RESUMO
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 (SbIII)-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 SbIII-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 SbIII-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 SbIII-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host-parasite interaction, cellular process and other categories. The downregulated transcripts in the SbIII-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.
Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Resistência a Medicamentos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Proteínas de Protozoários/genética , Animais , Leishmania infantum/metabolismo , Leishmania infantum/fisiologia , Proteínas de Protozoários/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.