Eukaryotic translation initiation factor 5A and its hypusination play an important role in proliferation and differentiation of the human enteric protozoan parasite Entamoeba histolytica

Abstract

The eukaryotic translation initiation factor 5A (eIF5A) is highly conserved and essential in all eukaryotes. However, the specific roles of eIF5A in translation and in other biological processes remain elusive. In the present study, we described the role of eIF5A, its posttranslational modifications (PTM), and the biosynthetic pathway needed for the PTM in Entamoeba histolytica, the protozoan parasite responsible for amoebic dysentery and liver abscess in humans. E. histolytica encodes two isotypes of eIF5A and two isotypes of enzymes, deoxyhypusine synthase (DHS), responsible for their PTM. Both of the two eIF5A are functional, whereas only one DHS (EhDHS1), but not EhDHS2, is catalytically active. The DHS activity increased {bsim}2000 fold when EhDHS1 was coexpressed with EhDHS2 in Escherichia coli, suggesting that the formation of a heteromeric complex is needed for full enzymatic activity. Both EhDHS1 and 2 genes were required for in vitro growth of E. histolytica trophozoites, indicated by small antisense RNA-mediated gene silencing. In trophozoites, only eIF5A2, but not eIF5A1, gene was actively transcribed. Gene silencing of eIF5A2 caused compensatory induction of expression of eIF5A1 gene, suggesting interchangeable role of two eIF5A isotypes and also reinforcing the importance of eIF5As for parasite proliferation and survival. Furthermore, using a sibling species, Entamoeba invadens, we found that eIF5A1 gene was upregulated during excystation, while eIF5A2 was downregulated, suggesting that eIF5A1 gene plays an important role during differentiation. Taken together, these results have underscored the essentiality of eIF5A and DHS, for proliferation and differentiation of this parasite, and suggest that the hypusination associated pathway represents a novel rational target for drug development against amebiasis. Author summaryEukaryotic initiation factor 5A is a ubiquitous protein that is essential for cell proliferation. We examined the maturation, regulation, and function of eIF5A in E. histolytica. We found by small antisense RNA-mediated gene silencing that EhDHS1/2 and EheIF5A2 are essential for growth of E. histolytica trophozoites. We further found that only one eIF5A, EheIF5A2, of two isotypes was constitutively expressed in the trophozoites stage and silencing of EheIF5A2 gene caused overexpression of the other eIF5A isotype (EheIF5A1) to partially rescue the growth defect in this parasites. Furthermore, we found that transcription of eIF5A1 gene was stage-specifically upregulated during excystation in E. invadens. Taken together, we have demonstrated for the first time that the two eIF5As play important and distinct roles in Entamoeba biology. This study has also provided an answer to a long standing conundrum on the biological importance of polyamines: spermidine is essential for eIF5A hypusination essential for protein translation in Entamoeba. Our work should also help our understanding of the physiological significance of eIF5A and its post-translational modifications in other pathogenic eukaryotes and potentially lead to formulation of control measures against parasitic diseases.