LeishIF4E1 deletion affects the promastigote proteome, morphology, and infectivity

Leishmania parasites cycle between sand-fly vectors and mammalian hosts,adapting to changing environmental conditions by driving a stage-specific program ofgene expression, which is tightly regulated by translation processes. Leishmania encodessix eIF4E orthologs (LeishIF4Es) and five eIF4G candidates, forming different cap-bindingcomplexes with potentially varying functions. Most LeishIF4E paralogs display temperaturesensitivity in their cap-binding activity, except for LeishIF4E1, which maintainsits cap-binding activity under all conditions. We used the CRISPR-Cas9 system tosuccessfully generate a null mutant of LeishIF4E1 and examine how its eliminationaffected parasite physiology. Although the LeishIF4E1-/-null mutant was viable,its growth was impaired, in line with a reduction in global translation. As a resultof the mutation, the null LeishIF4E1-/-mutant had a defective morphology, as thecells were round and unable to grow a normal flagellum. This was further emphasizedwhen the LeishIF4E1-/-cells failed to develop the promastigote morphologyonce 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 circularstructure. Finally, the LeishIF4E1-/-null mutant displayed difficulty in infecting culturedmacrophages. The morphological changes and reduced infectivity of the mutantmay be related to differences in the proteomic profile of LeishIF4E1-/-cellsfrom that of controls. All defects monitored in the LeishIF4E1-/-null mutant were reversedin 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.