RESUMO
The protozoan Trypanosoma cruzi is a parasite exposed to several environmental stressors inside its invertebrate and vertebrate hosts. Although stress conditions are involved in its differentiation processes, little information is available about the stress response proteins engaged in these activities. This work reports the first known association of the stress-inducible protein 1 (STI1) with the cellular differentiation process in a unicellular eukaryote. Albeit STI1 expression is constitutive in epimastigotes and metacyclic trypomastigotes, higher protein levels were observed in late growth phase epimastigotes subjected to nutritional stress. Analysis by indirect immunofluorescence revealed that T. cruzi STI1 (TcSTI1) is located throughout the cell cytoplasm, with some cytoplasmic granules appearing in greater numbers in late growing epimastigotes and late growing epimastigotes subjected to nutritional stress. We observed that part of the fluorescence signal from both TcSTI1 and TcHSP70 colocalized around the nucleus. Gene silencing of sti1 in Trypanosoma brucei did not affect cell growth. Similarly, the growth of T. cruzi mutant parasites with a single allele sti1 gene knockout was not affected. However, the differentiation of epimastigotes in metacyclic trypomastigotes (metacyclogenesis) was compromised. Lower production rates and numbers of metacyclic trypomastigotes were obtained from the mutant parasites compared with the wild-type parasites. These data indicate that reduced levels of TcSTI1 decrease the rate of in vitro metacyclogenesis, suggesting that this protein may participate in the differentiation process of T. cruzi.
Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Protozoários/genética , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Inativação de Genes , Inativação Gênica , Proteínas de Choque Térmico/genética , Estágios do Ciclo de Vida/genética , Estágios do Ciclo de Vida/fisiologia , Mutação , Proteínas de Protozoários/metabolismo , Estresse Fisiológico , Trypanosoma cruzi/química , Trypanosoma cruzi/ultraestruturaRESUMO
Chagas disease is caused by Trypanosoma cruzi and affects 18 million people in Central and South America. Here we analyzed the exposure of phosphatidylserine by the different forms of the parasite life cycle. Only the infective trypomastigotes, but not the epimastigotes or intracellular amastigotes, expose this phospholipid. This triggers a transforming growth factor beta signaling pathway, based on phosphorylated Smad 2 nuclear translocation, leading to iNOS disappearance in infected macrophages. This macrophage deactivation favors the survival of this intracellular parasite. Thus, phosphatidylserine exposure may be used by T. cruzi to evade innate immunity and be a common feature of obligate intracellular parasites that have to deal with activated macrophages.
Assuntos
Macrófagos/parasitologia , Fosfatidilserinas/metabolismo , Trypanosoma cruzi/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
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 VeroRESUMO
Kinetoplast DNA (kDNA) of trypanosomatid protozoa consists of an unusual arrangement of two types of circular molecules catenated into a single network: (1) a few maxicircles that encode various mitochondrial enzyme subunits and rRNA in a cryptic pattern and (2) thousands of minicircles that encode guide RNAs (gRNAs). kDNA is associated with proteins, known as kinetoplast-associated proteins (KAPs), which condense the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasite that displays different kDNA condensation patterns during its complex morphogenetic development. We cloned the T. cruzi gene encoding TcKAP3 (kinetoplast-associated protein 3). TcKAP3 is a single-copy gene that is transcribed into a 1.8-kb mRNA molecule and expressed in all stages of the parasite. Mouse antiserum raised against recombinant TcKPA3 recognized a 21.8kDa protein, which was found, by indirect immunofluorescence and immunoelectron microscopy, to be associated with the T. cruzi kinetoplast. Several features of TcKAP3, such as its small size, basic nature and similarity with KAP3 from the insect trypanosomatid Crithidia fasciculata, are consistent with a role in DNA charge neutralization and condensation. This suggests that this protein is involved in organizing the kDNA network. Gene deletion was used to investigate TcKAP3 function. Here we investigated the T. cruzi KAP3 null mutant, analyzing its fitness during proliferation, differentiation and infectivity.
Assuntos
DNA de Cinetoplasto/metabolismo , Técnicas de Inativação de Genes , Substâncias Macromoleculares/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/fisiologia , Clonagem Molecular , DNA de Protozoário/química , DNA de Protozoário/genética , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Peso Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Análise de Sequência de DNA , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
We used differential display to select genes differentially expressed during differentiation of epimastigotes into metacyclic trypomastigotes in the protozoan parasite Trypanosoma cruzi. One of the selected clones had a sequence similar to that of the small-subunit (SSU) processome protein Sof1p, which is involved in rRNA processing. The corresponding T. cruzi protein, TcSof1, displayed a nuclear localization and is downregulated during metacyclogenesis. Heterologous RNA interference assays showed that depletion of this protein impaired growth but did not affect progression through the cell cycle, suggesting that ribosome synthesis regulation and the cell cycle are uncoupled in this parasite. Quantitative PCR (qPCR) assays of several SSU processome-specific genes in T. cruzi also showed that most of them were regulated posttranscriptionally. This process involves the accumulation of mRNA in the polysome fraction of metacyclic trypomastigotes, where TcSof1 cannot be detected. Metacyclic trypomastigote polysomes were purified and separated by sucrose gradient sedimentation. Northern blot analysis of the sucrose gradient fractions showed the association of TcSof1 mRNA with polysomes, confirming the qPCR data. The results suggest that the mechanism of regulation involves the blocking of translation elongation and/or termination.