Abstract
The biological function of the post‐translational modification hypusine in the eukaryotic initiation factor 5A (EIF‐5A) in eukaryotes is still not understood. Hypusine is formed by two sequential enzymatic steps at a specific lysine residue in the precursor protein EIF‐5A. One important biological function of EIF‐5A which was recently identified is the translation of polyproline‐rich mRNA, suggesting its biological relevance in a variety of biological processes. Hypusinated eIF‐5A controls the proliferation of cancer cells and inflammatory processes in malaria. It was shown that pharmacological inhibition of the enzymes involved in this pathway, deoxyhypusine synthase (DHS) and the deoxyhypusine hydroxylase (DOHH), arrested the growth of malaria parasites. Down‐regulation of both the malarial eIF‐5A and dhs genes by in vitro and in vivo silencing led to decreased transcript levels and protein expression and, in turn, to low parasitemia, confirming a critical role of hypusination in eIF‐5A for proliferation in Plasmodium. To further investigate whether eIF‐5A and the activating enzyme DHS are essential for Plasmodium erythrocytic stages, targeted gene disruption was performed in the rodent malaria parasite Plasmodium berghei. Full disruption of both genes was not successful; instead parasites harboring the episome for eIF‐5A and dhs genes were obtained, suggesting that these genes may perform vital functions during the pathogenic blood cell stage. Next, a knock‐in strategy was pursued for both endogenous genes eIF‐5A and dhs from P. berghei. The latter resulted in viable recombinant parasites, strengthening the observation that they might be essential for proliferation during asexual development of the malaria parasite.
Highlights
The biological function of the post-translational modification hypusine in the eukaryotic initiation factor 5A (EIF-5A) in eukaryotes is still not understood
Successful genetic replacement of both the eukaryotic initiation factor 5A (eIF-5A) and dhs genes was checked in a PCR-specific amplification with a set of three different primer combinations which were located outside the coding region of the dihydrofolate reductase gene in b3d backbone vector and outside the 30 UTR of the eIF-5A and dhs genes, respectively (Fig. 3A,B)
Genotypical analysis for the 30 integration of the eIF-5A revealed an episomal integration of the recombinant b3D vector when the primer combination #T. gondii dihydrofolate reductase forward (Tg for) and eIF-5A 30UTR rev were employed detecting a fragment of a size of 1111 bp (Fig. 4A, lanes parental generation 1c, 2c)
Summary
The biological function of the post-translational modification hypusine in the eukaryotic initiation factor 5A (EIF-5A) in eukaryotes is still not understood. To further investigate whether eIF-5A and the activating enzyme DHS are essential for Plasmodium erythrocytic stages, targeted gene disruption was performed in the rodent malaria parasite Plasmodium berghei. A vital function for eIF-5A and dhs genes in malaria parasites demonstrating that heme biosynthesis is essential for the malaria parasite in the erythrocytic stages were recently challenged by knock-out parasite lines, lacking 5-aminolevulinic acid synthase and/or ferrochelatase (FC) [6]. These knock-out parasites grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs [6] due to their expression in the pre-erythrocytic liver stages
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