Caracterização funcional de fatores de virulência relacionados a N- glicosilação em Leishmania

Abstract

Leishmaniasis presents a wide range of clinical manifestations depending on the Leishmania species that causes the disease and the host immune response. However, the understanding of the species-specific adaptations responsible for parasite virulence and tropism is still limited. Several studies have shown that glycans are important factors that mediate several Leishmania-host interactions, being, therefore, an interesting object of study for a better understanding of the biology of the parasite. Comparative genomic analysis among Leishmania species, previously performed by our group, revealed that some genes involved in glycan biosynthesis are absent in L. tarentolae, a non-infective species for mammals. Flow cytometric analysis corroborated these in silico findings. Thus, the aim of this work was to carry out the functional characterization of two genes associated with the glycosylation process in Leishmania. Among the species evaluated in comparative genomics, one gene was selected for being absent only in L. tarentolae (LbrM2903_340062700), while another is absent in L. braziliensis and L. tarentolae (LINF_120006700). The mRNA expression levels of these genes were quantified by RT-qPCR in promastigotes and axenic and intracellular amastigotes of L. braziliensis, L. donovani, L. infantum and L. tarentolae. The gene LbrM2903_340062700 presented a higher expression level in the promastigotes in L. braziliensis and as expected, no expression was detected in L. tarentolae. For LINF_120006700, expression results confirmed the absence of the gene in L. braziliensis and L. tarentolae, revealed higher expression levels in amastigotes of the viscerotropic species. Mutants of L. braziliensis were generate by deleting the LbrM2903_340062700 gene, using CRISPR-Cas9 genome editing technique, and knock-in the LINF_120006700 gene. The mutant parasites were confirmed by conventional PCR and by RT-qPCR. Flow cytometry and fluorophore-labeled carbohydrate electrophoresis (FACE) assays suggest a possible phenotype associated with alternative pathways to transfer mannose residues to the parasite surface in knockout clones. Analyzes of mRNA expression of other genes related to the N-glycosylation in mutant and wild-type parasites reinforce this hypothesis. We hope that this work can contribute to a better understanding of the mechanisms of Leishmania infection and that these enzymes may be good candidates for therapeutic interventions.