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BackgroundDue to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms.ResultsHere we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect’s life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage.ConclusionsA large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1563-8) contains supplementary material, which is available to authorized users.

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Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/ FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle.

Kasatkina, T.N.; Okhapkina, V.F. Use of Robolact and Linolac dry milk mixtures in the overall therapy of infants with acute intestinal infections. Vopr. Pitan., v. 4, p. 45 -47, 1980. 7 Kubo, M.; Odani, T.; Nakamura S. Pharmacological study on kefir -a fermented milk product in Caucasus. I. On tumor activity. Yakugaku Zsshi, v. 112, n. 7, p. 489 -495, 1992 AbstractExtracts of 13 Brazilian medicinal plants were screened for their antimicrobial activity against bacteria and yeast. Of these, 10 plant extracts showed varied levels of antibacterial activity. Five of the plant extracts presented compounds with Rf values similar to the antibacterial compounds visible on bioautogram. Of these, three plants belong to the Compositae family. This may mean that the same compounds are responsible for the antibacterial activity in these plants. Anticandidal activity was detected in 9 plant extracts. The results might explain the ethnobotanical use of the studied species for the treatment of various infectious diseases.O uso de plantas medicinais especialmente na América do Sul contribui significantemente para os cuidados básicos com a saúde. Para o tratamento de infecções comuns, muitas plantas são utilizadas no Brasil na forma de extrato bruto, infusões ou emplastos, sem nenhuma evidência científica de sua eficácia. Recentemente demonstramos a atividade antibacteriana do óleo essencial e do composto ativo purificado (eugenol) extraído do Ocimum gratissimum, tradicionalmente usado na medicina popular brasileira para o tratamento de várias doenças

In trypanosomatids, the regulation of gene expression occurs mainly at the post-transcriptional level. Previous studies have revealed nontranslated mRNA in the Trypanosoma cruzi cytoplasm. Previously, we have identified and cloned the TcDHH1 protein, a DEAD box RNA helicase. It has been reported that Dhh1 is involved in multiple RNA-related processes in various eukaryotes. It has also been reported to accumulate in stress granules and processing bodies of yeast, animal cells, Trypanosoma brucei and T. cruzi. TcDHH1 is localized to discrete cytoplasmic foci that vary depending on the life cycle status and nutritional conditions. To study the composition of mRNPs containing TcDHH1, we carried out immunoprecipitation assays with anti-TcDHH1 using epimastigote lysates. The protein content of mRNPs was determined by MS and pre-immune serum was used as control. We also carried out a ribonomic approach to identify the mRNAs present within the TcDHH1 immunoprecipitated complexes. For this purpose, competitive microarray hybridizations were performed against negative controls, the nonprecipitated fraction. Our results showed that mRNAs associated with TcDHH1 in the epimastigote stage are those mainly expressed in the other forms of the T. cruzi life cycle. These data suggest that mRNPs containing TcDHH1 are involved in mRNA metabolism, regulating the expression of at least epimastigote-specific genes. Structured digital abstractl MINT-7909478: DHH1 (uniprotkb:Q4DIE1) physically interacts (MI:0915) with PABP2 (uniprotkb:Q27335) by anti bait coimmunoprecipitation (MI:0006) l MINT-7909338: DHH1 (uniprotkb:Q4DIE1) physically interacts (MI:0914) with ATP-dependent DEAD ⁄ H RNA helicase, putative (uniprotkb:Q4DIE1), Actin, putative (uniprotkb: Q4D7A6), Actin, putative (uniprotkb:Q4CLA9), Chaperonin HSP60, mitochondrial (uniprotkb:Q4DYP6), ATP-dependent Clp protease subunit, heat shock protein 100 (HSP100), putative (uniprotkb:Q4CNM5), Elongation factor 2, putative (uniprotkb:Q4D5X0), Elongation factor 1-alpha (EF-1-alpha), putative (uniprotkb:Q4CU73), Heat shock protein 85, putative (uniprotkb:Q4CQS6), Glutamate dehydrogenase, putative (uniprotkb:Q4DWV8), Putative uncharacterized protein (uniprotkb:Q4CNI8), 40S ribosomal protein S11, putative (uniprotkb:Q4CRH9), Sterol 24-c-methyltransferase, putative (uniprotkb:Q4CMB7), Heat shock protein 70 (HSP70), putative (uniprotkb:Q4DTM9), Glutamate dehydrogenase, putative (uniprotkb:Q4D5C2) and Calpain-like cysteine peptidase, putative (uniprotkb:Q4CYU3) by anti bait coimmunoprecipitation (MI:0006) l MINT-7909469: DHH1 (uniprotkb:Q4DIE1) physically interacts (MI:0915) with PABP1(uniprotkb:Q4E4I9) by anti bait coimmunoprecipitation (MI:0006)Abbreviations eIF, eukaryotic initiation factor; IP, immunoprecipitated; MASP, mucin-associated surface protein; PABP, poly(A)-binding protein; P-bodies, processing bodies; SG, stress granule; SP, supernatant.

Gene regulation is mainly post-transcriptional in trypanosomatids. The stability of mRNA and access to polysomes are thought to be tightly regulated, allowing Trypanosoma cruzi to adapt to the different environmental conditions during its life cycle. Post-transcriptional regulation requires the association between mRNAs and certain proteins to form mRNP complexes. We investigated the dynamic association between proteins and mRNAs, using poly(T) beads to isolate and characterize proteins and protein complexes bound to poly-A+ mRNAs. The protein content of these fractions was analyzed by mass spectrometry (LC-MS/MS). We identified 542 protein component of the mRNP complexes associated with mRNAs. Twenty-four of the proteins obtained were present in all fractions, whereas some other proteins were exclusive to a particular fraction: epimastigote polysomal (0.37%) and post-polysomal (2.95%) fractions; stress polysomal (13.8%) and post-polysomal (40.78%) fractions. Several proteins known to be involved in mRNA metabolism were identified, and this was considered important as it made it possible to confirm the reliability of our mRNP isolation approach. This procedure allowed us to have a first insight into the composition and dynamics of mRNPs in T. cruzi.