Fig 1.
Differentially expressed genes in response to heme treatment.
(A) RNAseq analysis showing the number of genes identified and differentially expressed. (B) Upregulated and downregulated genes modulated by heme.
Table 1.
Illumina deep sequencing reads map.
RNA was extracted from samples by using TRIzol method (Invitrogen) and its degradation and contamination were monitored on 1% agarose gels. The cDNA synthesis was performed with 3 μg of total RNA using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer's recommendations. The Deep Seq transcriptome sequencing analysis was carried out using polyA selected RNA at 30M PE reads per sample in an Illumina NextSeq500 sequencer. Reference genomes and gene model annotation files were used from TriTryp database. Raw sequencing reads (raw data) were processed and the read counts were extracted. The raw reads were then filtered to remove low-quality sequences and empty reads to gain clean reads. All subsequent analyses were based on the clean reads with high quality.
Fig 2.
Gene ontology (GO) classification, KEGG/MetaCyc analysis of the differentially expressed genes (DEGs) of heme-treated parasites.
The percentage regulated genes based on GO annotations and KEGG/MetaCyc pathways. The (A) biological process and (B) metabolic pathways. % regulated genes is the percentage of the background or GO/pathway-related genes that were observed as differentially expressed in RNA-Seq data. FDR ≤ 0,05.
Fig 3.
Differently expressed genes related to energy metabolism in epimastigotes.
(A) RNAseq heatmap of selected genes expressed of control and heme treated samples. Columns represent each technical replicate from two independent experiment. The colors vary from light green (very low expression) to light red (extremely high expression). Data were scaled by the ratio of FPKM of each sample by the total of gene multiplied by 10. (B) Transcripts levels of energy metabolism genes presented as fold change compared to control samples. ST: sugar transporter; GALKg: galactokinase, glycosomal HKg: hexokinase, glycosomal; PMIg: phosphomannose isomerase, glycosomal; PFKg: phosphofructokinase, glycosomal; ALDg: fructose bisphosphate aldolase, glycosomal; ENOc: enolase, cytosolic; PPDKg: pyruvate phosphate dikinase, glycosomal; PEPCKg: phosphoenolpyruvate carboxykinase, glycosomal; MDHg: malate dehydrogenase, glycosomal; FRDg*: NADH-dependent fumarate reductase, glycosomal; KBL: 2-amino-3-ketobutyrate CoA ligase; P5CDH: δ-1-pyrroline-5-carboxylate dehydrogenase; ASAT: aspartate aminotransferase; AS: asparagine synthetase A; AHADH: aromatic L-2-hydroxyacid dehydrogenase; ME: malic enzyme; COMPLEX I: NADH-ubiquinone oxidoreductase; SDH: succinate dehydrogenase subunit; ABCt: ABC transporter; PdxK: pyridoxal kinase. These graphs present DEGs in “non-esmeraldo- like” haplotype and some exclusive in “esmeraldo-like” were marked with (*).
Fig 4.
Schematic representation of the metabolic map of gene expression regulated by heme.
Trypanosoma cruzi epimastigotes metabolic pathway illustrating differentially expressed genes in response to heme treatment involved in energy metabolism measured by RNAseq analysis. Values inbox are expressed as fold change of heme treated parasites in relation to control sample. Enzymes reactions were presented as known (black solid arrow), downregulated suggested-routes (black dashed arrow) and downregulated (gray solid arrow) routes by RNAseq data. Yellow circles indicate pyridoxal 5-phosphate binding enzymes. The enzymes of each proposed pathway are presented as specific colors. Glucose metabolism (blue): ST: sugar transporter; GALKg: galactokinase, glycosomal; HKg: hexokinase, glycosomal; PMIg: phosphomannose isomerase, glycosomal; PFKg: phosphofructokinase, glycosomal; ALDg: fructose bisphosphate aldolase, glycosomal; ENOc: enolase, cytosolic. Fermentation process (green): PPDKg: pyruvate phosphate dikinase, glycosomal; PEPCKg: phosphoenolpyruvate carboxykinase, glycosomal; MDHg: malate dehydrogenase, glycosomal; FRDg*: NADH-dependent fumarate reductase, glycosomal. Amino acids metabolism (purple): KBL: 2-amino-3-ketobutyrate CoA ligase; P5CDH: δ-1-pyrroline-5-carboxylate dehydrogenase; ASAT: aspartate aminotransferase; AS: asparagine synthetase A. AHADH: aromatic L-2-hydroxyacid dehydrogenase; Malate metabolism (orange): ME: malic enzyme. Mitochondrial metabolism (pink): COMPLEX I: NADH-ubiquinone oxidoreductase; SDH: succinate dehydrogenase subunit. Other metabolic pathways (gray): PdxK: pyridoxal kinase; ABCt: ABC transporter. Metabolytes: G-6-P: glucose-6-phosphate; F-6-P: fructose-6-phosphate; F-1,6-P: fructose-1,6-biphosphate; DHAP: dihydroxyacetone phosphate; GA3P: glyceraldehyde-3-phosphate; 1,3-BPGA: 1,3-bisphosphoglycerate; 3-PGA: 3-phosphoglycerate; 2-PGA: 2-phosphoglycerate; PEP: phosphoenolpyruvate; OXA: oxaloacetate; Glu: glutamate; Gln: glutamine; Phe: phenylalanine; Trp: tryptophan; Tyr: tyrosine; P5C: δ-1-pyrroline-5-carboxylate; AOB: amino-oxobutyrate.
Fig 5.
RNAseq and qPCR data of selected genes modulated by heme.
For qPCR validation, total RNA was extracted using TRIzol method (Invitrogen) and then was reverse-transcripted to single strand cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer's instructions. All quantitative measurements were carried out in triplicate and normalized to the internal TCZ control. Results were expressed as mean value ± standard error (SE). The mRNA fold change was calculated as described in the Material and Methods section. qPCR and RNAseq data were plotted for each gene for analysis. ST: sugar transporter; GALK: galactokinase; HK: hexokinase; PMI: phosphomannose isomerase; PFK: phosphofructokinase; ALD: fructose bisphosphate aldolase, glycosomal; ENO: enolase; PPDKg: pyruvate phosphate dikinase, glycosomal; PEPCKg: phosphoenolpyruvate carboxykinase, glycosomal; MDHg: malate dehydrogenase, glycosomal; FRD*: NADH-dependent fumarate reductase; KBL: 2-amino-3-ketobutyrate CoA ligase; P5CDH: δ-1-pyrroline-5-carboxylate dehydrogenase; ASAT: aspartate aminotransferase; AS: asparagine synthetase A; AHADH: aromatic L-2-hydroxyacid dehydrogenase; ME: malic enzyme, cytosolic; COMPLEX I: NADH-ubiquinone oxidoreductase; SDH: succinate dehydrogenase subunit; ABCt: ABC transporter; PdxK: pyridoxal kinase. This graphic presents DEGs in “non-esmeraldo-like” haplotype and some only in “esmeraldo-like” that were marked with (*).