Comparative transcriptomics in Leishmania braziliensis: disclosing differential gene expression of coding and putative noncoding RNAs across developmental stages

Abstract

Leishmaniasis is a worldwide public health problem caused by protozoan parasites of the genus Leishmania. Leishmania braziliensis is the most important species responsible for tegumentary leishmaniases in Brazil. An understanding of the molecular mechanisms underlying the success of this parasite is urgently needed. An in-depth study on the modulation of gene expression across the life cycle stages of L. braziliensis covering coding and noncoding RNAs (ncRNAs) was missing and is presented herein. Analyses of differentially expressed (DE) genes revealed that most prominent differences were observed between the transcriptomes of insect and mammalian proliferative forms (6,576 genes). Gene ontology (GO) analysis indicated stage-specific enriched biological processes. A computational pipeline and 5 ncRNA predictors allowed the identification of 11,372 putative ncRNAs. Most of the DE ncRNAs were found between the transcriptomes of insect and mammalian proliferative stages (38%). Of the DE ncRNAs, 295 were DE in all three stages and displayed a wide range of lengths, chromosomal distributions and locations; many of them had a distinct expression profile compared to that of their protein-coding neighbors. Thirty-five putative ncRNAs were submitted to northern blotting analysis, and one or more hybridization-positive signals were observed in 22 of these ncRNAs. This work presents an overview of the L. braziliensis transcriptome and its adjustments throughout development. In addition to determining the general features of the transcriptome at each life stage and the profile of protein-coding transcripts, we identified and characterized a variety of noncoding transcripts. The novel putative ncRNAs uncovered in L. braziliensis might be regulatory elements to be further investigated.

Keywords: Noncoding RNAs; comparative transcriptomics; differential gene expression; gene expression.

Figure 1.

Time course for selection of life cycle stages and morphology of cells used for RNA extraction. (a) Growth curve illustrative of the study design and time points. Growth curves of promastigote forms of L. braziliensis. Culture-derived procyclic forms (PRO) were obtained on day 2, metacyclic forms (META) were purified from a stationary-phase culture (day 5), and amastigotes (AMA) were obtained from differentiated cultures maintained in fetal bovine serum (FBS) at 33°C in a 1% CO₂ atmosphere on day 3. Data are shown as the means ± SD, n = 3. (b) Morphology of the different L. braziliensis developmental stages, as indicated above each panel, under scanning electron microscopy. White bars: 5 µm.

Figure 2.

Length distribution of gene elements in Leishmania braziliensis. (a) CDSs (Leishmania braziliensis 2903-TriTrypDB version 30). (b) 5ʹUTRs. (c) 3ʹUTR. For the methodology used to define the main SL acceptor and polyA sites, refer to Methods. The size of each bin is 100 nt.

Figure 3.

Gene ontology enrichment analysis. Biological processes enriched in genes upregulated in the life cycle stages of L. braziliensis (adjusted p value ≤ 0.05) are shown. Red bars: biological processes upregulated in PRO compared to META. Green bars: biological processes upregulated in META compared to AMA, and blue bars: biological processes upregulated in AMA compared to PRO. The x-axis percentage represents the fraction (%) of genes upregulated within the set of all genes from Leishmania assigned to the corresponding biological process.

Figure 4.

Profiles of differentially expressed protein-coding genes during life cycle progression. The set of genes encompasses only those DE in all three comparisons analyzed (PROvsMETA, METAvsAMA and AMAvsPRO). Total number of DE protein-coding genes = 216, distributed into 6 groups. Group 1: 176 genes, group 2: 17 genes, group 3: no representative, group 4: 21 genes, group 5: 1 gene, group 6: 1 gene. Reads per counts per million (CPM), values in log2.

Figure 5.

Graphic representation of predicted putative ncRNAs. The in silico identified putative ncRNAs were submitted to five ncRNAs predictors (see Methods). Results from Portrait, which estimates coding and noncoding potential of the transcript, and RNAcon, which discriminates between coding and noncoding RNAs and classifies the ncRNAs under 18 different ncRNAs classes, are presented. (a) On the left, the 11,372 putative ncRNAs that had been predicted by at least one of the 5 predictors are distributed in PORTRAIT classes (pie on the top) and RNAcon classes (pie on the bottom). On the right, in a similar organization, the 9,561ncRNAs predicted by at least two of the 5 programs are depicted and distributed among the different classes. (b) Those differentially expressed (DE) putative ncRNAs (3,602, FC ≥ 1.5) predicted at least by two of the 5 programs are depicted under the RNAcon 18 classes. Portrait and RNAcon classes are color-coded and presented in the figure. The gray ‘no score attributed’ indicates that the transcript is neither ncRNA nor mRNA according to Portrait classification.

Figure 6.

Profiles of differentially expressed putative ncRNAs during life cycle progression. The set of transcripts encompasses only those DE in all three comparisons analyzed (PROvsMETA, METAvsAMA and AMAvsPRO). Total number of DE ncRNAs = 295, distributed into 6 groups. Group 1: 180 ncRNAs, Group 2: 15 ncRNAs, Group 3: 4 ncRNAs, Group 4: 67 ncRNAs, Group 5: 7 ncRNAs, Group 6: 22 ncRNAs. Reads per counts per million (CPM), values in log2.

Figure 7.

Genomic distribution of DE ncRNA identified in Leishmania braziliensis, represented in a circos plot. Sectors are equivalent to parasite chromosomes. Each sector contains 4 tracks, three of which are divided into two subtracks. From outside to inside, the tracks are named CDS, CDS_DE, ncRNA_DE and Size_&_Loc. The subtracks correspond to the positive and negative strands, transcription oriented clockwise or counterclockwise, respectively. Track 1 (‘CDS’) shows all CDS throughout the parasite chromosomes. Each CDS is represented by a vertical line painted with alternation of 3 grayscale colors to help discriminate individual CDSs and to highlight the annotation and orientation of the polycistronic transcription units (PTUs). Tracks 2 and 3 show the DE protein-coding and ncRNA transcripts, respectively. All of them are DE in all three comparisons. The color of each transcript line depicts the expression profile of each group, as shown in the graphical representation in figure 6 (P, M and A are procyclic, metacyclic and amastigote, respectively). Track 4 shows the DE ncRNAs smaller than 2000 bp. The subtrack delimits the ncRNA length (below or above 200 nucleotides long). The colors are related to the transcript location (orange – 5´UTR, gray – undetermined, yellow – 3´UTR, dark brown – antisense-CDS).

Figure 8.

Identification and validation of 2 putative ncRNAs in L. braziliensis. (a) and (d): Regions of chromosomes 33 and 32, enclosing the putative ncRNAs LbrM2903_33_lncRNA177 and LbrM2903_32_lncRNA243, respectively. (b) and (e): extracted and zoomed areas for the corresponding lncRNAs depicting the number of reads in L. braziliensis procyclic, metacyclic and amastigote stages. (c) and (f): Northern blot of L. braziliensis total RNA using specific antisense oligonucleotides to each putative ncRNA. Arrows show multiple bands with approximate sizes to those predicted for the putative ncRNA. Ama: amastigote; Meta: metacyclic; Pro: procyclic; Gene: annotated genes; ncRNA: noncoding RNAs. Thin colorful regions (in a and b) represent divergence between reads and the reference genome.