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1.
Mem Inst Oswaldo Cruz ; 116: e200538, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34468503

RESUMO

BACKGROUND: Trypanosoma cruzi is an important human pathogen in Latin America with nearly seven million people infected. It has a large degree of genetic diversity, classified into six discrete typing units (DTUs), which probably influences its physiological behavior and clinical manifestations. Several genotyping methods are available, with distinct performance on easiness, cost, resolution and applicability; no method excels in all parameters. OBJECTIVES AND METHODS: To devise a molecular method for T. cruzi genotyping, based on polymerase chain reaction (PCR) amplification of a single target with multiple copies in the nuclear genome by large scale sequencing. We have applied this method to 29 T. cruzi isolates, comprising all described DTUs. FINDINGS: We were able to classify all samples into sub DTU level with high robustness. Evolutionary relationship between DTUs were ascertained, suggesting that TcIII and TcIV DTUs are non-hybrid, and DTU IV is more similar to the common ancestral. CONCLUSION: As the TS-LSS method is based on a single PCR reaction, comprising several copies of the target, it is probably useful for clinical samples, when the amount of DNA is a limiting factor. As large scale sequencing systems become more common, the TS-LSS method can be increasingly applied for T. cruzi genotyping.


Assuntos
Doença de Chagas , Trypanosoma cruzi , Evolução Biológica , Variação Genética/genética , Genótipo , Técnicas de Genotipagem , Humanos , Trypanosoma cruzi/genética
2.
Mem Inst Oswaldo Cruz ; 113(5): e170404, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29668769

RESUMO

BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins.


Assuntos
Estágios do Ciclo de Vida/genética , Transcriptoma/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Cultura Axênica , Western Blotting , Polirribossomos/genética , Análise de Sequência de RNA , Trypanosoma cruzi/genética
3.
Mem. Inst. Oswaldo Cruz ; 113(5): e170404, 2018. graf
Artigo em Inglês | LILACS | ID: biblio-894928

RESUMO

BACKGROUND Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. OBJECTIVES We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. METHODS RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. FINDINGS We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins.


Assuntos
Humanos , Análise de Sequência de RNA , Transcriptoma/genética , Cultura Axênica , Estágios do Ciclo de Vida/genética
4.
BMC Genomics ; 18(1): 793, 2017 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-29037144

RESUMO

BACKGROUND: Trypanosomatids are a group of protozoan parasites that includes the etiologic agents of important human illnesses as Chagas disease, sleeping sickness and leishmaniasis. These parasites have a significant distinction from other eukaryotes concerning mRNA structure, since all mature mRNAs have an identical species-specific sequence of 39 nucleotides at the 5' extremity, named spliced leader (SL). Considering this peculiar aspect of trypanosomatid mRNA, the aim of the present work was to develop a Trypanosoma cruzi specific in vitro transcription (IVT) linear mRNA amplification method in order to improve parasite transcriptomics analyses. METHODS: We designed an oligonucleotide complementary to the last 21 bases of T. cruzi SL sequence, bearing an upstream T7 promoter (T7SL primer), which was used to direct the synthesis of second-strand cDNA. Original mRNA was then amplified by IVT using T7 RNA polymerase. T7SL-amplified RNA from two distinct T. cruzi stages (epimastigotes and trypomastigotes) were deep sequenced in SOLiD platform. Usual poly(A) + RNA and and T7-oligo(dT) amplified RNA (Eberwine method) were also sequenced. RNA-Seq reads were aligned to our new and improved T. cruzi Dm28c genome assembly (PacBio technology) and resulting transcriptome pattern from these three RNA preparation methods were compared, mainly concerning the conservation of mRNA transcritional levels and DEGs detection between epimastigotes and trypomastigotes. RESULTS: T7SL IVT method detected more potential differentially expressed genes in comparison to either poly(A) + RNA or T7dT IVT, and was also able to produce reliable quantifications of the parasite transcriptome down to 3 ng of total RNA. Furthermore, amplification of parasite mRNA in HeLa/epimastigote RNA mixtures showed that T7SL IVT generates transcriptome quantification with similar detection of differentially expressed genes when parasite RNA mass was only 0.1% of the total mixture (R = 0.78 when compared to poly(A) + RNA). CONCLUSIONS: The T7SL IVT amplification method presented here allows the detection of more potential parasite differentially expressed genes (in comparison to poly(A) + RNA) in host-parasite mixtures or samples with low amount of RNA. This method is especially useful for trypanosomatid transcriptomics because it produces less bias than PCR-based mRNA amplification. Additionally, by simply changing the complementary region of the T7SL primer, the present method can be applied to any trypanosomatid species.


Assuntos
Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Transcrição Gênica , Trypanosoma cruzi/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
5.
Parasitol Int ; 66(3): 236-239, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28137669

RESUMO

New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (>90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce.


Assuntos
Transfecção/métodos , Trypanosoma cruzi/genética , DNA de Protozoário/genética , Eletroporação , Edição de Genes , Genes Reporter , Proteínas de Fluorescência Verde , Ensaios de Triagem em Larga Escala , Trypanosoma cruzi/crescimento & desenvolvimento
6.
Mol Microbiol ; 104(5): 712-736, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28240790

RESUMO

Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell-derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long-term cultured epimastigotes: resistance to complement-mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up-regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host-parasite interactions, showing that rdEpi can be infective to the mammalian host.


Assuntos
Doença de Chagas/parasitologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/patogenicidade , Animais , Diferenciação Celular/fisiologia , Interações Hospedeiro-Parasita , Estágios do Ciclo de Vida/fisiologia , Camundongos , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo
7.
Parasitology ; 143(4): 434-43, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26818093

RESUMO

Trypanosoma cruzi, the etiological agent of Chagas disease, is ingested by triatomines during their bloodmeal on an infected mammal. Aiming to investigate the development and differentiation of T. cruzi inside the intestinal tract of Rhodnius prolixus at the beginning of infection we fed insects with cultured epimastigotes and blood trypomastigotes from infected mice to determine the amount of recovered parasites after ingestion. Approximately 20% of the ingested parasites was found in the insect anterior midgut (AM) 3 h after feeding. Interestingly, a significant reduction (80%) in the numbers of trypomastigotes was observed after 24 h of infection suggesting that parasites were killed in the AM. Moreover, few parasites were found in that intestinal portion after 96 h of infection. The evaluation of the numbers of parasites in the posterior midgut (PM) at the same periods showed a reduced parasite load, indicating that parasites were not moving from the AM. Additionally, incubation of blood trypomastigotes with extracts from R. prolixus AMs revealed that components of this tissue could induce significant death of T. cruzi. Finally, we observed that differentiation from trypomastigotes to epimastigotes is not completed in the AM; instead we suggest that trypomastigotes change to intermediary forms before their migration to the PM, where differentiation to epimastigotes takes place. The present work clarifies controversial points concerning T. cruzi development in insect vector, showing that parasite suffers a drastic decrease in population size before epimastigonesis accomplishment in PM.


Assuntos
Doença de Chagas/parasitologia , Insetos Vetores/parasitologia , Rhodnius/parasitologia , Trypanosoma cruzi/crescimento & desenvolvimento , Análise de Variância , Animais , Doença de Chagas/sangue , Doença de Chagas/transmissão , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Camundongos , Ninfa/parasitologia , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi/genética
8.
PLoS One ; 8(6): e67441, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840703

RESUMO

Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.


Assuntos
Proteínas de Fluorescência Verde/biossíntese , Trypanosoma cruzi/fisiologia , Animais , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos/métodos , Citometria de Fluxo , Fluorometria , Genes Reporter , Proteínas de Fluorescência Verde/genética , Interações Hospedeiro-Parasita , Concentração Inibidora 50 , Viabilidade Microbiana , Nitroimidazóis/farmacologia , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Organismos Geneticamente Modificados/fisiologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Células Vero
9.
PLoS One ; 8(4): e60209, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23560078

RESUMO

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.


Assuntos
Genes de Protozoários , Filogenia , Proteínas de Protozoários/genética , Simbiose/genética , Trypanosomatina/genética , Bactérias/metabolismo , Composição de Bases , Sequência de Bases , Evolução Biológica , Leishmania major/genética , Redes e Vias Metabólicas , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Trypanosomatina/classificação , Trypanosomatina/metabolismo , Trypanosomatina/microbiologia
10.
PLoS One ; 8(1): e55497, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383204

RESUMO

The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs) ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC(50)/72 h) or killing all cells within 24 hours (EC(100)/24 h). Incubation with inhibitors at the EC(50)/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC(50)/72 h. By contrast, treatment with SBIs at the EC(100)/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP), culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the "point of no return" in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite.


Assuntos
Vias Biossintéticas/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Esteróis/biossíntese , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Morte Celular/efeitos dos fármacos , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Cetoconazol/farmacologia , Lovastatina/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Permeabilidade , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestrutura
11.
Mem Inst Oswaldo Cruz ; 107(6): 790-9, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22990970

RESUMO

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trypanosoma cruzi/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Estabilidade de RNA , Trypanosoma cruzi/crescimento & desenvolvimento
12.
Mem. Inst. Oswaldo Cruz ; 107(6): 790-799, set. 2012. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-649496

RESUMO

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trypanosoma cruzi/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Estabilidade de RNA , Trypanosoma cruzi/crescimento & desenvolvimento
13.
Proteomics ; 12(17): 2694-703, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22761176

RESUMO

Trypanosoma cruzi is the etiologic agent of Chagas disease, which is estimated to affect over eight million people around the world. Trypanosoma cruzi has a complex life cycle, involving insect and mammalian hosts and four distinct developmental stages: epimastigotes, metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes. Metacyclogenesis is the process by which T. cruzi epimastigotes differentiate into metacyclic trypomastigotes and acquire infectivity, and involves differential gene expression associated with acquisition of virulence. In T. cruzi, gene expression regulation is achieved mainly posttranscriptionally. Therefore, proteomics-based approaches are extremely useful for gaining a better understanding of the changes that occur in the stage-regulated gene expression program of the parasite at the molecular level. Here, we performed an in-depth quantitative MS-based proteomic study of T. cruzi metacyclogenesis and quantified almost 3000 proteins expressed during the process of differentiation. To the best of our knowledge, this work is the most comprehensive quantitative proteomics study of different cell populations of T. cruzi available so far. We identified relevant proteins and pathways involved in the parasite's differentiation and infectivity acquisition, opening new perspectives for further studies that could, ultimately, lead to the identification of new targets for chemotherapy.


Assuntos
Doença de Chagas/parasitologia , Regulação da Expressão Gênica no Desenvolvimento , Proteômica/métodos , Proteínas de Protozoários/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Humanos , Proteínas de Protozoários/metabolismo , Espectrometria de Massas em Tandem/métodos , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo
14.
Cytokine ; 60(1): 76-82, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22795294

RESUMO

UNLABELLED: Chronic kidney disease (CKD) and periodontitis (PD) are complex inflammatory disturbances, influenced by genetic factors. Interleukin (IL)-1 genes code for inflammatory mediators involved in the physiopathogenesis of both diseases. Functional polymorphisms in IL1 genes modulate cytokine levels and have been associated with susceptibility to immune-inflammatory conditions. OBJECTIVES: The aim of this study was investigate the association of functional IL1 gene polymorphisms and transcript levels with susceptibility to CKD and PD. DESIGN: The sample consisted of 246 individuals, mean age 44.8 years, divided into: group 1 (64 patients without CKD and without PD), group 2 (58 without CKD and with PD), group 3 (52 with CKD and without PD) and group 4 (72 with CKD and with PD). DNA was obtained from cells of oral mucosa and polymorphisms IL1AC-889T, IL1BC-511T, IL1BC+3954T and IL1RN (intron 2) were analyzed by PCR-RFLP. Transcript levels from gingival tissues were analyzed by real-time PCR. RESULTS: IL1RN(*)1 allele was associated with almost 4-fold increased risk for CKD (OR 3.92 95% CI=1.6-9.4, p=0.002). IL1RN(*)2 allele was associated with 3-fold increased risk for PD in CKD patients (OR 3.08 95% CI=1.2-7.9, p=0.019). Allele T for polymorphism IL1B+3954 was associated with CKD in PD patients (OR 2.28 95% CI=1.1-4.7, p=0.019). Significantly increased levels of transcripts of IL1A, IL1B and IL1RN genes were found in PD patients. CONCLUSIONS: It was observed an evidence for association of IL1B and IL1RN alleles with susceptibility to CKD and PD. Higher levels of IL1 gene transcripts were found in PD patients.


Assuntos
Interleucina-1/genética , Periodontite/genética , Polimorfismo Genético , Insuficiência Renal Crônica/genética , Transcrição Gênica , Adulto , Idoso , Alelos , Análise de Variância , Distribuição de Qui-Quadrado , Feminino , Expressão Gênica , Frequência do Gene , Predisposição Genética para Doença/genética , Genótipo , Haplótipos , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Interleucina-1alfa/genética , Interleucina-1beta/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Adulto Jovem
15.
J Biotechnol ; 156(1): 56-8, 2011 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-21810448

RESUMO

The goal of the present work was to develop reagents with potential for tuberculosis diagnosis. Genetic sequences of Mycobacterium tuberculosis secretion antigens were amplified by PCR, cloned into the Gateway(®) system, and expressed in Escherichia coli. The recombinant M. tuberculosis proteins were purified by metal affinity chromatography and preparative gel SDS-PAGE electrophoresis followed by electroelution and removal of endotoxins using Triton X-114. In total, seven recombinant proteins were obtained (ESAT-6, CFP10, TB10.3, TB10.4, MTSP11, MPT70, and MPT83). Delayed hypersensitivity reactions (DHR) was evaluated in Cavia porcellus and compared to the response using a standard purified protein derivative (PPD). All seven recombinant proteins produced a positive induration reaction in an intradermal test in guinea pigs previously sensitized with M. tuberculosis. When applied together, at a concentration of each recombinant protein 0.04 mg/mL, the intradermoreaction in C. porcellus was significantly higher than that obtained by standard PPD (p-value=0.00386).


Assuntos
Antígenos de Bactérias/biossíntese , Mycobacterium tuberculosis/imunologia , Proteínas Recombinantes/biossíntese , Testes Cutâneos/métodos , Tuberculose/diagnóstico , Animais , Antígenos de Bactérias/genética , Cromatografia de Afinidade , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Cobaias , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/genética , Tuberculina , Tuberculose/imunologia
16.
Mol Biosyst ; 6(12): 2403-16, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20644878

RESUMO

Spiders of the Loxosceles genus are cosmopolitan, and their venom components possess remarkable biological properties associated with their ability to act upon different molecules and receptors. Accidents with Loxosceles intermedia specimens are recognized as a public health problem in the south of Brazil. To describe the transcriptional profile of the L. intermedia venom gland, we generated a wide cDNA library, and its transcripts were functionally and structurally analyzed. After initial analyses, 1843 expressed sequence tags (ESTs) produced readable sequences that were grouped into 538 clusters, 281 of which were singletons. 985 reads (53% of total ESTs) matched to known proteins. Similarity searches showed that toxin-encoding transcripts account for 43% of the total library and comprise a great number of ESTs. The most frequent toxins were from the LiTx family, which are known for their insecticidal activity. Both phospholipase D and astacin-like metalloproteases toxins account for approximately 9% of total transcripts. Toxins components such as serine proteases, hyaluronidases and venom allergens were also found but with minor representation. Almost 10% of the ESTs encode for proteins involved in cellular processes. These data provide an important overview of the L. intermedia venom gland expression scenario and revealed significant differences from profiles of other spiders from the Loxosceles genus. Furthermore, our results also confirm that this venom constitutes an amazing source of novel compounds with potential agrochemical, industrial and pharmacological applications.


Assuntos
Estruturas Animais/metabolismo , Perfilação da Expressão Gênica , Venenos de Aranha/genética , Aranhas/anatomia & histologia , Aranhas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Peptídeos/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação , Venenos de Aranha/metabolismo
17.
BMC Microbiol ; 9: 120, 2009 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-19497120

RESUMO

BACKGROUND: The kinetoplast DNA (kDNA) of trypanosomatids consists of an unusual arrangement of circular molecules catenated into a single network. The diameter of the isolated kDNA network is similar to that of the entire cell. However, within the kinetoplast matrix, the kDNA is highly condensed. Studies in Crithidia fasciculata showed that kinetoplast-associated proteins (KAPs) are capable of condensing the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasitic protozoon that shows distinct patterns of kDNA condensation during their complex morphogenetic development. In epimastigotes and amastigotes (replicating forms) the kDNA fibers are tightly packed into a disk-shaped kinetoplast, whereas trypomastigotes (non-replicating) present a more relaxed kDNA organization contained within a rounded structure. It is still unclear how the compact kinetoplast disk of epimastigotes is converted into a globular structure in the infective trypomastigotes. RESULTS: In this work, we have analyzed KAP coding genes in trypanosomatid genomes and cloned and expressed two kinetoplast-associated proteins in T. cruzi: TcKAP4 and TcKAP6. Such small basic proteins are expressed in all developmental stages of the parasite, although present a differential distribution within the kinetoplasts of epimastigote, amastigote and trypomastigote forms. CONCLUSION: Several features of TcKAPs, such as their small size, basic nature and similarity with KAPs of C. fasciculata, are consistent with a role in DNA charge neutralization and condensation. Additionally, the differential distribution of KAPs in the kinetoplasts of distinct developmental stages of the parasite, indicate that the kDNA rearrangement that takes place during the T. cruzi differentiation process is accompanied by TcKAPs redistribution.


Assuntos
Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Genoma de Protozoário , Estágios do Ciclo de Vida/genética , Dados de Sequência Molecular , Filogenia , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Sintenia , Trypanosoma cruzi/genética
18.
Mem Inst Oswaldo Cruz ; 103(5): 483-8, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18797763

RESUMO

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.


Assuntos
Vírus da Dengue/genética , Dengue/virologia , Surtos de Doenças , Brasil/epidemiologia , Dengue/epidemiologia , Vírus da Dengue/classificação , Genótipo , Humanos , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Mem. Inst. Oswaldo Cruz ; 103(5): 483-488, Aug. 2008. ilus, mapas, tab
Artigo em Inglês | LILACS | ID: lil-491971

RESUMO

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.


Assuntos
Humanos , Surtos de Doenças , Vírus da Dengue/genética , Dengue/virologia , Brasil/epidemiologia , Vírus da Dengue/classificação , Dengue/epidemiologia , Genótipo , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Viral/genética
20.
Blood Purif ; 25(5-6): 411-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17914260

RESUMO

BACKGROUND/AIMS: Chronic kidney disease (CKD) and periodontitis (PD) are serious public-health concerns. Vitamin D is a fat-soluble steroid hormone that interacts with its nuclear receptor (VDR) to regulate a variety of biological processes, such as bone metabolism, immune response modulation and transcription of several genes involved in CKD and PD disease mechanisms. The aim of this work was to investigate the association between polymorphisms in the VDR gene and end-stage renal disease (ESRD) and PD. METHODS: 222 subjects with and without ESRD (in hemodialysis) were divided into groups with and without PD. Polymorphisms TaqI and BsmI in the VDR gene were analyzed by PCR restriction fragment length polymorphism. The significance of differences in allele, genotype and haplotype frequencies between groups was assessed by the chi2 test (p value <0.05) and odds ratio (OR). RESULTS: Allele G was associated with protection against ESRD: groups without versus with ESRD (GG) x (GA+AA): OR = 2.5, 95% CI = 1.4-4.6, p = 0.00; (G x A): OR = 1.5, 95% CI = 1.0-2.3, p = 0.02; (TG + CG) x (TA + CA): OR = 1.5, 95% CI = 1.0-2.3, p = 0.02. No association was observed between the study polymorphisms and susceptibility to or protection against PD. CONCLUSION: Allele G of the VDR BsmI polymorphism was associated with protection against ESRD.


Assuntos
Predisposição Genética para Doença , Falência Renal Crônica/genética , Periodontite/genética , Polimorfismo Genético , Receptores de Calcitriol/genética , Adulto , Idoso , Alelos , Estudos de Casos e Controles , Desoxirribonucleases de Sítio Específico do Tipo II , Frequência do Gene , Genótipo , Haplótipos , Humanos , Falência Renal Crônica/etiologia , Pessoa de Meia-Idade , Periodontite/etiologia , Polimorfismo de Fragmento de Restrição
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