RESUMEN

Recombinant filamentous fd bacteriophages (rfd) expressing antigenic peptides were shown to induce cell-mediated immune responses in the absence of added adjuvant, being a promising delivery system for vaccination. Here, we tested the capacity of rfd phages to protect against infection with the human protozoan Trypanosoma cruzi, the etiologic agent of Chagas Disease. For this, C57BL/6 (B6) and Tlr9-/- mice were vaccinated with rfd phages expressing the OVA257-264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 and challenged with either the T. cruzi Y-OVA or Y-strain, respectively. We found that vaccination with rfd phages induces anti-PA8 and anti-TSKB20 IgG production, expansion of Ag-specific IFN-γ, TNF-α, and Granzyme B-producing CD8+ T cells, as well as in vivo Ag-specific cytotoxic responses. Moreover, the fd-TSKB20 vaccine was able to protect against mortality induced by a high-dose inoculum of the parasite. Although vaccination with rfd phages successfully reduced both parasitemia and parasite load in the myocardium of WT B6 mice, Tlr9-/- animals were not protected against infection. Thus, our data extend previous studies, demonstrating that rfd phages induce Ag-specific IgG and CD8+ T cell-mediated responses and confer protection against an important human parasite infection, through a TLR9-dependent mechanism.

Asunto(s)

Bacteriófago M13 , Enfermedad de Chagas , Regulación de la Expresión Génica , Vacunas Antiprotozoos , Receptor Toll-Like 9 , Trypanosoma cruzi , Vacunación , Animales , Bacteriófago M13/genética , Bacteriófago M13/inmunología , Enfermedad de Chagas/genética , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/prevención & control , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Ratones , Ratones Noqueados , Vacunas Antiprotozoos/genética , Vacunas Antiprotozoos/inmunología , Vacunas Antiprotozoos/farmacología , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunología , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología

RESUMEN

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'-deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

RESUMEN

Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5'-chloro-2'-deoxyuridine (CldU) and 5'-iodo-2'-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.

RESUMEN

In recent years, proteasome involvement in the damage response induced by ionizing radiation (IR) became evident. However, whether proteasome plays a direct or indirect role in IR-induced damage response still unclear. Trypanosoma cruzi is a human parasite capable of remarkable high tolerance to IR, suggesting a highly efficient damage response system. Here, we investigate the role of T. cruzi proteasome in the damage response induced by IR. We exposed epimastigotes to high doses of gamma ray and we analyzed the expression and subcellular localization of several components of the ubiquitin-proteasome system. We show that proteasome inhibition increases IR-induced cell growth arrest and proteasome-mediated proteolysis is altered after parasite exposure. We observed nuclear accumulation of 19S and 20S proteasome subunits in response to IR treatments. Intriguingly, the dynamic of 19S particle nuclear accumulation was more similar to the dynamic observed for Rad51 nuclear translocation than the observed for 20S. In the other hand, 20S increase and nuclear translocation could be related with an increase of its regulator PA26 and high levels of proteasome-mediated proteolysis in vitro. The intersection between the opposed peaks of 19S and 20S protein levels was marked by nuclear accumulation of both 20S and 19S together with Ubiquitin, suggesting a role of ubiquitin-proteasome system in the nuclear protein turnover at the time. Our results revealed the importance of proteasome-mediated proteolysis in T. cruzi IR-induced damage response suggesting that proteasome is also involved in T. cruzi IR tolerance. Moreover, our data support the possible direct/signaling role of 19S in DNA damage repair. Based on these results, we speculate that spatial and temporal differences between the 19S particle and 20S proteasome controls proteasome multiple roles in IR damage response.

Asunto(s)

Complejo de la Endopetidasa Proteasomal/metabolismo , Radiación Ionizante , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/efectos de la radiación , Ubiquitina/metabolismo , Reparación del ADN , Proteolisis , Respuesta de Proteína Desplegada

RESUMEN

BACKGROUND: During Trypanosoma cruzi infection, macrophages produce reactive oxygen species (ROS) in a process called respiratory burst. Several works have aimed to elucidate the role of ROS during T. cruzi infection and the results obtained are sometimes contradictory. T. cruzi has a highly efficiently regulated antioxidant machinery to deal with the oxidative burst, but the parasite macromolecules, particularly DNA, may still suffer oxidative damage. Guanine (G) is the most vulnerable base and its oxidation results in formation of 8-oxoG, a cellular marker of oxidative stress. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the contribution of ROS in T. cruzi survival and infection, we utilized mice deficient in the gp91phox (Phox KO) subunit of NADPH oxidase and parasites that overexpress the enzyme EcMutT (from Escherichia coli) or TcMTH (from T. cruzi), which is responsible for removing 8-oxo-dGTP from the nucleotide pool. The modified parasites presented enhanced replication inside murine inflammatory macrophages from C57BL/6 WT mice when compared with control parasites. Interestingly, when Phox KO macrophages were infected with these parasites, we observed a decreased number of all parasites when compared with macrophages from C57BL/6 WT. Scavengers for ROS also decreased parasite growth in WT macrophages. In addition, treatment of macrophages or parasites with hydrogen peroxide increased parasite replication in Phox KO mice and in vivo. CONCLUSIONS: Our results indicate a paradoxical role for ROS since modified parasites multiply better inside macrophages, but proliferation is significantly reduced when ROS is removed from the host cell. Our findings suggest that ROS can work like a signaling molecule, contributing to T. cruzi growth inside the cells.

Asunto(s)

Macrófagos Peritoneales/parasitología , Especies Reactivas de Oxígeno/metabolismo , Trypanosoma cruzi/fisiología , Animales , Células Cultivadas , Enfermedad de Chagas/parasitología , Nucleótidos de Desoxiguanina/metabolismo , Modelos Animales de Enfermedad , Interacciones Huésped-Parásitos , Peróxido de Hidrógeno/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Estrés Oxidativo , Rodaminas/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrollo

RESUMEN

Schistosomiasis is a neglected tropical disease, and after malaria, is the second most important tropical disease in public health. A vaccine that reduces parasitemia is desirable to achieve mass treatment with a low cost. Although potential antigens have been identified and tested in clinical trials, no effective vaccine against schistosomiasis is available. Y-box-binding proteins (YBPs) regulate gene expression and participate in a variety of cellular processes, including transcriptional and translational regulation, DNA repair, cellular proliferation, drug resistance, and stress responses. The Schistosoma mansoni ortholog of the human YB-1, SMYB1, is expressed in all stages of the parasite life cycle. Although SMYB1 binds to DNA or RNA oligonucleotides, immunohistochemistry assays demonstrated that it is primarily localized in the cytoplasm of parasite cells. In addition, SMYB1 interacts with a protein involved in mRNA processing, suggesting that SMYB1 functions in the turnover, transport, and/or stabilization of RNA molecules during post-transcriptional gene regulation. Here we report the potential of SMYB1 as a vaccine candidate. We demonstrate that recombinant SMYB1 stimulates the production of high levels of specific IgG1 antibodies in a mouse model. The observed levels of specific IgG1 and IgG2a antibodies indicate an actual protection against cercariae challenge. Animals immunized with rSMYB1 exhibited a 26% reduction in adult worm burden and a 28% reduction in eggs retained in the liver. Although proteins from the worm tegument are considered optimal targets for vaccine development, this study demonstrates that unexposed cytoplasmic proteins can reduce the load of intestinal worms and the number of eggs retained in the liver.

RESUMEN

Nucleotide excision repair (NER) is a highly conserved genome repair pathway acting on helix distorting DNA lesions. NER is divided into two subpathways: global genome NER (GG-NER), which is responsible for repair throughout genomes, and transcription-coupled NER (TC-NER), which acts on lesions that impede transcription. The extent of the Trypanosoma brucei genome that is transcribed is highly unusual, since most genes are organized in multigene transcription units, each transcribed from a single promoter. Given this transcription organization, we have addressed the importance of NER to T. brucei genome maintenance by performing RNAi against all predicted contributing factors. Our results indicate that TC-NER is the main pathway of NER repair, but only CSB, XPBz and XPG contribute. Moreover, we show that UV lesions are inefficiently repaired in T. brucei, perhaps due to preferential use of RNA polymerase translesion synthesis. RNAi of XPC and DDB was found to be lethal, and we show that these factors act in inter-strand cross-link repair. XPD and XPB appear only to act in transcription, not repair. This work indicates that the predominance of multigenic transcription in T. brucei has resulted in pronounced adaptation of NER relative to the host and may be an attractive drug target.

Asunto(s)

Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Transcripción Genética , Trypanosoma brucei brucei/fisiología , Enzimas Reparadoras del ADN/genética , Genes Esenciales , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo

RESUMEN

THE SPLICED LEADER (SL) IS A GENE THAT GENERATES A FUNCTIONAL NCRNA THAT IS COMPOSED OF TWO REGIONS: an intronic region of unknown function (SLi) and an exonic region (SLe), which is transferred to the 5' end of independent transcripts yielding mature mRNAs, in a process known as spliced leader trans-splicing (SLTS). The best described function for SLTS is to solve polycistronic transcripts into monocistronic units, specifically in Trypanosomatids. In other metazoans, it is speculated that the SLe addition could lead to increased mRNA stability, differential recruitment of the translational machinery, modification of the 5' region or a combination of these effects. Although important aspects of this mechanism have been revealed, several features remain to be elucidated. We have analyzed 157 SLe sequences from 148 species from seven phyla and found a high degree of conservation among the sequences of species from the same phylum, although no considerable similarity seems to exist between sequences of species from different phyla. When analyzing case studies, we found evidence that a given SLe will always be related to a given set of transcripts in different species from the same phylum, and therefore, different SLe sequences from the same species would regulate different sets of transcripts. In addition, we have observed distinct transcript categories to be preferential targets for the SLe addition in different phyla. This work sheds light into crucial and controversial aspects of the SLTS mechanism. It represents a comprehensive study concerning various species and different characteristics of this important post-transcriptional regulatory mechanism.

RESUMEN

The main consequence of oxidative stress is the formation of DNA lesions, which can result in genomic instability and lead to cell death. Guanine is the base that is most susceptible to oxidation, due to its low redox potential, and 8-oxoguanine (8-oxoG) is the most common lesion. These characteristics make 8-oxoG a good cellular biomarker to indicate the extent of oxidative stress. If not repaired, 8-oxoG can pair with adenine and cause a G:C to T:A transversion. When 8-oxoG is inserted during DNA replication, it could generate double-strand breaks, which makes this lesion particularly deleterious. Trypanosoma cruzi needs to address various oxidative stress situations, such as the mammalian intracellular environment and the triatomine insect gut where it replicates. We focused on the MutT enzyme, which is responsible for removing 8-oxoG from the nucleotide pool. To investigate the importance of 8-oxoG during parasite infection of mammalian cells, we characterized the MutT gene in T. cruzi (TcMTH) and generated T. cruzi parasites heterologously expressing Escherichia coli MutT or overexpressing the TcMTH enzyme. In the epimastigote form, the recombinant and wild-type parasites displayed similar growth in normal conditions, but the MutT-expressing cells were more resistant to hydrogen peroxide treatment. The recombinant parasite also displayed significantly increased growth after 48 hours of infection in fibroblasts and macrophages when compared to wild-type cells, as well as increased parasitemia in Swiss mice. In addition, we demonstrated, using western blotting experiments, that MutT heterologous expression can influence the parasite antioxidant enzyme protein levels. These results indicate the importance of the 8-oxoG repair system for cell viability.

Asunto(s)

Daño del ADN , Guanina/análogos & derivados , Estrés Oxidativo , Trypanosoma cruzi/fisiología , Animales , Supervivencia Celular , Células Cultivadas , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/patología , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Femenino , Fibroblastos/parasitología , Expresión Génica , Guanina/metabolismo , Peróxido de Hidrógeno/toxicidad , Macrófagos/parasitología , Ratones , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Parasitemia/parasitología , Parasitemia/patología , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo

RESUMEN

SMYB1 is a Schistosoma mansoni protein highly similar to members of the Y-box binding protein family. Similar to other homologues, SMYB1 is able to bind double- and single-stranded DNA, as well as RNA molecules. The characterization of proteins involved in the regulation of gene expression in S. mansoni is of great importance for the understanding of molecular events that control morphological and physiological changes in this parasite. Here we demonstrate that SMYB1 is located in the cytoplasm of cells from different life-cycle stages of S. mansoni, suggesting that this protein is probably acting in mRNA metabolism in the cytoplasm and corroborating previous findings from our group that showed its ability to bind RNA. Protein-protein interactions are important events in all biological processes, since most proteins execute their functions through large supramolecular structures. Yeast two-hybrid screenings using SMYB1 as bait identified a partner in S. mansoni similar to the SmD3 protein of Drosophila melanogaster (SmRNP), which is important in the assembly of small nuclear ribonucleoprotein complexes. Also, pull-down assays were conducted using immobilized GST-SMYB1 proteins and confirmed the SMYB1-SmRNP interaction. The interaction of SMYB1 with a protein involved in mRNA processing suggests that it may act in processes such as turnover, transport and stabilization of RNA molecules.

Asunto(s)

Proteínas del Helminto/metabolismo , ARN de Helminto/metabolismo , ARN Mensajero/metabolismo , Schistosoma mansoni/metabolismo , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , Transporte Biológico , Citoplasma/metabolismo , Femenino , Biblioteca de Genes , Proteínas del Helminto/genética , Inmunohistoquímica , Masculino , ARN de Helminto/genética , ARN Mensajero/genética , Conejos , Schistosoma mansoni/genética , Técnicas del Sistema de Dos Híbridos

RESUMEN

The protozoan Trypanosoma cruzi, its mammalian reservoirs, and vectors have existed in nature for millions of years. The human infection, named Chagas disease, is a major public health problem for Latin America. T. cruzi is genetically highly diverse and the understanding of the population structure of this parasite is critical because of the links to transmission cycles and disease. At present, T. cruzi is partitioned into six discrete typing units (DTUs), TcI-TcVI. Here we focus on the current status of taxonomy-related areas such as population structure, phylogeographical and eco-epidemiological features, and the correlation of DTU with natural and experimental infection. We also summarize methods for DTU genotyping, available for widespread use in endemic areas. For the immediate future multilocus sequence typing is likely to be the gold standard for population studies. We conclude that greater advances in our knowledge on pathogenic and epidemiological features of these parasites are expected in the coming decade through the comparative analysis of the genomes from isolates of various DTUs.

Asunto(s)

Enfermedad de Chagas/epidemiología , Enfermedad de Chagas/parasitología , Trypanosoma cruzi/clasificación , Animales , Quirópteros/parasitología , Evolución Molecular , Variación Genética , Genoma de Protozoos , Genómica , Humanos , Tipificación de Secuencias Multilocus , Filogeografía , Terminología como Asunto , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad

RESUMEN

BACKGROUND: During its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite. METHODOLOGY/PRINCIPAL FINDINGS: In this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p

Asunto(s)

Proteínas del Helminto/metabolismo , Schistosoma mansoni/metabolismo , Esquistosomiasis mansoni/parasitología , Factores de Transcripción/metabolismo , Animales , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Femenino , Proteínas del Helminto/genética , Humanos , Masculino , Unión Proteica , Transporte de Proteínas , Schistosoma mansoni/genética , Schistosoma mansoni/crecimiento & desarrollo , Esquistosomiasis mansoni/metabolismo , Factores de Transcripción/genética

RESUMEN

Virus-host biological interaction is a continuous coevolutionary process involving both host immune system and viral escape mechanisms. Flaviviridae family is composed of fast evolving RNA viruses that infects vertebrate (mammals and birds) and/or invertebrate (ticks and mosquitoes) organisms. These host groups are very distinct life forms separated by a long evolutionary time, so lineage-specific anti-viral mechanisms are likely to have evolved. Flaviviridae viruses which infect a single host lineage would be subjected to specific host-induced pressures and, therefore, selected by them. In this work we compare the genomic evolutionary patterns of Flaviviridae viruses and their hosts in an attempt to uncover coevolutionary processes inducing common features in such disparate groups. Especially, we have analyzed dinucleotide and codon usage patterns in the coding regions of vertebrate and invertebrate organisms as well as in Flaviviridae viruses which specifically infect one or both host types. The two host groups posses very distinctive dinucleotide and codon usage patterns. A pronounced CpG under-representation was found in the vertebrate group, possibly induced by the methylation-deamination process, as well as a prominent TpA decrease. The invertebrate group displayed only a TpA frequency reduction bias. Flaviviridae viruses mimicked host nucleotide motif usage in a host-specific manner. Vertebrate-infecting viruses possessed under-representation of CpG and TpA, and insect-only viruses displayed only a TpA under-representation bias. Single-host Flaviviridae members which persistently infect mammals or insect hosts (Hepacivirus and insect-only Flavivirus, respectively) were found to posses a codon usage profile more similar to that of their hosts than to related Flaviviridae. We demonstrated that vertebrates and mosquitoes genomes are under very distinct lineage-specific constraints, and Flaviviridae viruses which specifically infect these lineages appear to be subject to the same evolutionary pressures that shaped their host coding regions, evidencing the lineage-specific coevolutionary processes between the viral and host groups.

Asunto(s)

Evolución Biológica , Flaviviridae/genética , Interacciones Huésped-Patógeno , Animales , Secuencia de Bases , Codón , Vectores de Enfermedades , Flaviviridae/clasificación , Filogenia

RESUMEN

We have previously demonstrated that both parasite genetic variability and host genetic background were important in determining the differential tissue distribution of the Col1.7G2 and JG T. cruzi monoclonal strains after artificial infections in mice. We observed that the JG strain was most prevalent in hearts of mouse lineages with the MHC haplotype H-2(d) (BALB/c and DBA2), while Col1.7G2 was predominant in hearts from C57BL/6 mice, which have the H-2(b) haplotype. To assess whether the MHC gene region indeed influenced tissue tropism of T. cruzi, we used the same two parasite strains to infect C57BL/6 (H-2(b)) and C57BLKS/J (H-2(d)) mice; the latter strain results from the introgression of DBA2 MHC region into the C57BL/6 background. We also performed ex vivo infections of cardiac explants from four congenic mice lineages with the H-2(b) and H-2(d) haplotypes arranged in two different genetic backgrounds: C57BLKS/J (H-2(d)) versus C57BL/6 (H-2(b)) and BALB/c (H-2(d)) versus BALB/B10-H2(b) (H-2(b)). In agreement with our former observations, Col1.7G2 was predominant in hearts from C57BL/6 mice (H-2(b)), but we observed a clear predominance of the JG strain in hearts from C57BLKS/J animals (H-2(d)). In the ex vivo experiments Col1.7G2 also prevailed in explants from H-2(b) animals while no predominance of any of the strains was observed in H-2(d) mice explants, regardless of the genetic background. These observations clearly demonstrate that the MHC region influences the differential tissue distribution pattern of infecting T. cruzi strains, which by its turn may be in a human infection the determinant for the clinical forms of the Chagas disease.

Asunto(s)

Interacciones Huésped-Parásitos/genética , Complejo Mayor de Histocompatibilidad/genética , Tropismo , Trypanosoma cruzi/patogenicidad , Animales , Secuencia de Bases , Cartilla de ADN , Haplotipos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Especificidad de Órganos , Reacción en Cadena de la Polimerasa

RESUMEN

The SCF (Skp1-Cul1-F-box) complex is one of the several E3 ligase enzymes and it catalyzes protein ubiquitination and degradation by the 26S proteasome. Rbx1 is a member of the SCF complex in humans and HRT1 is its yeast orthologue. A cDNA encoding a Schistosoma mansoni Rbx1 homolog was cloned and functionally characterized. Heterologous functional complementation in yeast showed that the worm SmRbx gene was able to complement the HRT1yeast null mutation. Gene deletion constructs for N- and C-termini truncated proteins were used to transform hrt1(-) yeast mutant strains, allowing us to observe that regions reported to be involved in the interaction with cullin1 (Cul1) were essential for SmRbx function. Yeast two-hybrid assays using SmRbx and yeast Cul1 confirmed that SmRbx, but not the mutant SmRbxDelta24N, lacking the N-terminus of the protein, was capable of interacting with Cul1. These results suggest that SmRbx protein is involved in the SCF complex formation.

Asunto(s)

Proteínas del Helminto/genética , Schistosoma mansoni/genética , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , ADN Complementario/química , ADN de Helmintos/química , Etiquetas de Secuencia Expresada , Femenino , Prueba de Complementación Genética , Vectores Genéticos , Proteínas del Helminto/química , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ligasas SKP Cullina F-box , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schistosoma mansoni/metabolismo

RESUMEN

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.

Asunto(s)

Animales , Humanos , Cafeína/farmacología , Proteína Quinasa C/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Schistosoma mansoni/genética , Proteínas de Unión al GTP rho/genética , Genes de Helminto , Mutación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Schistosoma mansoni/metabolismo , Transducción de Señal/genética , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

In a previous study, the Schistosoma mansoni Rho1 protein was able to complement Rho1 null mutant Saccharomyces cerevisiae cells at restrictive temperatures and under osmotic stress (low calcium concentration) better than the human homologue (RhoA). It is known that under osmotic stress, the S. cerevisiae Rho1 triggers two distinct pathways: activation of the membrane 1,3-beta-glucan synthase enzymatic complex and activation of the protein kinase C1 signal transduction pathway, promoting the transcription of response genes. In the present work the SmRho1 protein and its mutants smrho1E97P, smrho1L101T, and smrho1E97P, L101T were used to try to clarify the basis for the differential complementation of Rho1 knockout yeast strain by the human and S. mansoni genes. Experiments of functional complementation in the presence of caffeine and in the presence of the osmotic regulator sorbitol were conducted. SmRho1 and its mutants showed a differential complementation of the yeast cells in the presence of caffeine, since smrho1E97P and smrho1E97P, L101T mutants showed a delay in the growth when compared to the yeast complemented with the wild type SmRho1. However, in the presence of sorbitol and caffeine the wild type SmRho1 and mutants showed a similar complementation phenotype, as they allowed yeast growth in all caffeine concentrations tested.

Asunto(s)

Cafeína/farmacología , Proteína Quinasa C/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Schistosoma mansoni/genética , Proteínas de Unión al GTP rho/genética , Animales , Genes de Helminto , Humanos , Mutación , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schistosoma mansoni/metabolismo , Transducción de Señal/genética , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

Chagas disease, caused by the protozoan Trypanosoma cruzi, has a variable clinical course, ranging from symptomless infection to severe chronic disease with cardiovascular or gastrointestinal involvement or, occasionally, overwhelming acute episodes. The factors influencing this clinical variability have not been elucidated, but it is likely that the genetic variability of both the host and the parasite are of importance. In this work we review the the genetic structure of T. cruzi populations and analyze the importance of genetic variation of the parasite in the pathogenesis of the disease under the light of the histotropic-clonal model.

Asunto(s)

Enfermedad de Chagas/genética , Variación Genética , Trypanosoma cruzi/genética , Animales , Enfermedad de Chagas/parasitología , Interacciones Huésped-Parásitos , Humanos , Trypanosoma cruzi/patogenicidad

RESUMEN

Chagas disease, caused by the protozoan Trypanosoma cruzi, has a variable clinical course, ranging from symptomless infection to severe chronic disease with cardiovascular or gastrointestinal involvement or, occasionally, overwhelming acute episodes. The factors influencing this clinical variability have not been elucidated, but it is likely that the genetic variability of both the host and the parasite are of importance. In this work we review the the genetic structure of T. cruzi populations and analyze the importance of genetic variation of the parasite in the pathogenesis of the disease under the light of the histotropic-clonal model.

Asunto(s)

Animales , Humanos , Enfermedad de Chagas , Trypanosoma cruzi , Enfermedad de Chagas , Variación Genética , Interacciones Huésped-Parásitos , Trypanosoma cruzi

Asunto(s)

Humanos , Fractura Craneal Deprimida/cirugía , Protocolos Clínicos