Análise do perfil de splicing alternativo de genes do patógeno oportunista Cryptococcus neoformans durante o processo infeccioso em modelo murino

Abstract

The yeast Cryptococcus neoformans is an environmental basidiomycete, that when inhaled by an immunocompromised person, becomes an opportunistic pathogen, causing the cryptococcosis. This disease manifests itself causing moderate conditions, such as pneumonias, and severe, such as meningitis - the latter being the cause of many deaths. As it is an eukaryote, C. neoformans presents Alternative Splicing (AS) as a way to produce more than one protein from the same gene and also as a form of gene regulation in response to environmental influences. The study of the profiles of AS in response to the infection environment, as well as its consequences for this pathogenic fungus, is essential to understanding the molecular systems necessary for its survival in the host. In this sense, the aim of this work was to analyze the occurrence, the profile and role of alternative splicing of C. neoformans genes, after 6 hours and 10 days of infection in experimental murine host. To this end, 24 C57BL/6 mice were intratracheally infected with 1x106 cells of C. neoformans. Bronchoalveolar lavage (BAL) was collected after 6 hours of infection in half of the animals (n=12) and after 10 days in the other half (n=12), so that it was possible to analyze the fungal transcriptome and compare with the control condition, which consisted of cultivating the fungus in YPD medium overnight. RNA extraction and transcriptome sequencing were performed and, then, the RNA-seq data were analyzed using the DEXseq and rMATS tools, to investigate how alternative splicing occurs in genes expressed by C. neoformans, in the condition of initial infection (after 6 hours) and late infection (10 days), compared to the control in YPD culture medium. The results show 221 differential exon usage (DEU) events and 1281 events of differential alternative splicing (DAS), among them: Intron retention (IR), Mutually exclusive exons (MXE), Alternative splice site 3' and 5' (A3 'SS and A5'SS) and exon skipping (ES), the latter being the most frequent in the conditions of infection, in comparison to the control condition. Nevertheless, the results indicated possible unannotated AS events in at least two C. neoformans genes. Furthermore, the analyzes provide a good correlation between these AS events and the increased expression of genes involved in pathways known to be important in the adaptation, survival and virulence of the C. neoformans pathogen in the context of infection, such as energy harvesting, cell replication and copper uptake – which evidences the biological role of AS for the microorganism, acting as a post-transcriptional gene control mechanism in response to environmental stimuli. Thus, works like this one – which provide relevant information regarding the splicing differences present in the opportunistic pathogen C. neoformans between conditions of infection and non-infection – are crucial to understand the dynamics of the interaction C. neoformans-mammal, as well as to generate income for proposing strategies to control this pathogen, as well as cryptococcosis.