Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros










Tipo de estudo
Intervalo de ano de publicação
1.
Int J Genomics ; 2019: 8458263, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31531340

RESUMO

The HIV-1 virus (human immunodeficiency virus) affects 36.9 million people worldwide, with approximately 900000 deaths in 2017. The virus carrier can develop severe immunodeficiency since CD4+ T lymphocytes are the main target, leading to acquired immunodeficiency syndrome (AIDS). Despite advances in pharmacological treatment, it is still difficult to eliminate latent reservoirs, becoming one of the main obstacles for viral eradication. The CRISPR- (clustered regularly interspaced short palindromic repeat-) Cas system is a genome-editing method which uses a guide RNA, a complementary sequence to the interested site, recruiting a nuclease that can break the viral or the host cell genetic material. From this double-stranded break, cellular repair mechanisms are activated being able to generate deletions, insertions, or substitutions, in order to inactivate specific gene loci, leading to loss of function. The objective of this minireview is to synthesize the current knowledge on the application of CRISPR-Cas-based gene therapy for HIV-1. The strategies encompass all steps of the viral infection cycle, from inhibition of cell invasion, through viral replication and integration inhibition, to excision of the latent provirus. Off-target effects and ethical implications were also discussed to evaluate the safety of the approach and viability of its application in humans, respectively. Although preclinical and clinical tests are still needed, the recent results establish an exciting possibility of applying this technology for prophylaxis and treatment of HIV-1.

2.
Sci Rep ; 6: 24610, 2016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27113535

RESUMO

Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals, and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway components and proteins related to actin cytoskeleton modifications. We hypothesized that TcMVK is a bifunctional enzyme that in addition to playing a classical role in isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for T. cruzi invasion.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , Microcorpos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais , Trypanosoma cruzi/enzimologia , Citoesqueleto de Actina , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/imunologia , Dimerização , Células HeLa , Humanos , Estágios do Ciclo de Vida , Camundongos , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Simulação de Dinâmica Molecular , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Trypanosoma cruzi/fisiologia
5.
PLoS Negl Trop Dis ; 8(9): e3176, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25233456

RESUMO

BACKGROUND: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts. METHODOLOGY/PRINCIPAL FINDINGS: The T. rangeli haploid genome is ∼ 24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins. CONCLUSIONS/SIGNIFICANCE: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets.


Assuntos
Genoma de Protozoário , Filogenia , Trypanosoma rangeli/genética , Animais , Sequência de Bases , DNA de Protozoário/genética , Haploidia , Humanos
6.
Infect Genet Evol ; 25: 157-65, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24727645

RESUMO

Chagas disease is caused by the protozoan Trypanosoma cruzi which affects 10 million people worldwide. Very few kinases have been characterized in this parasite, including the phosphatidylinositol kinases (PIKs) that are at the heart of one of the major pathways of intracellular signal transduction. Recently, we have classified the PIK family in T. cruzi using five different models based on the presence of PIK conserved domains. In this study, we have mapped PIK genes to the chromosomes of two different T. cruzi lineages (G and CL Brener) and determined the cellular localization of two PIK members. The kinases have crucial roles in metabolism and are assumed to be conserved throughout evolution. For this reason, they should display a conserved localization within the same eukaryotic species. In spite of this, there is an extensive polymorphism regarding PIK localization at both genomic and cellular levels, among different T. cruzi isolates and between T. cruzi and Trypanosomabrucei, respectively. We showed in this study that the cellular localization of two PIK-related proteins (TOR1 and 2) in the T. cruzi lineage is distinct from that previously observed in T. brucei. In addition, we identified a new PIK gene with peculiar feature, that is, it codes for a FYVE domain at N-terminal position. FYVE-PIK genes are phylogenetically distant from the groups containing exclusively the FYVE or PIK domain. The FYVE-PIK architecture is only present in trypanosomatids and in virus such as Acanthamoeba mimivirus, suggesting a horizontal acquisition. Our Bayesian phylogenetic inference supports this hypothesis. The exact functions of this FYVE-PIK gene are unknown, but the presence of FYVE domain suggests a role in membranous compartments, such as endosome. Taken together, the data presented here strengthen the possibility that trypanosomatids are characterized by extensive genomic plasticity that may be considered in designing drugs and vaccines for prevention of Chagas disease.


Assuntos
Fosfatidilinositol 3-Quinases/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/classificação , Trypanosoma cruzi/enzimologia , Teorema de Bayes , Doença de Chagas/epidemiologia , Evolução Molecular , Transferência Genética Horizontal , Genoma de Protozoário , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Filogenia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética
7.
Mol Biochem Parasitol ; 193(2): 71-4, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24583081

RESUMO

A growing body of evidence in mammalian cells indicates that secreted vesicles can be used to mediate intercellular communication processes by transferring various bioactive molecules, including mRNAs and microRNAs. Based on these findings, we decided to analyze whether Trypanosoma cruzi-derived extracellular vesicles contain RNA molecules and performed a deep sequencing and genome-wide analysis of a size-fractioned cDNA library (16-40nt) from extracellular vesicles secreted by noninfective epimastigote and infective metacyclic trypomastigote forms. Our data show that the small RNAs contained in these extracellular vesicles originate from multiple sources, including tRNAs. In addition, our results reveal that the variety and expression of small RNAs are different between parasite stages, suggesting diverse functions. Taken together, these observations call attention to the potential regulatory functions that these RNAs might play once transferred between parasites and/or to mammalian host cells.


Assuntos
RNA de Protozoário , Vesículas Transportadoras/genética , Trypanosoma cruzi/genética , Espaço Extracelular/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , RNA de Transferência/genética
8.
PLoS One ; 8(5): e63738, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23667668

RESUMO

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.


Assuntos
Cromossomos/genética , Rearranjo Gênico/genética , Variação Genética , Cariotipagem , Trypanosoma cruzi/citologia , Trypanosoma cruzi/genética , Alelos , Animais , Células Clonais , Sequência Conservada/genética , Loci Gênicos/genética , Marcadores Genéticos , Tamanho do Genoma/genética , Genoma de Protozoário/genética , Técnicas de Genotipagem , Repetições de Microssatélites/genética , Gambás , Polimorfismo Genético , Sintenia/genética , Telômero/metabolismo , Tubulina (Proteína)/metabolismo
9.
Acta Trop ; 120(3): 231-7, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21925137

RESUMO

A new genotype of Trypanosoma cruzi, associated with bats from anthropic areas, was recently described. Here we characterized a T. cruzi strain from this new genetic group, which could be a potential source of infection to humans. Metacyclic trypomastigotes (MT) of this strain, herein designated BAT, were compared to MT of well characterized CL and G strains, as regards the surface profile and infectivity toward human epithelial HeLa cells. BAT strain MT expressed gp82, the surface molecule recognized by monoclonal antibody 3F6 and known to promote CL strain invasion by inducing lysosomal exocytosis, as well as mucin-like molecules, but lacked gp90, which functions as a negative regulator of invasion in G strain. A set of experiments indicated that BAT strain internalization is gp82-mediated, and requires the activation of host cell phosphatidylinositol 3-kinase, protein kinase C and the mammalian target of rapamycin. MT of BAT strain were able to migrate through a gastric mucin layer, a property associated with p82 and relevant for oral infection. Gp82 was found to be a highly conserved molecule. Analysis of the BAT strain gp82 domain, containing the cell binding- and gastric mucin-binding sites, showed 91 and 93% sequence identity with G and CL strains, respectively. Hela cell invasion by BAT strain MT was inhibited by purified mucin-like molecules, which were shown to affect lysosome exocytosis required for MT internalization. Although MT of BAT strain infected host cells in vitro, they were less effective than G or CL strains in infecting mice either orally or intraperitoneally.


Assuntos
Doença de Chagas/parasitologia , Doença de Chagas/veterinária , Quirópteros/parasitologia , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genética , Animais , DNA de Protozoário/química , DNA de Protozoário/genética , Endocitose , Células Epiteliais/parasitologia , Feminino , Perfilação da Expressão Gênica , Células HeLa , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Análise de Sequência de DNA , Trypanosoma cruzi/isolamento & purificação
10.
Biochem Biophys Res Commun ; 390(3): 963-70, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19852933

RESUMO

Phosphatidylinositol (PI) kinases are at the heart of one of the major pathways of intracellular signal transduction. Herein, we present the first report on a survey made by similarity searches against the five human pathogenic trypanosomatids Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Leishmania braziliensis and Leishmania infantum genomes available to date for phosphatidylinositol- and related-kinases (TryPIKs). In addition to generating a panel called "The TryPIKinome", we propose a model of signaling pathways for these TryPIKs. The involvement of TryPIKs in fundamental pathways, such as intracellular signal transduction and host invasion processes, makes the study of TryPIKs an important area for further inquiry. New subtype-specific inhibitors are expected to work on individual members of the PIK family and, therefore, can presumably neutralize trypanosomatid invasion processes.


Assuntos
1-Fosfatidilinositol 4-Quinase/classificação , Desenho de Drogas , Inibidores Enzimáticos/química , Tripanossomicidas/química , Trypanosomatina/enzimologia , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , 1-Fosfatidilinositol 4-Quinase/genética , Sequência de Aminoácidos , Androstadienos/química , Androstadienos/farmacologia , Autofagia , Membrana Celular/enzimologia , Citocinese/efeitos dos fármacos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Humanos , Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/enzimologia , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/enzimologia , Leishmania infantum/crescimento & desenvolvimento , Leishmania major/efeitos dos fármacos , Leishmania major/enzimologia , Leishmania major/crescimento & desenvolvimento , Lisina/genética , Dados de Sequência Molecular , Filogenia , Tripanossomicidas/isolamento & purificação , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/crescimento & desenvolvimento , Wortmanina
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA