LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity

Abstract

ABSTRACT Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to changing environmental conditions by driving a stage-specific program of gene expression, which is tightly regulated by translation processes. Leishmania encodes six eIF4E orthologs (LeishIF4Es) and five eIF4G candidates, forming different cap-binding complexes with potentially varying functions. Most LeishIF4E paralogs display temperature sensitivity in their cap-binding activity, except for LeishIF4E1, which maintains its cap-binding activity under all conditions. We used the CRISPR-Cas9 system to successfully generate a null mutant of LeishIF4E1 and examine how its elimination affected parasite physiology. Although the LeishIF4E1–/– null mutant was viable, its growth was impaired, in line with a reduction in global translation. As a result of the mutation, the null LeishIF4E1–/– mutant had a defective morphology, as the cells were round and unable to grow a normal flagellum. This was further emphasized when the LeishIF4E1–/– cells failed to develop the promastigote morphology once they shifted from conditions that generate axenic amastigotes (33°C, pH 5.5) back to neutral pH and 25°C, and they maintained their short flagellum and circular structure. Finally, the LeishIF4E1–/– null mutant displayed difficulty in infecting cultured macrophages. The morphological changes and reduced infectivity of the mutant may be related to differences in the proteomic profile of LeishIF4E1–/– cells from that of controls. All defects monitored in the LeishIF4E1–/– null mutant were reversed in the add-back strain, in which expression of LeishIF4E1 was reconstituted, establishing a strong link between the cellular defects and the absence of LeishIF4E1 expression. IMPORTANCE 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.

Keywords

• CRISPR
• Leishmania
• cap-binding protein
• eIF4E