Drug screening and development cascade for Chagas disease: an update of in vitro and in vivo experimental models.

Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years.

Trypanosoma cruzi XRNA granules colocalise with distinct mRNP granules at the nuclear periphery.

BACKGROUND Eukaryotic ribonucleoprotein (RNP) granules are important for the regulation of RNA fate. RNP granules exist in trypanosomatids; however, their roles in controlling gene expression are still not understood. XRNA is a component of granules in Trypanosoma brucei but has not been investigated in Trypanosoma cruzi. OBJECTIVES This study aimed to investigate the TcXRNA dynamic assembly and its interaction with RNP components under conditions that affect the mRNA availability. METHODS We used in vitro metacyclogenesis of T. cruzi to observe changes in RNP granules during the differentiation process. TcXRNA expression was analysed by Western blot and immunofluorescence. Colocalisation assays were performed to investigate the interaction of TcXRNA with other RNP components. FINDINGS TcXRNA is constantly present during metacyclogenesis and is localised in cytoplasmic granules. TcXRNA does not colocalise with TcDHH1 and TcCAF1 granules in the cytoplasm. However, TcXRNA granules colocalise with mRNP granules at the nuclear periphery when mRNA processing is inhibited. MAIN CONCLUSIONS TcXRNA plays a role in mRNA metabolism as a component of mRNP granules whose assembly is dependent on mRNA availability. TcXRNA granules colocalise with distinct RNP granules at the nuclear periphery, suggesting that the perinuclear region is a regulatory compartment in T. cruzi mRNA metabolism.

Modulation of Virulence Factors during Trypanosoma cruzi Differentiation.

Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. This protozoan developed several mechanisms to infect, propagate, and survive in different hosts. The specific expression of proteins is responsible for morphological and metabolic changes in different parasite stages along the parasite life cycle. The virulence strategies at the cellular and molecular levels consist of molecules responsible for mediating resistance mechanisms to oxidative damage, cellular invasion, and immune evasion, performed mainly by surface proteins. Since parasite surface coat remodeling is crucial to invasion and infectivity, surface proteins are essential virulence elements. Understanding the factors involved in these processes improves the knowledge of parasite pathogenesis. Genome sequencing has opened the door to high-throughput technologies, allowing us to obtain a deeper understanding of gene reprogramming along the parasite life cycle and identify critical molecules for survival. This review therefore focuses on proteins regulated during differentiation into infective forms considered virulence factors and addresses the current known mechanisms acting in the modulation of gene expression, emphasizing mRNA signals, regulatory factors, and protein complexes.

Long Non-Coding RNAs Associated with Ribosomes in Human Adipose-Derived Stem Cells: From RNAs to Microproteins.

Ribosome profiling reveals the translational dynamics of mRNAs by capturing a ribosomal footprint snapshot. Growing evidence shows that several long non-coding RNAs (lncRNAs) contain small open reading frames (smORFs) that are translated into functional peptides. The difficulty in identifying bona-fide translated smORFs is a constant challenge in experimental and bioinformatics fields due to their unconventional characteristics. This motivated us to isolate human adipose-derived stem cells (hASC) from adipose tissue and perform a ribosome profiling followed by bioinformatics analysis of transcriptome, translatome, and ribosome-protected fragments of lncRNAs. Here, we demonstrated that 222 lncRNAs were associated with the translational machinery in hASC, including the already demonstrated lncRNAs coding microproteins. The ribosomal occupancy of some transcripts was consistent with the translation of smORFs. In conclusion, we were able to identify a subset of 15 lncRNAs containing 35 smORFs that likely encode functional microproteins, including four previously demonstrated smORF-derived microproteins, suggesting a possible dual role of these lncRNAs in hASC self-renewal.

Analysis of the In Vivo Translation Process in Trypanosoma cruzi Using Ribosome Profiling.

The technique of ribosome profiling is based on the isolation of sequences around 30 nucleotides in size protected by mRNA-associated ribosomes, following digestion with specific nucleases, generating a footprint. After isolation and purification, these 30-nucleotide sequences are converted to a cDNA library and analyzed by deep sequencing, providing a high-precision picture of the translation process in vivo. In addition, this powerful technique allows for the study of several biological phenomena such as alternative splicing, alternative codon usage and initiation of translation by non-AUG codons. Furthermore, the ribosome footprinting technique has proved to be very efficient for studies of ribosome pause sites on mRNAs, which could act as key regulators in the translation process. Here we describe a modified protocol of the ribosome footprinting technique for translation efficiency analysis in Trypanosoma cruzi.

GCN2-Like Kinase Modulates Stress Granule Formation During Nutritional Stress in Trypanosoma cruzi.

The integrated stress response in eukaryotic cells is an orchestrated pathway that leads to eukaryotic Initiation Factor 2 alpha subunit (eIF2α) phosphorylation at ser51 and ultimately activates pathways to mitigate cellular damages. Three putative kinases (Tck1, Tck2, and Tck3) are found in the Trypanosoma cruzi genome, the flagellated parasite that causes Chagas disease. These kinases present similarities to other eukaryotic eIF2α kinases, exhibiting a typical insertion loop in the kinase domain of the protein. We found that this insertion loop is conserved among kinase 1 of several T. cruzi strains but differs among various Kinetoplastidae species, suggesting unique roles. Kinase 1 is orthologous of GCN2 of several eukaryotes, which have been implicated in the eIF2α ser51 phosphorylation in situations that mainly affects the nutrients levels. Therefore, we further investigated the responses to nutritional stress of T. cruzi devoid of TcK1 generated by CRISPR/Cas9 gene replacement. In nutrient-rich conditions, replicative T. cruzi epimastigotes depleted of TcK1 proliferate as wild type cells but showed increased levels of polysomes relative to monosomes. Upon nutritional deprivation, the polysomes decreased more than in TcK1 depleted line. However, eIF2α is still phosphorylated in TcK1 depleted line, as in wild type parasites. eIF2α phosphorylation increased at longer incubations times, but KO parasites showed less accumulation of ribonucleoprotein granules containing ATP-dependent RNA helicase involved in mRNA turnover (DHH1) and Poly-A binding protein (PABP1). Additionally, the formation of metacyclic-trypomastigotes is increased in the absence of Tck1 compared to controls. These metacyclics, as well as tissue culture trypomastigotes derived from the TcK1 knockout line, were less infective to mammalian host cells, although replicated faster inside mammalian cells. These results indicate that GCN2-like kinase in T. cruzi affects stress granule formation, independently of eIF2α phosphorylation upon nutrient deprivation. It also modulates the fate of the parasites during differentiation, invasion, and intracellular proliferation.

Improvements on the quantitative analysis of Trypanosoma cruzi histone post translational modifications: Study of changes in epigenetic marks through the parasite's metacyclogenesis and life cycle.

Trypanosome histone N-terminal sequences are very divergent from the other eukaryotes, although they are still decorated by post-translational modifications (PTMs). Here, we used a highly robust workflow to analyze histone PTMs in the parasite Trypanosoma cruzi using mass spectrometry-based (MS-based) data-independent acquisition (DIA). We adapted the workflow for the analysis of the parasite's histone sequences by modifying the software EpiProfile 2.0, improving peptide and PTM quantification accuracy. This workflow could now be applied to the study of 141 T. cruzi modified histone peptides, which we used to investigate the dynamics of histone PTMs along the metacyclogenesis and the life cycle of T. cruzi. Global levels of histone acetylation and methylation fluctuates along metacyclogenesis, however most critical differences were observed between parasite life forms. More than 66 histone PTM changes were detected. Strikingly, the histone PTM pattern of metacyclic trypomastigotes is more similar to epimastigotes than to cellular trypomastigotes. Finally, we highlighted changes at the H4 N-terminus and at H3K76 discussing their impact on the trypanosome biology. Altogether, we have optimized a workflow easily applicable to the analysis of histone PTMs in T. cruzi and generated a dataset that may shed lights on the role of chromatin modifications in this parasite. SIGNIFICANCE: Trypanosomes are unicellular parasites that have divergent histone sequences, no chromosome condensation and a peculiar genome/gene regulation. Genes are transcribed from divergent polycistronic regions and post-transcriptional gene regulation play major role on the establishment of transcripts and protein levels. In this regard, the fact that their histones are decorated with multiple PTMs raises interesting questions about their role. Besides, this digenetic organism must adapt to different environments changing its metabolism accordingly. As metabolism and epigenetics are closely related, the study of histone PTMs in trypanosomes may enlighten this strikingly, and not yet fully understood, interplay. From a biomedical perspective, the comprehensive study of molecular mechanisms associated to the metacyclogenesis process is essential to create better strategies for controlling Chagas disease.

Trypanosoma cruzi XRNA granules colocalise with distinct mRNP granules at the nuclear periphery

BACKGROUND Eukaryotic ribonucleoprotein (RNP) granules are important for the regulation of RNA fate. RNP granules exist in trypanosomatids; however, their roles in controlling gene expression are still not understood. XRNA is a component of granules in Trypanosoma brucei but has not been investigated in Trypanosoma cruzi. OBJECTIVES This study aimed to investigate the TcXRNA dynamic assembly and its interaction with RNP components under conditions that affect the mRNA availability. METHODS We used in vitro metacyclogenesis of T. cruzi to observe changes in RNP granules during the differentiation process. TcXRNA expression was analysed by Western blot and immunofluorescence. Colocalisation assays were performed to investigate the interaction of TcXRNA with other RNP components. FINDINGS TcXRNA is constantly present during metacyclogenesis and is localised in cytoplasmic granules. TcXRNA does not colocalise with TcDHH1 and TcCAF1 granules in the cytoplasm. However, TcXRNA granules colocalise with mRNP granules at the nuclear periphery when mRNA processing is inhibited. MAIN CONCLUSIONS TcXRNA plays a role in mRNA metabolism as a component of mRNP granules whose assembly is dependent on mRNA availability. TcXRNA granules colocalise with distinct RNP granules at the nuclear periphery, suggesting that the perinuclear region is a regulatory compartment in T. cruzi mRNA metabolism.

Evidence of P-body-like structures in Trypanosoma cruzi.

Gene expression in trypanosomatids is mainly regulated post-transcriptionally. One of the mechanisms involves the differential stability of mRNAs. However, the existence of other mechanisms involving the accessibility of mRNAs to the translation machinery cannot be ruled out. Defined cytoplasmic foci containing non-translating mRNPs, known as P-bodies, have been discovered in recent years. P-bodies are sites where mRNA can be decapped and 5'-3' degraded or stored for subsequent return to polysomes. The highly conserved DEAD box helicase Dhh1p is a marker protein of P-body functions. Here, we report the identification and cloning of a Trypanosoma cruzi Dhh1 homolog gene. TcDhh1 expression is not regulated through the parasite life cycle or under stress conditions. We show that TcDhh1 is present in polysome-independent complexes and is localized to discrete cytoplasmic foci, resembling P-bodies; these foci vary in number according to nutritional stress conditions and cycloheximide/puromycin treatment.

Biological effects of extracts obtained from Stryphnodendron adstringens on Herpetomonas samuelpessoai.

We report the effect of Stryphnodendron adstringens on the trypanosomatid Herpetomonas samuelpessoai. The parasites were grown at 28 degrees C in a chemically defined medium containing crude extract and fractions at concentrations from 100 to 5000 microg/ml obtained from S. adstringens. Concentrations of 500, 1000, 2500, and 5000 microg/ml both crude extract and semi-purified fraction progressively inhibited the protozoans' growth. At a concentration of 100 microg/ml, crude extract or a semi-purified (F3) fraction did not affect the growth of the protozoans. The F3-9 - F3-12 sub-fractions, at a concentration of 1000 microg/ml, also showed increased inhibitory activity on H. samuelpessoai. The IC50 of the crude extract and the F3 fraction were 538 and 634 microg/ml, respectively. Ultrastructural and enzymatic alterations in the trypanosomatids were also evaluated. H. samuelpessoai cultivated in the presence of IC50 crude extract showed considerable ultrastructural alterations, such as marked mitochondrial swelling with a large number of cristae and evident Golgi complex vesiculation, as observed by transmission electron microscopy. Cells exposed to 538 microg/ml of crude extract at 28 degrees C for 72 h, showed decreased activity of the enzyme succinate cytochrome c reductase, a typical mitochondrion marker, as compared to untreated cells.

Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases.

Extracts of 13 Brazilian medicinal plants were screened for their antimicrobial activity against bacteria and yeasts. Of these, 10 plant extracts showed varied levels of antibacterial activity. Piper regnellii presented a good activity against Staphylococus aureus and Bacillus subtilis, a moderate activity on Pseudomonas aeruginosa, and a weak activity against Escherichia coli. Punica granatum showed good activity on S. aureus and was inactive against the other standard strains. Eugenia uniflora presented moderate activity on both S. aureus and E. coli. Psidium guajava,Tanacetum vulgare, Arctium lappa, Mikania glomerata, Sambucus canadensis, Plantago major and Erythrina speciosa presented some degree of antibacterial activity. Spilanthes acmella, Lippia alba, and Achillea millefolium were considered inactive. 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 Asteraceae family. This may mean that the same compounds are responsible for the antibacterial activity in these plants. Anticandidal activity was detected in nine plant extracts (P. guajava, E. uniflora, P. granatum, A. lappa, T. vulgare, M. glomerata, L. alba, P. regnellii, and P. major). The results might explain the ethnobotanical use of the studied species for the treatment of various infectious diseases.

Biological effects of extracts obtained from Stryphnodendron adstringens on Herpetomonas samuelpessoai

We report the effect of Stryphnodendron adstringens on the trypanosomatid Herpetomonas samuelpessoai. The parasites were grown at 28ºC in a chemically defined medium containing crude extract and fractions at concentrations from 100 to 5000 µg/ml obtained from S. adstringens. Concentrations of 500, 1000, 2500, and 5000 µg/ml both crude extract and semi-purified fraction progressively inhibited the protozoans' growth. At a concentration of 100 µg/ml, crude extract or a semi-purified (F3) fraction did not affect the growth of the protozoans. The F3-9 - F3-12 sub-fractions, at a concentration of 1000 µg/ml, also showed increased inhibitory activity on H. samuelpessoai. The IC50 of the crude extract and the F3 fraction were 538 and 634 µg/ml, respectively. Ultrastructural and enzymatic alterations in the trypanosomatids were also evaluated. H. samuelpessoai cultivated in the presence of IC50 crude extract showed considerable ultrastructural alterations, such as marked mitochondrial swelling with a large number of cristae and evident Golgi complex vesiculation, as observed by transmission electron microscopy. Cells exposed to 538 µg/ml of crude extract at 28ºC for 72 h, showed decreased activity of the enzyme succinate cytochrome c reductase, a typical mitochondrion marker, as compared to untreated cells.

Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases

Extracts of 13 Brazilian medicinal plants were screened for their antimicrobial activity against bacteria and yeasts. Of these, 10 plant extracts showed varied levels of antibacterial activity. Piper regnellii presented a good activity against Staphylococus aureus and Bacillus subtilis, a moderate activity on Pseudomonas aeruginosa, and a weak activity against Escherichia coli. Punica granatum showed good activity on S. aureus and was inactive against the other standard strains. Eugenia uniflora presented moderate activity on both S. aureus and E. coli. Psidium guajava,Tanacetum vulgare, Arctium lappa, Mikania glomerata, Sambucus canadensis, Plantago major and Erythrina speciosa presented some degree of antibacterial activity. Spilanthes acmella, Lippia alba, and Achillea millefolium were considered inactive. 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 Asteraceae family. This may mean that the same compounds are responsible for the antibacterial activity in these plants. Anticandidal activity was detected in nine plant extracts (P. guajava, E. uniflora, P. granatum, A. lappa, T. vulgare, M. glomerata, L. alba, P. regnellii, and P. major). The results might explain the ethnobotanical use of the studied species for the treatment of various infectious diseases

Effect of plant extracts used in folk medicine on cell growth and differentiation of Herpetomonas samuelpessoai (Kinetoplastida, Trypanosomatidae) cultivated in defined medium

Neste trabalho, verificou-se o efeito de 15 plantas medicinais no crescimento e diferenciação celular de Herpetomonas samuelpessoai, um tripanosomatídeo não patogênico utilizado como modelo biológico, que apresenta antígenos semelhantes aos do Trypanosoma cruzi. Extratos brutos (1.000 mg/ml) ou óleo essencial (250 mg/ml) foram adicionados ao meio definido. O crescimento celular foi determinado pela contagem em câmara de Newbauer e a diferenciação celular examinada por microscopia ótica. Ocimum gratissimum, Lippia alba, Piper regnellii, Stryphnodendron adstringens, e Tanacetum vulgare mostraram atividade antiprotozoário, Psidium guajava e Punica granatum menor atividade e Achillea millefolium, Eugenia uniflora, Mikania glomerata, Plantago major, e Spilanthes acmella não apresentaram atividade. Por outro lado, Arctium lappa, Erythrina speciosa, e Sambucus canadensis estimularam o crescimento de H. samuelpessoai e L. alba e S. acmella a diferenciação celular deste flagelado. Estes resultados indicam que plantas medicinais possuem princípios ativos contra H. samuelpessoai, o qual parece ser útil como modelo para seleção de plantas que contém drogas tripanomicidas

Grânulos de processamento de mRNAs em Trypanosoma cruzi / Granules of processing of mRNAs in Trypanosoma cruzi

Em tripanossomatídeos a regulação da expressão gênica ocorre principalmente em nível pós-transcricional por mecanismos que envolvem mudanças na estabilidade e no acesso dos mRNAs aos polissomos. Estes mecanismos permitem uma rápida adaptação destes parasitas a diferentes condições as quais são expostos durante o ciclo de vida. Estudos recentes têm demonstrado que grânulos de mRNA, presentes em diversos eucariotos têm papel fundamental na regulação da expressão gênica em nível pós-transcricional. Estes grânulos são divididos em diferentes classes (entre elas os P-bodies e os grânulos de estresse), contém mRNPs não comprometidas com a tradução, compartilham algumas proteínas e podem utilizar mecanismos semelhantes para regular o metabolismo de mRNAs. Os P-bodies são sítios de estocagem e/ou degradação de mRNAs, enquanto os grânulos de estresse estão envolvidos na triagem e estocagem de diversos transcritos. Como existe evidência de que moléculas de mRNA podem ser mantidas estáveis e não associadas à polissomos em T. cruzi, nós conjecturamos a possibilidade de que estas estruturas estejam presentes e sejam funcionais neste parasita. Os genes que codificam alguns componentes de P-bodies estão presentes em T. cruzi e embora algumas proteínas que constituem o cerne destas estruturas pareçam estar ausentes neste parasita, a maioria das funções inerentes aos P-bodies estão representadas. Nós identificamos e clonamos a proteína TcDhh1, uma DEAD box RNA helicase altamente conservada entre os eucariotos e considerada marcadora de P-bodies em mamíferos e leveduras. Esta proteína é constitutivamente expressa durante todo o ciclo de vida do parasita, está presente em complexos independentes de polissomos e está localizada em foci citoplasmáticos que variam em número quando as células são submetidas a condições de estresse ou ao tratamento com as drogas cicloheximida e puromicina. Para determinar a composição destes grânulos, nós realizamos ensaios de...

Efeito de extratos de plantas medicinais no crescimento, diferenciação e ultraestrutura de Herpetomonas samuelpessoai / Extract effect of medicinal plants in the growth, differentiation and ultrastructure of Herpetomonas samuelpessoai

O objetivo do presente estudo foi avaliar os efeitos dos extratos brutos de quatorze plantas e do óleo essencial de acimum gratissimum, no crescimento e diferenciação celular de Herpetomonas samuelpessoai cultivada em meio quimicamente definido e meio complexo. Alterações ultraestruturais e enzimatica do tripanosomatídeo foram avaliadas utilizando extrato bruto e algumas frações de Striphnodendron adstringens e o óleo essencial. As plantas selecionadas através de informações etnobotânicas foram: Arctium lappa, Achillea millefolium, Erythrina speciosa, Eugenia uniflora, Lippia alba, Mikania glomerata, Piper regnellii, Plantago major, Psidium guajava, Punica granatum, Sambucus canadensis, Spilanthes acmella, Stryphnodendron adstringens, Tanacetum vulgare, e o óleo essencial de Acimum gratissimum. Um efeito antiprotozoário positivo foi encontrado nos extratos de L. alba, T. vulgare e P. regnellii, na concentração de 1.000 ug/mL, com 90,7 por cento, 97,4 por cento e 99,5 por cento de inibição de crescimento, respectivamente. Os extratos de A. millefolium, E. uniflora, M. glomerata, P. major, P. guajava, e P. granatum, mostraram fraca atividade inibitória. Por outro lado, A. lappa, E. speciosa, S. canadensis, e S. acmella demonstraram um estímulo no crescimento de H. samuelpessoai...