Do Plasmodium and human translational machineries fully interact? – Trans-tRNAs

Do Plasmodium and human translational machineries fully interact? The aim of this proposal is to study enzymes and RNAs from the translation machinery in Plasmodium falciparum with the objective to gain fundamental knowledge on protein synthesis in the pathogen and to search for novel anti-malarial strategies. Indeed, except sequence data from the genome, knowledge on Plasmodium aminoacyl-tRNA synthetases (aaRS) and transfer RNAs (tRNA) is essentially lacking. To reach this goal, the two partners with complementary skills, molecular biology (M. Frugier) and parasitology (E. Candolfi) will collaborate to explore putative intimate interactions between the parasite and its human host. In particular, we will explore tRNA expression and accumulation in Plasmodium. The reason for that relies on a peculiarity of the tRNA content in P. falciparum. Indeed, and in contrast to most organisms where abundance of tRNA species is correlated with codon usage, tRNA redundancy is minimal in Plasmodium. Therefore we want to shed light on the apparent contradiction of a need of efficient translation during the differentiation and multiplication stages characterizing the Plasmodium life cycle in human liver and the limited number of tRNA genes in this organism. As working hypothesis we consider the possible import of human tRNAs during the intracellular stages of the parasite with independently published supporting observations: P. falciparum is characterized by (i) a life cycle in human with two distinct rates of multiplication, (ii) the nature and availability of tRNAs, (iii) the existence of a not understood liver stage called transmigration and (iv) the inhibition of host translation during transmigration. Two lines of experiments are proposed to investigate the tRNA fate in Plasmodium during the liver stage. First, we will test the capacity of Plasmodium aaRSs to aminoacylate human tRNAs. We therefore will investigate structural and functional properties of three model aaRSs, namely aspartyl-, asparaginyl- and tyrosyl-tRNA synthetases (AspRS, AsnRS and TyrRS, respectively). This first strategy consists to compare aminoacylation systems from Plasmodium and from human to determine if Plasmodium can use efficiently human tRNAs to compensate for the restricted number of tRNA genes encoded in its genome. In parallel, we will try to unravel the putative mechanism used by Plasmodium to import human tRNA during the liver stage. We intend to conduct a series of experiments where, conditions can be adapted to detect a possible transfer of nucleic acids from the host cell to the pathogen by comparing the tRNA concentrations in parasites before and after migration through hepatocytes. We have also orientated our research towards the study of a unique gene coding for a putative 'tRNA receptor' in Plasmodium. Indeed, based on bio-informatics predictions this gene would code for a surface protein with a N-terminal trans-membrane helix and a soluble domain sharing a strong sequence homology with the C-terminal tRNA binding module of AIMP1 or Arc1p (tRNA binding proteins in mammals and yeast, respectively). Moreover, it is noteworthy that this protein is present only in Plasmodium (all different strains sequenced at this date) and could, for example, increase the human tRNAs concentration locally. Access to Plasmodium biological material including its tRNAs, expression of the parasite proteins in Escherichia coli and purification of their corresponding recombinant forms constitute a prerequisite essential for the access to the structural and functional properties not only for the three selected Plasmodium tRNA aminoacylation systems but also for the newly identified putative 'tRNA receptor'. The final purpose of the project will be the exploitation of the gained basic knowledge to select inhibitory compounds of Plasmodium development.