Comparative transcriptomic analysis of antimony resistant and susceptible Leishmania infantum lines

Abstract

Background: One of the major challenges to leishmaniasis treatment is the emergence of parasites resistant to antimony. To study differentially expressed genes associated with drug resistance, we performed a comparative transcriptomic analysis between wild-type and potassium antimonyl tartrate (Sb^III)-resistant Leishmania infantum lines using high-throughput RNA sequencing.

Methods: All the cDNA libraries were constructed from promastigote forms of each line, sequenced and analyzed using STAR for mapping the reads against the reference genome (L. infantum JPCM5) and DESeq2 for differential expression statistical analyses. All the genes were functionally annotated using sequence similarity search.

Results: The analytical pipeline considering an adjusted p-value < 0.05 and fold change > 2.0 identified 933 transcripts differentially expressed (DE) between wild-type and Sb^III-resistant L. infantum lines. Out of 933 DE transcripts, 504 presented functional annotation and 429 were assigned as hypothetical proteins. A total of 837 transcripts were upregulated and 96 were downregulated in the Sb^III-resistant L. infantum line. Using this DE dataset, the proteins were further grouped in functional classes according to the gene ontology database. The functional enrichment analysis for biological processes showed that the upregulated transcripts in the Sb^III-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host-parasite interaction, cellular process and other categories. The downregulated transcripts in the Sb^III-resistant line are assigned in the GO categories: ribonucleoprotein complex, ribosome biogenesis, rRNA processing, nucleosome assembly and translation.

Conclusions: The transcriptomic profile of L. infantum showed a robust set of genes from different metabolic pathways associated with the antimony resistance phenotype in this parasite. Our results address the complex and multifactorial antimony resistance mechanisms in Leishmania, identifying several candidate genes that may be further evaluated as molecular targets for chemotherapy of leishmaniasis.

Keywords: Differentially expressed genes; Leishmania infantum; RNA sequencing; Resistance; Transcriptome; Trivalent antimony.

Fig. 1

Venn diagram of shared and specific Gene Ontology terms for the differentially expressed transcripts. The 644 differentially expressed genes (FC ≥ 2) of antimony-resistant L. infantum were compared and grouped together using the Gene Ontology categories (BP biological process, MF molecular function, CC cellular component). The total amount of shared and specific sequences in each ontology group is depicted in the figure. In addition, 289 differentially expressed genes (FC ≥ 2) were not assigned to any gene ontology category (WGO)

Fig. 2

Gene Ontology enrichment analysis for the upregulated transcripts. The GO enrichment analysis (Fisher’s exact test) was performed using as test set the list of upregulated transcripts and as reference set the L. infantum JPCM5 predicted proteome. FDR < 0.05 was set as a threshold to define the functional enrichment significance. The percentage of sequences in each GO category is described in the Y axis. Red bars represent the percentage of sequences classified in each GO term for the “reference set” group, and the blue bars represent the percentage of sequences classified in each GO term for the “test set” group (DE genes)

Fig. 3

Gene Ontology enrichment analysis for the downregulated transcripts. The GO enrichment analysis (Fisher’s exact test) was performed using as test set the list of downregulated transcripts and as reference set the L. infantum JPCM5 predicted proteome. FDR < 0.05 was set as a threshold to define the functional enrichment significance. The percentage of sequences in each GO category is described in the Y axis. Red bars represent the percentage of sequences classified in each GO term for the “reference set” group, and the blue bars represent the percentage of sequences classified in each GO term for the “test set” group (DE genes)

Fig. 4

Differentially expressed (DE) genes for the most representative GO-enriched terms for the biological process category. The figure shows the most representative GO terms for the not enriched but differentially expressed (up- and downregulated) dataset. Blue bars represent the total number of genes with functional annotation for each term, and red bars represent the total number of hypothetical proteins for each category, in the same dataset