIF4G3 interacts with eIF4E4, but not with eIF4E1, eIF4E2, or eIF4E3 [86,87].…”

“…Later studies indicate that eIF4E3 (which has only a weak cap-binding activity) interacts through a non-canonical alpha-helix of eIF4G4. Upon nutritional stress, this interaction ceases and eIF4E3 enters stress granules [85,94].…”

“…While eIF4E4 predominates in promastigotes (the flagellated and elongated infective form of Leishmania), in amastigotes (the non-flagellated form replicating in infected cells) eIF4E4 decreases and is replaced by eIF4E1 which does not interact with eIF4G [90]. An eIF4E1-knockdown is viable but the parasite cannot form the infective flagellated promastigote form [85]. In vitro, Leishmania eIF4E1 forms a complex with the carboxyterminus of eIF3a in the absence of eIF4G3 [92].…”

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

“…However, upon nutritional stress LeishIF4E3 concentrates in cytoplasmic granules that function as storage sites for inactive mRNAs and ribosomal proteins [ 18 ]. Deletion of a single LeishIF4E3 copy by CRISPR-Cas9 led to changes in cell morphology and infectivity; it was not possible to generate a null mutant suggesting that LeishIF4E3 is an essential gene [ 19 ]. Two additional cap-binding proteins were identified in T .…”