Investigation of the role of telomeres and inositol pyrophosphates during the in vivo metacyclogenesis in Leishmania braziliensis

• Grant number: 24/13506-4
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: January 31, 2025
• End date: January 30, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Marcelo Santos da Silva
• Grantee: Vitor Luiz da Silva
• Supervisor: David Lawrence Sacks
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: National Institutes of Health, Bethesda (NIH), United States
• Associated to the scholarship: 22/00923-0 - Could telomeres and inositol pyrophosphates play important roles in the metacyclogenesis and virulence of Leishmania?, BP.DD

Abstract

Leishmaniases refer to a group of diseases caused by parasites of the Leishmania genus, which mainly affect people in a socially vulnerable state. Currently, the drugs available for the treatment of leishmaniasis have side effects and end up selecting persistent parasites, which makes the search for new therapeutic targets fundamental. In eukaryotes, inositol pyrophosphates (PP-IPs) - mainly IP7 - are involved in a wide range of cellular processes, such as telomere length regulation and fungal virulence. However, the mechanism of action of these metabolites is a question that remains open. We hypothesize that IP7 plays an important role in telomeric dynamics and pathogenicity in Leishmania spp., organisms that have a high proliferative capacity. Using the CRISPR/Cas9 system, we successfully depleted both TERT (responsible for telomere elongation) and IP6K (responsible for IP7 synthesis) alleles in Leishmania braziliensis (the causative agent of mucocutaneous leishmaniasis in the Americas), generating a double knockout lineage (TERT-/- + IP6K-/-). Our findings suggest that the depletion of TERT + IP6K decreases the proliferation rate and impairs cell cycle progression, generating an accumulation of cells in cytokinesis (2F2K2N). Also, preliminary assays suggest an impairment in the in vitro infection process in mammalian macrophages. Thus, in collaboration with Dr. David Sacks (NIH), we intend to use the generated L. braziliensis lineages to learn how to infect and dissect Sandflies (Lutzomyia longipalpis) to isolate the organs where the different forms of Leishmania are housed. Next, we will quantify and analyze the different transitional forms between procyclic promastigotes and metacyclic promastigotes (pro-meta transition) by confocal microscopy (morphology and host-localization), flow cytometry (DNA content and cell cycle analysis) and scRNA-seq (single-cell transcriptomics). This approach will benefit both groups, as it will allow expand and validate the pseudotime inference to reconstruct the temporal progression of Leishmania differentiation in sandfly infections - standardized so far only in L. major. Furthermore, the expected results will clarify the effects of TERT + IP6K loss during the in vivo pro-meta transition of L. braziliensis, which will contribute significantly to a better understanding of the pyrophosphorylation and telomerase dynamics during the metacyclogenesis of this human pathogen.