Epimune: Investigating epitranscriptomic alterations in Leishmania-host interaction

• Grant number: 25/06837-7
• 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: Anelise Gonçalves Marino
• 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

Leishmaniasis is a group of neglected tropical diseases caused by protozoan parasites of the genus Leishmania, with more than 20 species capable of infecting humans. During its life cycle, the parasite alternates between an invertebrate host, where it exists in the promastigote form, and a vertebrate host, where it resides as intracellular amastigotes within macrophages. In the intracellular phase, the parasite faces the host's immune defense machinery and has developed host-subversion mechanisms, which may include changes in the expression of specific genes and alterations in the host cell metabolism that support its replication. Recently, post-transcriptional RNA modifications have gained attention as novel regulatory mechanisms of gene expression and immune responses during infections. Over one hundred mRNA modifications have been described in recent years, occurring in different regions of the RNA molecule and affecting multiple stages of its lifecycle. Among these, N6-methyladenosine (m6A) is the most abundant and has been associated with immune evasion by viruses such as HIV, ZIKV, and DENV, as well as with the regulation of genes involved in the immune response during infection. Considering the ability of Leishmania to manipulate macrophages to promote infection and the regulatory roles of m6A in immune responses, this project aims to investigate how parasite infection affects m6A levels and its regulatory machinery in macrophages. Our preliminary data indicate a trend toward increased expression of the m6A writer METTL3 and decreased expression of the eraser ALKBH5. Moreover, we observed alterations in global m6A levels in macrophages during Leishmania infection. To identify which genes-such as those involved in the immune response-may be methylated by this modification, we performed direct RNA sequencing using Nanopore technology. The next phase of the project aims to analyze the generated data to identify and quantify m6A-modified transcripts during infection. We will also examine the expression of additional components of the m6A regulatory machinery beyond METTL3 and ALKBH5. Additionally, we intend to generate METTL3 and ALKBH5 knockout macrophage lines using the CRISPR/Cas9 system to investigate the functional role of m6A during L. amazonensis infection. The analyses will include in vitro infection rates, cytokine profiling, iNOS expression, and nitric oxide (NO) production. This project will contribute to a better understanding of post-transcriptional RNA modifications and their biological implications in the host-parasite interaction. (AU)