Inositol pyrophosphates (e.g., IP7 and IP8) are metabolites involved in a wide range of cellular processes, such as telomeric homeostasis, homologous recombination, microbial pathogenicity, among others. However, the mechanism of action and the involvement of these metabolites in possible species-specific pathways are open questions. IP7 and IP8 are synthesized through complementary pathways involving the kinases IP6K and PP-IP5K, respectively. Trypanosomatid parasites (order Kinetoplastida, family Trypanosomatidae), such as Trypanosoma cruzi and Leishmania spp., have orthologs genes for IP6K, but the genes that code for PP-IP5K have not yet been identified in the genome of these organisms. In other words, these parasites apparently do not synthesize IP8 via PP-IP5K, which makes them excellent models for the study of inositol pyrophosphates. Curiously, preliminary results from our group show that Bodo saltans (a free-living kinetoplastid organism) has an ortholog gene for PP-IP5K with 42% identity to human PP-IP5K. Thus, the main goal of this study is to investigate whether the hypothetical absence of PP-IP5K is mutually exclusive in relation to the parasitism developed by some organisms within the Kinetoplastida order. For this, we carried out in silico analyzes using the kinases IP6K and PP-IP5K to confirm the supposed absence of PP-IP5K. Using MUSCLE, TRIMAL, PHYML, MEGA X and HMMER software, we reconstructed phylogenetic trees and obtained preliminary evidence that points to the absence of PP-IP5K orthologs in all trypanosomatids but Paratrypanosoma confusum. Predictions of tertiary structures suggest that the catalytic domain of PP-IP5K from P. confusum, although it has 44% identity relative to human PP-IP5K, presents an unusual structure, which puts its annotation in check. These preliminary findings suggest that the transition from a free-living to parasitic lifestyle resulted in the loss of PP-IP5K. The next steps to achieve the objective of this project involve the inclusion of the PP-IP5K gene from Bodo saltans (BsPP-IP5K) in T. cruzi and Leishmania spp. using specific plasmids (episomal knock-in). Alternatively, we can use the CRISPR/Cas9 system to introduce the BsPP-IP5K gene into a constitutive gene locus (e.g., ²-tubulin). After confirmation by PCR, qPCR, and western blot, we will perform clonal dilutions to select homogeneous populations. Then, we intend to carry out a phenotypic characterization of the generated lineages, e.g., evaluate the presence of IP8, proliferation, and cell cycle phases profile. Also, we will evaluate the infection and DNA repair capacity using wild-type host cells (WT) and host cells knockout (KO) for PP-IP5K. Infection assays using host cells PP-IP5K KO will allow us to assess whether T. cruzi or Leishmania use host IP8 during infection. Thus, this project will clarify in an unprecedented way whether PP-IPs have any relationship with the parasitism developed by some kinetoplastids, contributing to the understanding of the function of these metabolites that seem to be important in several pathways.
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