RESUMEN
Writing and erasing of posttranslational modifications are crucial to phenotypic plasticity and antigenic variation of eukaryotic pathogens. Targeting pathogens' modification machineries, thus, represents a valid approach to fighting parasitic diseases. However, identification of parasitic targets and the development of selective anti-parasitic drugs still represent major bottlenecks. Here, we show that the zinc-dependent histone deacetylases (HDACs) of the protozoan parasite Trypanosoma cruzi are key regulators that have significantly diverged from their human counterparts. Depletion of T. cruzi class I HDACs tcDAC1 and tcDAC2 compromises cell-cycle progression and division, leading to cell death. Notably, tcDAC2 displays a deacetylase activity essential to the parasite and shows major structural differences with human HDACs. Specifically, tcDAC2 harbors a modular active site with a unique subpocket targeted by inhibitors showing substantial anti-parasitic effects in cellulo and in vivo. Thus, the targeting of the many atypical HDACs in pathogens can enable anti-parasitic selective chemical impairment.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Animales , Dominio Catalítico , Ciclo Celular , División Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Chlorocebus aethiops , ADN Protozoario , Femenino , Prueba de Complementación Genética , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/química , Interacciones Huésped-Parásitos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Filogenia , Conformación Proteica , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Eliminación de Secuencia , Trypanosoma cruzi/efectos de los fármacos , Células VeroRESUMEN
BACKGROUND: Due 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. RESULTS: Here 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. CONCLUSIONS: A 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.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Proteómica/métodos , Ribosomas/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Estadios del Ciclo de Vida , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/análisis , ARN Protozoario/análisis , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Regulación hacia ArribaRESUMEN
Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.
Asunto(s)
Transporte de ARN , ARN Mensajero/metabolismo , Trypanosoma cruzi/metabolismo , Regiones no Traducidas 3'/genética , Animales , Compartimento Celular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Citoplasma/metabolismo , Hibridación Fluorescente in Situ , Estadios del Ciclo de Vida , Luciferasas/metabolismo , Parásitos/crecimiento & desarrollo , Parásitos/metabolismo , Proteínas Protozoarias/metabolismo , Estrés Fisiológico , Fracciones Subcelulares/metabolismo , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Trypanosoma cruzi is the causative agent of Chagas disease, a neglected disorder that affects millions of people in the Americas. T. cruzi relies mostly upon post-transcriptional regulation to control stage specific gene expression. RNA binding proteins (RBPs) associate with functionally related mRNAs forming ribonucleoprotein complexes that define post-transcriptional operons. The RNA Recognition Motif (RRM) is the most common and ancient family of RBPs. This family of RBPs has been identified in trypanosomatid parasites and only a few of them have been functionally characterized. We describe here the functional characterization of TcRBP40, a T. cruzi specific RBP, and its associated mRNAs. We used a modified version of the recombinant RIP-Chip assay to identify the mRNAs with which it associates and in vivo TAP-tag assays to confirm these results. TcRBP40 binds to an AG-rich sequence in the 3'UTR of the associated mRNAs, which were found to encode mainly putative transmembrane proteins. TcRBP40 is differentially expressed in metacyclogenesis. Surprisingly, in epimastigotes, it is dispersed in the cytoplasm but is concentrated in the reservosomes, a T. cruzi specific organelle, which suggests a putative new function for this parasite organelle.