Characterization of the telomerase reverse transcriptase (TERT) component of Leishmania infantum and assessment of its role in telomere maintenance, genome integrity, cell cycle progression, and parasite infectivity

• Grant number: 23/16161-5
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: August 01, 2024
• End date: October 31, 2026
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Maria Isabel Nogueira Cano
• Grantee: Habtye Bisetegn Endalamaw
• Host Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil
• Associated research grant: 18/04375-2 - Studies about the biogenesis and composition of the Leishmania spp. ribonucleoprotein complex and its regulation, AP.TEM

Abstract

Leishmaniasis is a neglected vector-borne tropical disease caused by intracellular protozoan parasites of the genus Leishmania. The disease represents a major global public health problem, with 1.5 to 2 million cases annually worldwide. Leishmania infantum causes visceral leishmaniasis, which is the most fatal form of the disease and responsible for more than 90% of leishmaniasis deaths. As there is no efficient treatment or control method to eradicate the disease, the search for parasite-specific targets for drug design is urgent. Telomeres are nucleoprotein structures composed of repetitive DNA and associated proteins, which protect the ends of eukaryotic chromosomes from DNA damage response. They are vital for maintaining genome stability and cell proliferation. They are elongated with each cell cycle by the action of the telomerase ribonucleoprotein complex, minimally composed of the TERT (reverse transcriptase) and TER (telomerase RNA). In this project, we used the CRISPR-Cas9 system devised by Beneke et al (2017) to induce the knockout of the two alleles of the L. infantum TERT gene (LiTERT) and evaluate its role in parasite homeostasis. Preliminary results presented here show the success of gene editing with the deletion of both alleles of the TERT gene and adequate insertion of donor cassettes containing puromycin and neomycin resistance genes at the LiTERT locus on chromosome 36. Telomere southern blot with genomic DNA extracted from promastigote forms of early passages (P3) showed that the selected clones present shorter telomeres when compared to the parental control lineage. We selected one of the clones to continue the investigation to verify whether the absence of the LiTERT component can alter the parasite's lifespan and genomic integrity. Different in vivo and in vitro experiments will be carried out to evaluate the infective potential of the knockout strain and to verify if LiTERT could be a potential therapeutic target against the parasites.