A suggested vital function for eIF-5A and dhs genes during murine malaria blood-stage infection

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.

Keywords: Plasmodium; hypusine; malaria; reverse genetics.

Figure 1

Schematic of the biosynthetic pathway of the post‐translational modification hypusine. Hypusine is formed within two subsequent steps. In the first step DHS transfers an aminopropyl moiety from the triamine spermidine to a specific lysine residue in the EIF‐5A precursor protein to form the deoxyhypusine intermediate. DOHH introduces a hydroxyl group into the side chain and thus completes hypusine formation. Targeting was performed for the eIF‐5A precursor protein and the DHS.

Figure 2

Schematic representation of the design of the knock‐in construct. Through two PCR amplification steps the 5′ UTR and the ORF of the dhs gene were amplified and assembled into the appropriately digested b3D⁺ Cherry vector. The amplified 3′UTR of the dhs gene was finally assembled into recombinant b3D+ mCherry vector 30.

Figure 3

(A) Targeted gene disruption of the eIF‐5A gene in Plasmodium berghei ANKA strain by replacement strategy to generate a potential PbeIF‐5A knock‐out parasite. The PBA MRA‐871 ANKA strain cl15cy1 wild‐type strain eIF5A locus is targeted with a XbaI/HindIII linearized fragment containing the 5′UTR and the 3′UTR of the eIF‐5A gene and the Toxoplasma gondii Dhfr‐positive selectable marker. Upon a double crossover event the PbeIF5A ORF is replaced by T. gondii Dhfr‐positive selectable marker. Three replacement‐specific primer combinations marked with blue arrows were applied. (a) T. gondii ORF forward and 3′UTR eIF‐5A reverse primer (b) T. gondii ORF forward and eIF‐5A ORF reverse (c) eIF‐5A ORF forward and 3′UTR eIF‐5A reverse. (B) The replacement strategy for the generation of potential Pbdhs parasites was similar to the targeted gene disruption for the eIF‐5A gene. The same primer combinations were employed except that dhs ORF primers forward and reverse, and 3′UTR dhs primer was used.

Figure 4

Replacement specific analysis of targeted gene disruption of the eIF‐5A and dhs genes: (A) Check of the predicted gene targeting of the 3′ UTR of the eIF‐5A gene by PCR analysis was performed using a set of gene‐specific primer combinations. PCR amplificates were analyzed on 1% agarose gels. M = 1 kb plus ladder (ThermoScientific, Darmstadt, Germany); 1a–c amplificates obtained from the transfected parental mouse 1; 2a–c amplificates detected from mouse 2 and the wild‐type Plasmodium berghei ANKA strain. Listed primer combination, which can give only a signal from the recombinant locus. (a) Primer # ORF P. berghei eIF‐5A forward + primer # P. berghei eIF‐5A genotype 3′ UTR reverse (b) Primer # Toxoplasma gondii forward + primer# eIF‐5A genotype 3′UTR reverse (c) Primer # T. gondii forward + primer# eIF‐5A ORF primer reverse. The results were obtained from two independent transfection experiments. (B) Replacement test primer combinations for the 3′UTR of the dhs gene by PCR analysis. 1a–c amplificates obtained from parental mouse 1; 2a–c amplificates detected from mouse 2 and 3a–c the wild‐type P. berghei ANKA strain. (C) 1a–c amplificates from the parental population represented by mouse 1, amplificates 2a–c from the wild‐type and the recombinant b3d⁺ mCherry vector 3a‐c. Replacement‐specific primer combinations: (a) Primer # ORF dhs forward + primer # dhs genotype 3′ UTR reverse; (b) Primer # T. gondii forward + primer dhs genotype 3′ UTR reverse; (c) Primer # T. gondii forward + primer# dhs ORF primer reverse.

Figure 5

Schematic representation of the knock‐in constructs applied for a 1 : 1 substitution of the entire eIF‐5A (A) and dhs genes (B) from Plasmodium berghei ANKA strain. Constructs contained the coding region from either the eIF‐5A or dhs gene from P. berghei cloned in b3D+ mCherry vector behind the 5′ UTR of either gene. The full coding sequence of the dhfr (dihydrofolate resistance gene) with the 5′UTR and 3′ UTR is used for drug selection with pyrimethamine. Upon a double cross over event, the endogenous eIf‐5A or dhs gene from P. berghei is replaced by the cloned eIF‐5A or dhs genes from P. berghei involving the linearized 5′UTR and 3′ UTR, respectively.

Figure 6

Analysis of the Plasmodium berghei ANKA eIF‐5A knock‐in after homologous recombination by double cross over. (A) PCR analysis to investigate 5′ and 3′ integration of the eIF‐5A gene from P. berghei using three different primer combinations (given in Table 1) in the transfer population (T), the parental population (P) and the wild‐type (W). (B) Table 1: 5′ integration of the eIF‐5A and dhs knock‐out parasites. Calculated fragments (bp) for the primer combination primer# genotype 5′UTR forward and primer# genotype 3′UTR rev after PCR analysis.

Figure 7

Genotypical analysis of integration after knock‐in into the dhs locus after gene targeting by homologous recombination. PCR analysis from three different primer combinations was employed to prove 5′ and 3′ integration of the dhs gene from Plasmodium berghei. These primer combinations are given in Table 2. Table 2: Calculated fragments (bp) of integration after knock‐in into Plasmodium berghei dhs locus for three different primer combinations after PCR analysis.