Impact of m6A RNA modification on Leishmania drug resistance to antimonial

Abstract

Leishmania species have a complex life cycle involving migrating from an invertebrate to a vertebrate host, placing the parasite in adverse environmental conditions requiring rapid survival adaptation. Another scenario that requires the parasite to adapt is during stress caused by treatment drugs. To overcome these situations the parasite can use different mechanisms that involve for example changes in gene expression, which in Leishmania relies mainly on the post-transcriptional level. RNA nucleotide modifications, known as epitranscriptome, have been widely recognized as an additional mechanism for regulating gene expression and include N6-methyladenosine (m6A), N1-methyladenosine (m1A) and acetylcytidine (ac4C), present in rRNAs, tRNAs and mRNAs from different organisms. m6A is the most prevalent modification in mRNAs and depending on the region that is placed can affect the processing, stability and translation of the molecule. In order to investigate the presence and the possible function of m6A in the adaptation process of Leishmania, we performed Immuno-Northern Blotting analyses of five different parasite species. We found the presence of the modification in all species. Also, comparing m6A levels among the three main L. mexicana and L. infantum stages we detected higher m6A levels in amastigote than in metacyclic and procyclics. The presence of m6A was confirmed by Direct RNA sequencing using Oxford Nanopore experiments and revealed several mRNAs involved in stress response modified. Thus, with these initial data, we intend to this BEPE project to study how m6A could contribute to Leishmania antimonial-resistant phenotype, and for that, we will join our expertise with Dr. Fernandez-Prada's expertise to: i) investigate the changes in m6A levels in L. infantum under SbV pressure; ii) compare the m6A levels among L. infantum SbV-resistant and sensitive strains; iii) evaluate the presence of m6A-modified RNAs in the EVs released by L. infantum SbV-resistant versus sensible. The results generated by this project will contribute to our understating not only the mechanisms involved in Leishmania drug resistance but also the function of m6A as a whole.