CONICET | Buscador de Institutos y Recursos Humanos

Background Trypanosoma cruzi is the protozoan parasite responsible for Chagas? disease. This is an endemic disease in Latin America that affects 18?20 million people. No vaccines are available at present and drugs used for treatment show undesirable side effects. The identification of new targets for chemotherapy is a major challenge in the control of this disease. In this regard, protein synthesis machinery has been proved to be a good target for drugs in many organisms, and many evidences suggest that translation in trypanosomatids show important differences with that of model organisms such as yeast and mammals [reviewed in 1]. The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the elongation step of protein synthesis through interaction with the elongation factor 2 (EF2). In T. cruzi, five components of the stalk have been identified, and their interaction with the EF2 was assessed showing that all P1/P2 proteins interacted with TcEF2 with similar affinities [2]. Interestingly, antibodies against the conserved C-terminal region of ribosomal P proteins from T. cruzi inhibits the elongation step of protein synthesis in trypanosomatids but not in mammals [3], probably by blocking the binding of EF2 to the ribosome. These evidences suggest that the interaction between EF2 and the P proteins could be targeted specifically by drugs. Methods & Results Because Trypanosoma cruzi can be typed into six major groups or Discrete Typing Units (DTUs) [4], we first decided to assess if there were differences between the T. cruzi EF2 (TcEF2) genes from the different DTUs. Primers to amplify TcEF2 from all the different strains were designed based on the putative T. cruzi EF2 gene from the strain CL Brener [5]. Because CL Brener belongs to DTU VI, we cloned the genes from the remaining five DTUs (I: strain G; II: strain Tul8; III: strain M5631; IV: strain CanIII; V: TEV41). Sequencing was performed by Macrogen Inc. and analysis with CLUSTAL omega [6] showed identities of >99% between all the genes at the nucleotide level. The resulting topology of the Neighbor-joining tree built with MEGA5 [7] from this alignment, was better explained by the ?Two-Hybridization? evolutionary model [8]. We then performed complementation assays in Saccharomyces cerevisiae using a double knock-out strain for both endogenous EF2 [9]. All genes were cloned to both p416-GPD and p426-GPD constitutive shuttle vectors. EF2 from S. cerevisiae (ScEF2) and the empty vectors were used as positive and negative controls, respectively. In these experiments we saw that TcEF2 wasn´t able to rescue the lethal phenotype, showing there´s a difference in the functional characteristics of both EF2s. To better identify and characterize the interaction surfaces between TcEF2 and the other proteins from the ribosome, a homology model was built using the MODELLER 9.12 pipeline [10]. Although the new model still needs further refinement, it already is better than the old one built with PHYRE [2], as observed by ERRAT2 validation analysis [11] and PROCHECK [12]. Conclusions Our results show that the TcEF2 genes are highly conserved among the different T. cruzi DTUs. This fact suggests that results obtained in one strain can be extrapolated to others. Also, the complementation assays show that TcEF2 differs functionally from its yeast counterpart; ScEF2, suggesting it could be possible to find specific drugs that target this factor. We propose this new model as the starting point for studying the TcEF2 interactions. These data will be used to tune up a BRET assay to measure protein-protein interactions, and the effect of different drugs on the system.