Authors

CR Espada¹; JC Quilles¹; RM Magalhães¹; TP Defina¹; AK Cruz¹;  ¹ University of São Paulo, Brazil

Discussion

Leishmania (Viannia) braziliensis is an important causative agent of cutaneous and mucocutaneous leishmaniasis in Americas. During its life cycle, this parasite colonizes two different hosts facing dramatic changes in physiological conditions which requires a fast and dynamic gene expression modulation in order to survive. The genetic organization of Leishmania, where unrelated genes are transcribed by RNA Polymerase II as polycistronic units in the absence of canonical promoters, suggests that the regulation of gene expression in these organisms results mainly from other processes rather than transcription itself. Non-protein coding RNAs (ncRNAs) have been identified in different trypanosomatids parasites, including L. braziliensis. In the latter, the transcriptome of the main morphologies across the Leishmania lifecycle(procyclic promastigotes, metacyclic promastigotes and axenic amastigotes) were compared and revealed the presence of 11,372 putative ncRNAs of which at least 295 were differentially expressed in all three stages. Using CRISPR/Cas9, we are investigating the functional role of these elements in L. braziliensis. Up to date, 14 ncRNAs (including short and long ncRNAS) were successfully knocked out from L. braziliensis M2903 genome by our group and phenotypically screened for phenotypic alterations. Herein, we present the results obtained for three potential long ncRNAs (lncRNAs) each of them enrolled in different phenotypes of L. braziliensis. The fitness of each knocked out line was compared to the parental wild-type line (WT) in experiments mimicking important points of Leishmania life cycle such as parasite multiplication, survival to oxidative and nutritional stresses, metacyclogenesis and infectivity. We found that deletion of lncRNA66 led to a significant reduction in parasite growth as promastigotes whereas the deletion of lncRNA31 increased the doubling time of axenic amastigotes from 8.7 hours (WT) to 11.6 hours.  Deletion of lncRNA52 resulted in a lower percentage of metacyclic parasites recovered after Ficoll enrichment, suggesting that this lncRNA may be enrolled in metacyclogenesis in L. braziliensis. Characterization of these transcripts by northern blotting, determination of ncRNA extremities and posttranscriptional processing (presence or absence of CAP and PolyA tail) by RNA circularization coupled with sequencing, and identification of possible ligands using a S1M tag-mediated pull down are currently ongoing to better understand the biogenesis and role of these putative regulatory ncRNAs in the parasite. Our results will help to understand the regulation of Leishmania gene expression and may result in the discovery of ncRNAs enrolled in parasite fitness and pathways essentials for parasite survival.