Investigating the role of m6A in the biology and stress response of Leishmania

• Grant number: 25/06325-6
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: June 01, 2025
• End date: April 30, 2029
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Nilmar Silvio Moretti
• Grantee: Artur Honorato Reis
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 22/03075-0 - Unraveling the regulatory mechanisms of host-parasite interaction of Leishmania: focus on post-transcriptional and post-translational changes, AP.PNGP.PI

Abstract

The life cycle of Leishmania involves promastigotes in the insect vector and amastigotes in the mammalian host. Adaptation to these distinct environments requires not only morphological changes but also alterations in translation and gene expression. In Leishmania, gene expression is predominantly regulated at the post-transcriptional level through mechanisms involving mRNA stability and degradation. Various post-transcriptional RNA modifications have been described and are widely recognized as additional mechanisms for gene regulation. Among these, N6-methyladenosine (m6A) and N1-methyladenosine (m1A) are the most abundant and are present in RNAs from a wide range of organisms. m6A is added by the methyltransferases METTL3/METTL14 and removed by the demethylases FTO/ALKBH5. Although a METTL3 ortholog has not yet been identified in trypanosomatids, m6A has been detected in Trypanosoma brucei, where it impacts the regulation of VSG genes.Additionally, our group has confirmed the presence and quantified the levels of m6A in five Leishmania species, observing variation across the three major life cycle stages of L. mexicana and L. infantum (procyclic, metacyclic, and amastigote), with a marked increase in the amastigote form. Preliminary data using direct RNA sequencing with the Oxford Nanopore platform, we identified more than 4,000 m6A-modified genes. Based on these findings, this project aims to investigate the role of the m6A epitranscriptomic modification in L. infantum, focusing on cellular differentiation throughout the life cycle and on resistance to antimonials. The proposal includes analysis of m6A levels during metacyclogenesis and amastigogenesis, as well as under oxidative stress and exposure to drugs used in leishmaniasis treatment. Direct RNA sequencing data will be used to map m6A-modified transcripts in Sb-sensitive and Sb-resistant strains, with particular attention to genes associated with drug resistance, such as AQP1 and MRPA.In addition, we aim to identify and characterize the methyltransferase responsible for m6A deposition in L. infantum using computational and experimental approaches, including the generation of knockout and overexpressing mutants for functional assessment. The expected results will contribute to elucidating the role of m6A as a potential regulatory mechanism for parasite adaptation to the host environment and to drug pressure. (AU)