The evolutionary history was inferred using the Maximum Parsimony method.

<p>The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). The analysis involved 21 amino acid sequences. There were a total of 289 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164650#pone.0164650.ref034" target="_blank">34</a>].</p

Cruzipain mRNA within reservosomes and colocalization of TcRBP40 protein and cruzipain mRNA.

<p>A) Plane Z reconstruction from confocal images obtained with cruzipain probes labeled with Cy-5 in epimastigotes. B) Merged image counterstaining with DAPI (green); Differential interference contrast (DIC) images are shown for identification of the cellular body of the parasite and the flagellum. Scale bar  = 10 µm. White arrows indicate the position of the flagellum. Colocalization of C) cruzipain mRNA labeled with Cy-5 and D) TcRBP40 protein. E) Merged image counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). F) Western blot of protein extracts from the same fractions using antibodies against TcRBP40 and Histone H2AZ. G) RT-PCR of RNA obtained from the different cellular fractions of <i>T. cruzi</i> epimastigote form, S – soluble cytoplasm, P – pellet, R – reservosome enriched fraction. H) Quantitative PCR for Cruzipain, TcRPB15, TcRBP40 and L9 transcripts enrichment in the reservosome compared to the soluble cytoplasm fractions. The reference used was RNA Pol II and the error bars are indicated. *p-value <0.0035. I) Western blot of total (T), intact (I-R) or disrupted (D-R) reservosomal protein extracts using antibodies against Cruzipain, 40S ribosomal S7 and 60S ribosomal L26 proteins. J) Bioanalyzer's electropherograms of RNAs extracted from intact (I-R) and disrupted (D-R) reservosomal fractions. Peaks corresponding to rRNAs are shown in fraction I-R.</p

Subcellular localization of specific mRNAs in stressed epimastigotes.

<p>A) Poly-A (Cy-5-labeled); B) β-tubulin (Cy-3-labeled); C) PFR2 (Cy-3-labeled); D) Cruzipain (Cy-5-labeled); E) to H) Merged images, counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). Differential interference contrast images are shown for identification of the cellular body of the parasite and the flagellum. Scale bar  = 10 µm. White arrows indicate the position of the flagellum.</p

Luciferase mRNA localization in <i>T. cruzi</i> with various UTRs.

<p>A) Luciferase probes labeled with Cy-5, showing the distribution of UTR-GAPDH as a control. B) Luciferase probes labeled with Cy-5, showing the distribution of UTR-β-tubulin. C) Luciferase probes labeled with Cy-5, showing the distribution of UTR-PFR2. D) to F) Merged images. G) Luciferase probes labeled with Cy-3, showing the distribution of UTR-Cruzipain. H) Cruzipain probes labeled with Cy-5, showing the distribution of cruzipain mRNA. I) Merged images. Counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). Differential interference contrast images are shown for identification of the cellular body of the parasite and the flagellum. Scale bar  = 10 µm. White arrows indicate the position of the flagellum.</p

Subcellular localization of specific mRNAs in <i>T. brucei</i>.

<p>A) β-tubulin probes labeled with Cy-5 in procyclic forms. B) PFR2 probes labeled with Cy-5 in procyclic forms. C) and D) Merged images, counterstaining with DAPI (blue) was used to identify the nuclei (n) and kinetoplast (k), flagellum (f). Differential interference contrast images are shown for identification of the cellular body of the parasite and the flagellum. Scale bar  = 10 µm. White arrows indicate the position of the flagellum.</p

Subcellular localization of <i>T. cruzi</i> mRNAs.

<p>A) Cruzipain (Cy-5-labeled); B) β-tubulin (Cy-3-labeled); C) PFR2 (Cy-5-labeled); D) merged image of the β-tubulin (Cy-3-labeled) and PFR2 (Cy-5-labeled) probes; E) to G) merged images, counterstaining with DAPI (blue) was used to identify nuclei (n) and kinetoplast (k), flagellum (f); H) Poly-A mRNA (Cy-5-labeled). Differential interference contrast images are shown for identification of the cellular body of the parasite and the flagellum. F) to I) Scale bar  = 10 µm. White arrows indicate the position of the flagellum.</p

Controls used for FISH validation.

<p>A) DNase I treatment before poly-T probe incubation. B) RNase A treatment before poly-T probe incubation. C) Cruzipain sense probes Cy-5 labeled in epimastigotes. D) β-tubulin sense probes Cy-3 labeled in epimastigotes. E) PFR2 sense probes Cy-3 labeled in epimastigotes. F) to J) Merged images, counterstaining with DAPI (blue) was used to identify nuclei (n), kinetoplast (k). Scale bars  = 10 µm.</p

Post-translational Modifications of <i>Trypanosoma cruzi</i> Canonical and Variant Histones

Chagas disease, caused by <i>Trypanosoma cruzi</i>, still affects millions of people around the world. No vaccines nor treatment for chronic Chagas disease are available, and chemotherapy for the acute phase is hindered by limited efficacy and severe side effects. The processes by which the parasite acquires infectivity and survives in different hosts involve tight regulation of gene expression, mainly post-transcriptionally. Nevertheless, chromatin structure/organization of trypanosomatids is similar to other eukaryotes, including histone variants and post-translational modifications. Emerging evidence suggests that epigenetic mechanisms also play an important role in the biology/pathogenesis of these parasites, making epigenetic targets suitable candidates to drug discovery. Here, we present the first comprehensive map of post-translational modifications of <i>T. cruzi</i> canonical and variant histones and show that its histone code can be as sophisticated as that of other eukaryotes. A total of 13 distinct modification types were identified, including rather novel and unusual ones such as alternative lysine acylations, serine/threonine acetylation, and N-terminal methylation. Some histone marks correlate to those described for other organisms, suggesting that similar regulatory mechanisms may be in place. Others, however, are unique to <i>T. cruzi</i> or to trypanosomatids as a group and might represent good candidates for the development of antiparasitic drugs

Additional file 1: of Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi

T cruzi candidate genes tested for UDP-GlcNAc transport by in vivo complementation assays. K. lactis mutant (Kl3) cells were transfected with TcCLB.504057.120 (TcCLB120), TcCLB.504085.60 (TcCLB60) and TcCLB.511277.400 (TcCLB400), the K. lactis UDP-GlcNAc transporter (Kl UGT, positive control) or empty vector (pE4, negative control). Cells were grown as described in the Methods section. After labeling with GS-II lectin (Alexa Fluor 488 conjugate), yeast cells were separated by flow cytometry in a FACS Canto II flow cytometer (Becton & Dickinson). (TIF 1867 kb

Additional file 3: of Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi

Expression of GFP-TcNST1 in T. cruzi epimastigotes. Western blot analysis of T. cruzi parasites transfected with GFP-TcNST1 and wild-type cells. Expression of the fusion protein (arrow) was detected with a monoclonal antibody against GFP. Expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ~ 37 kDa) was used as an internal control. (TIF 69 kb