RESUMEN
Trypanosoma cruzi is the etiological agent of Chagas disease and a peculiar eukaryote with unique biological characteristics. DNA damage can block RNA polymerase, activating transcription-coupled nucleotide excision repair (TC-NER), a DNA repair pathway specialized in lesions that compromise transcription. If transcriptional stress is unresolved, arrested RNA polymerase can activate programmed cell death. Nonetheless, how this parasite modulates these processes is unknown. Here, we demonstrate that T. cruzi cell death after UV irradiation, a genotoxic agent that generates lesions resolved by TC-NER, depends on active transcription and is signaled mainly by an apoptotic-like pathway. Pre-treated parasites with α-amanitin, a selective RNA polymerase II inhibitor, become resistant to such cell death. Similarly, the gamma pre-irradiated cells are more resistant to UV when the transcription processes are absent. The Cockayne Syndrome B protein (CSB) recognizes blocked RNA polymerase and can initiate TC-NER. Curiously, CSB overexpression increases parasites' cell death shortly after UV exposure. On the other hand, at the same time after irradiation, the single-knockout CSB cells show resistance to the same treatment. UV-induced fast death is signalized by the exposition of phosphatidylserine to the outer layer of the membrane, indicating a cell death mainly by an apoptotic-like pathway. Furthermore, such death is suppressed in WT parasites pre-treated with inhibitors of ataxia telangiectasia and Rad3-related (ATR), a key DDR kinase. Signaling for UV radiation death may be related to R-loops since the overexpression of genes associated with the resolution of these structures suppress it. Together, results suggest that transcription blockage triggered by UV radiation activates an ATR-dependent apoptosis-like mechanism in T. cruzi, with the participation of CSB protein in this process.
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Introduction. Avian reovirus (ARV) is associated with arthritis/tenosynovitis and malabsorption syndrome in chickens. The σC and σB proteins, both exposed to the virus capsid, are highly immunogenic and could form the basis for diagnostic devices designed to assess the immunological status of the flock.Gap Statement. Commercial ARV ELISAs cannot distinguish between vaccinated and infected animals and might not detect circulating ARV strains.Aim. We aimed to develop a customized test to detect the circulating field ARV strains as well as distinguish between vaccinated and unvaccinated animals.Methodology. We developed ELISA assays based on recombinant (r) σB, σC and the nonstructural protein σNS and tested them using antisera of vaccinated and unvaccinated chickens as well as negative controls. Fragments of σB and σC proteins were also used to study regions that could be further exploited in diagnostic tests.Results. Vaccinated and unvaccinated birds were positive by commercial ELISA, with no difference in optical density values. In contrast, samples of unvaccinated animals showed lower absorbance in the rσB and rσC ELISA tests and higher absorbance in the rσNS ELISA test than the vaccinated animals. Negative control samples were negative in all tests. Fragmentation of σB and σC proteins showed that some regions can differentiate between vaccinated and unvaccinated animals. For example, σB amino acids 128-179 (σB-F4) and σC amino acids 121-165 (σC-F4) exhibited 85 and 95% positivity among samples of vaccinated animals but only 5% and zero positivity among samples of unvaccinated animals, respectively.Conclusion. These data suggest that unvaccinated birds might have been exposed to field strains of ARV. The reduction in absorbance in the recombinant tests possibly reflects an increased specificity of our test since unvaccinated samples showed less cross-reactivity with the vaccine proteins immobilized on ELISAs. The discrepant results obtained with the protein fragment tests between vaccinated and unvaccinated animals are discussed in light of the diversity between ARV strains.
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Pollos , Ensayo de Inmunoadsorción Enzimática , Orthoreovirus Aviar , Enfermedades de las Aves de Corral , Proteínas Recombinantes , Infecciones por Reoviridae , Animales , Orthoreovirus Aviar/inmunología , Orthoreovirus Aviar/genética , Orthoreovirus Aviar/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/diagnóstico , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/diagnóstico , Proteínas Recombinantes/inmunología , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/genética , Proteínas Virales/inmunología , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. OBJECTIVES: In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. METHODS: Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. FINDINGS: The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. MAIN CONCLUSIONS: Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.
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Proteínas Fluorescentes Verdes , Tripanocidas , Trypanosoma cruzi , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Proteínas Fluorescentes Verdes/genética , Tripanocidas/farmacología , Nitroimidazoles/farmacología , Pruebas de Sensibilidad Parasitaria , Animales , Concentración 50 Inhibidora , Evaluación Preclínica de Medicamentos , Supervivencia Celular/efectos de los fármacosRESUMEN
BACKGROUND Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. OBJECTIVES In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. METHODS Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. FINDINGS The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. MAIN CONCLUSIONS Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.
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Leishmaniasis represents a complex of diseases with a broad clinical spectrum and epidemiological diversity, considered a major public health problem. Although there is treatment, there are still no vaccines for cutaneous leishmaniasis. Because Leishmania spp. is an intracellular protozoan with several escape mechanisms, a vaccine must provoke cellular and humoral immune responses. Previously, we identified the Leishmania homolog of receptors for activated C kinase (LACK) and phosphoenolpyruvate carboxykinase (PEPCK) proteins as strong immunogens and candidates for the development of a vaccine strategy. The present work focuses on the in silico prediction and characterization of antigenic epitopes that might interact with mice or human major histocompatibility complex class I. After immunogenicity prediction on the Immune Epitope Database (IEDB) and the Database of MHC Ligands and Peptide Motifs (SYFPEITHI), 26 peptides were selected for interaction assays with infected mouse lymphocytes by flow cytometry and ELISpot. This strategy identified nine antigenic peptides (pL1-H2, pPL3-H2, pL10-HLA, pP13-H2, pP14-H2, pP15-H2, pP16-H2, pP17-H2, pP18-H2, pP26-HLA), which are strong candidates for developing a peptide vaccine against leishmaniasis.
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Leishmania mexicana , Leishmania , Leishmaniasis Cutánea , Humanos , Animales , Ratones , Epítopos , Antígenos de Histocompatibilidad Clase I , Antígenos HLA , Leishmania/metabolismo , Péptidos/química , Vacunas de Subunidad , Complejo Mayor de HistocompatibilidadRESUMEN
Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the T. cruzi plasma membrane, which hydrolyze a wide range of tri-/-diphosphate nucleosides. In this work, we used previously developed genetically modified strains of Trypanosoma cruzi (T. cruzi), hemi-knockout (KO +/−) and overexpressing (OE) the TcNTPDase-1 gene to evaluate the parasite infectivity profile in a mouse model of acute infection (n = 6 mice per group). Our results showed significantly higher parasitemia and mortality, and lower weight in animals infected with parasites OE TcNTPDase-1, as compared to the infection with the wild type (WT) parasites. On the other hand, animals infected with (KO +/−) parasites showed no mortality during the 30-day trial and mouse weight was more similar to the non-infected (NI) animals. In addition, they had low parasitemia (45.7 times lower) when compared with parasites overexpressing TcNTPDase-1 from the hemi-knockout (OE KO +/−) group. The hearts of animals infected with the OE KO +/− and OE parasites showed significantly larger regions of cardiac inflammation than those infected with the WT parasites (p < 0.001). Only animals infected with KO +/− did not show individual electrocardiographic changes during the period of experimentation. Together, our results expand the knowledge on the role of NTPDases in T. cruzi infectivity, reenforcing the potential of this enzyme as a chemotherapy target to treat Chagas disease (CD).
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Enfermedad de Chagas , Trypanosoma cruzi , Ratones , Animales , Enfermedad de Chagas/genética , Enfermedad de Chagas/parasitología , Corazón , Modelos Animales de EnfermedadRESUMEN
Histone variants play a crucial role in chromatin structure organization and gene expression. Trypanosomatids have an unusual H2B variant (H2B.V) that is known to dimerize with the variant H2A.Z generating unstable nucleosomes. Previously, we found that H2B.V protein is enriched in tissue-derived trypomastigote (TCT) life forms, a nonreplicative stage of Trypanosoma cruzi, suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among parasite life forms. Here, we performed the first genome-wide profiling of histone localization in T. cruzi using epimastigotes and TCT life forms, and we found that H2B.V was preferentially located at the edges of divergent transcriptional strand switch regions, which encompass putative transcriptional start regions; at some tDNA loci; and between the conserved and disrupted genome compartments, mainly at trans-sialidase, mucin and MASP genes. Remarkably, the chromatin of TCT forms was depleted of H2B.V-enriched peaks in comparison to epimastigote forms. Interactome assays indicated that H2B.V associated specifically with H2A.Z, bromodomain factor 2, nucleolar proteins and a histone chaperone, among others. Parasites expressing reduced H2B.V levels were associated with higher rates of parasite differentiation and mammalian cell infectivity. Taken together, H2B.V demarcates critical genomic regions and associates with regulatory chromatin proteins, suggesting a scenario wherein local chromatin structures associated with parasite differentiation and invasion are regulated during the parasite life cycle.
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Parásitos , Trypanosoma cruzi , Animales , Cromatina , Histonas/genética , Histonas/metabolismo , Mamíferos , Nucleosomas , Parásitos/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMEN
BACKGROUND: Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria. RESULTS: We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera. CONCLUSIONS: The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.
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Antígenos Virales/inmunología , Infecciones por Birnaviridae/veterinaria , Pollos , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Enfermedades de las Aves de Corral/virología , Proteínas Estructurales Virales/inmunología , Animales , Infecciones por Birnaviridae/epidemiología , Infecciones por Birnaviridae/virología , Brasil/epidemiología , Regulación Viral de la Expresión Génica , Enfermedades de las Aves de Corral/epidemiologíaRESUMEN
Chagas disease (ChD), caused by Trypanosoma cruzi, remains a challenge for the medical and scientific fields due to the inefficiency of the therapeutic approaches available for its treatment. Thiosemicarbazones and hydrazones present a wide spectrum of bioactivities and are considered a platform for the design of new anti-T. cruzi drug candidates. Herein, the potential antichagasic activities of [(E)-2-(1-(4-chlorophenylthio)propan-2-ylidene)-hydrazinecarbothioamides] (C1, C3), [(E)-N'-(1-((4-chlorophenyl)thio)propan-2-ylidene)benzohydrazide] (C2), [(E)-2-(1-(4-, and [(E)-2-(1-((4-chlorophenyl)thio)propan-2-ylidene)hydrazinecarboxamide] (C4) were investigated. Macrophages (MOs) from C57BL/6 mice stimulated with C1 and C3, but not with C2 and C4, reduced amastigote replication and trypomastigote release, independent of nitric oxide (NO) and reactive oxygen species production and indoleamine 2,3-dioxygenase activity. C3, but not C1, reduced parasite uptake by MOs and potentiated TNF production. In cardiomyocytes, C3 reduced trypomastigote release independently of NO, TNF, and IL-6 production. C1 and C3 were non-toxic to the host cells. A reduction of parasite release was found during infection of MOs with trypomastigotes pre-incubated with C1 or C3 and MOs pre-stimulated with compounds before infection. Moreover, C1 and C3 acted directly on trypomastigotes, killing them faster than Benznidazole, and inhibited T. cruzi proliferation at various stages of its intracellular cycle. Mechanistically, C1 and C3 inhibit parasite duplication, and this process cannot be reversed by inhibiting the DNA damage response. In vivo, C1 and C3 attenuated parasitemia in T. cruzi-infected mice. Moreover, C3 loaded in a lipid nanocarrier system (nanoemulsion) maintained anti-T. cruzi activity in vivo. Collectively, these data suggest that C1 and C3 are candidates for the treatment of ChD and present activity in both the host and parasite cells.
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Tiosemicarbazonas/química , Tripanocidas/química , Animales , Supervivencia Celular/efectos de los fármacos , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/patología , Cisteína Endopeptidasas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Diseño de Fármacos , Femenino , Estadios del Ciclo de Vida/efectos de los fármacos , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Conformación Molecular , Óxido Nítrico/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Ratas , Tiosemicarbazonas/farmacología , Tiosemicarbazonas/uso terapéutico , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/fisiologíaRESUMEN
The lipid mediators, platelet-activating factor (PAF) and lysophosphatidylcholine (LPC), play relevant pathophysiological roles in Trypanosoma cruzi infection. Several species of LPC, including C18:1 LPC, which mimics the effects of PAF, are synthesized by T. cruzi. The present study identified a receptor in T. cruzi, which was predicted to bind to PAF, and found it to be homologous to members of the progestin and adiponectin family of receptors (PAQRs). We constructed a three-dimensional model of the T. cruzi PAQR (TcPAQR) and performed molecular docking to predict the interactions of the TcPAQR model with C16:0 PAF and C18:1 LPC. We knocked out T. cruzi PAQR (TcPAQR) gene and confirmed the identity of the expressed protein through immunoblotting and immunofluorescence assays using an anti-human PAQR antibody. Wild-type and knockout (KO) parasites were also used to investigate the in vitro cell differentiation and interactions with peritoneal mouse macrophages; TcPAQR KO parasites were unable to react to C16:0 PAF or C18:1 LPC. Our data are highly suggestive that PAF and LPC act through TcPAQR in T. cruzi, triggering its cellular differentiation and ability to infect macrophages.
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Lisofosfatidilcolinas/metabolismo , Factor de Activación Plaquetaria/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Enfermedad de Chagas/parasitología , Técnicas de Inactivación de Genes/métodos , Interacciones Huésped-Parásitos , Humanos , Lisofosfatidilcolinas/química , Macrófagos , Ratones , Simulación del Acoplamiento Molecular , Filogenia , Factor de Activación Plaquetaria/química , Conformación Proteica , Proteínas Protozoarias/química , Receptores de Adiponectina/química , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Receptores de Progesterona/química , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Trypanosoma cruzi/químicaRESUMEN
DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3α KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.
RESUMEN
DNA topoisomerases are enzymes that modulate DNA topology. Among them, topoisomerase 3α is engaged in genomic maintenance acting in DNA replication termination, sister chromatid separation, and dissolution of recombination intermediates. To evaluate the role of this enzyme in Trypanosoma cruzi, the etiologic agent of Chagas disease, a topoisomerase 3α knockout parasite (TcTopo3α KO) was generated, and the parasite growth, as well as its response to several DNA damage agents, were evaluated. There was no growth alteration caused by the TcTopo3α knockout in epimastigote forms, but a higher dormancy rate was observed. TcTopo3α KO trypomastigote forms displayed reduced invasion rates in LLC-MK2 cells when compared with the wild-type lineage. Amastigote proliferation was also compromised in the TcTopo3α KO, and a higher number of dormant cells was observed. Additionally, TcTopo3α KO epimastigotes were not able to recover cell growth after gamma radiation exposure, suggesting the involvement of topoisomerase 3α in homologous recombination. These parasites were also sensitive to drugs that generate replication stress, such as cisplatin (Cis), hydroxyurea (HU), and methyl methanesulfonate (MMS). In response to HU and Cis treatments, TcTopo3α KO parasites showed a slower cell growth and was not able to efficiently repair the DNA damage induced by these genotoxic agents. The cell growth phenotype observed after MMS treatment was similar to that observed after gamma radiation, although there were fewer dormant cells after MMS exposure. TcTopo3α KO parasites showed a population with sub-G1 DNA content and strong γH2A signal 48 h after MMS treatment. So, it is possible that DNA-damaged cell proliferation due to the absence of TcTopo3α leads to cell death. Whole genome sequencing of MMS-treated parasites showed a significant reduction in the content of the multigene families DFG-1 and RHS, and also a possible erosion of the sub-telomeric region from chromosome 22, relative to non-treated knockout parasites. Southern blot experiments suggest telomere shortening, which could indicate genomic instability in TcTopo3α KO cells owing to MMS treatment. Thus, topoisomerase 3α is important for homologous recombination repair and replication stress in T. cruzi, even though all the pathways in which this enzyme participates during the replication stress response remains elusive.
RESUMEN
Ecto-Nucleoside Triphosphate Diphosphohydrolases are enzymes that hydrolyze tri- and/or diphosphate nucleosides. Evidences pointed out to their participation in Trypanosoma cruzi virulence, infectivity, and purine acquisition. In this study, recombinant T. cruzi knocking out or overexpressing the TcNTPDase-1 gene were built, and the role of TcNTPDase-1 in the in vitro interaction with VERO cells was investigated. Results show that epimastigote forms of hemi-knockout parasites showed about 50% lower level of TcNTPDase-1 gene expression when compared to the wild type, while the T. cruzi overexpressing this gene reach 20 times higher gene expression. In trypomastigote forms, the same decreasing in TcNTPDase-1 gene expression was observed to the hemi-knockout parasites. The in vitro infection assays showed a reduction to 51.6 and 59.9% at the adhesion and to 25.2 and 26.4% at the endocytic indexes to the parasites knockout to one or other allele (Hygro and Neo hemi-knockouts), respectively. In contrast, the infection assays with T. cruzi overexpressing TcNTPDase-1 from the WT or Neo hemi-knockout parasites showed an opposite result, with the increasing to 287.7 and 271.1% at the adhesion and to 220.4 and 186.7% at the endocytic indexes, respectively. The parasitic load estimated in infected VERO cells by quantitative real time PCR corroborated these findings. Taken together, the partial silencing and overexpression of the TcNTPDase-1 gene generated viable parasites with low and high infectivity rates, respectively, corroborating that the enzyme encoded for this gene plays an important role to the T. cruzi infectivity.
RESUMEN
The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects around 8 million people worldwide. Chagas disease can be divided into two stages: an acute stage with high parasitemia followed by a low parasitemia chronic stage. Recently, the importance of dormancy concerning drug resistance in T. cruzi amastigotes has been shown. Here, we quantify the percentage of dormant parasites from different T. cruzi DTUs during their replicative epimastigote and amastigote stages. For this study, cells of T. cruzi CL Brener (DTU TcVI); Bug (DTU TcV); Y (DTU TcII); and Dm28c (DTU TcI) were used. In order to determine the proliferation rate and percentage of dormancy in epimastigotes, fluorescent-labeled cells were collected every 24 h for flow cytometer analysis, and cells showing maximum fluorescence after 144 h of growth were considered dormant. For the quantification of dormant amastigotes, fluorescent-labeled trypomastigotes were used for infection of LLC-MK2 cells. The number of amastigotes per infected LLC-MK2 cell was determined, and those parasites that presented fluorescent staining after 96 h of infection were considered dormant. A higher number of dormant cells was observed in hybrid strains when compared to non-hybrid strains for both epimastigote and amastigote forms. In order to investigate, the involvement of homologous recombination in the determination of dormancy in T. cruzi, we treated CL Brener cells with gamma radiation, which generates DNA lesions repaired by this process. Interestingly, the dormancy percentage was increased in gamma-irradiated cells. Since, we have previously shown that naturally-occurring hybrid T. cruzi strains present higher transcription of RAD51-a key gene in recombination process -we also measured the percentage of dormant cells from T. cruzi clone CL Brener harboring single knockout for RAD51. Our results showed a significative reduction of dormant cells in this T. cruzi CL Brener RAD51 mutant, evidencing a role of homologous recombination in the process of dormancy in this parasite. Altogether, our data suggest the existence of an adaptive difference between T. cruzi strains to generate dormant cells, and that homologous recombination may be important for dormancy in this parasite.
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Recombinación Homóloga , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiología , Animales , Línea Celular , Macaca mulatta , Mutación , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Protozoario/genética , Recombinasa Rad51/genética , Especificidad de la Especie , Trypanosoma cruzi/citología , Trypanosoma cruzi/crecimiento & desarrolloRESUMEN
Replication protein A (RPA), a heterotrimeric complex, is the major single-stranded DNA binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a nuclear export signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.
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Núcleo Celular/metabolismo , Estadios del Ciclo de Vida , Morfogénesis , Proteína de Replicación A/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Simulación por Computador , Citoplasma/metabolismo , Morfogénesis/genética , Señales de Exportación Nuclear/genética , Señales de Exportación Nuclear/fisiología , Proteína de Replicación A/genética , Trypanosoma cruzi/citologíaRESUMEN
Replication Protein A (RPA), a heterotrimeric complex, is the major single-stranded DNA-binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a Nuclear Export Signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.
RESUMEN
Replication Protein A (RPA), a heterotrimeric complex, is the major single-stranded DNA-binding protein in eukaryotes. Recently, we characterized RPA from Trypanosoma cruzi, showing that it is involved in DNA replication and DNA damage response in this organism. Better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms was observed in TcRPA-2 subunit heterozygous knockout cells, suggesting that RPA is involved in this process. Here, we show that RPA cellular localization changes during the T. cruzi life cycle, with RPA being detected only in the cytoplasm of the metacyclic and bloodstream trypomastigotes. We also identify a Nuclear Export Signal (NES) in the trypanosomatid RPA-2 subunit. Mutations in the negatively charged residues of RPA-2 NES impair the differentiation process, suggesting that RPA exportation affects parasite differentiation into infective forms.
RESUMEN
DNA polymerase theta (Polθ), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Polθ plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Polθ modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Polθ in different genomic loci; one ortholog is homologous to the Polθ C-terminal polymerase domain, and the other is homologous to the Polθ helicase domain, called Polθ-polymerase and Polθ-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Polθ-helicase from the nuclear extract. Orc1/Cdc6 and Polθ-helicase directly interacted, and Polθ-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Polθ-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Polθ-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.
Asunto(s)
Cromatina/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/crecimiento & desarrollo , Cromatina/genética , ADN Helicasas/genética , ADN Polimerasa Dirigida por ADN/genética , Humanos , Complejo de Reconocimiento del Origen/genética , Proteínas Protozoarias/genética , Origen de Réplica , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , ADN Polimerasa thetaRESUMEN
DNA polymerase theta (Pol theta), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Pol theta plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Pol theta modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Pol theta in different genomic loci; one ortholog is homologous to the Pol theta C-terminal polymerase domain, and the other is homologous to the Pol theta helicase domain, called Pol theta-polymerase and Pol theta-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Pol theta-helicase from the nuclear extract. Orc1/Cdc6 and Pol theta-helicase directly interacted, and Pol theta-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Pol theta-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Pol theta-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.
RESUMEN
DNA polymerase theta (Pol theta), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Pol theta plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Pol theta modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Pol theta in different genomic loci; one ortholog is homologous to the Pol theta C-terminal polymerase domain, and the other is homologous to the Pol theta helicase domain, called Pol theta-polymerase and Pol theta-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Pol theta-helicase from the nuclear extract. Orc1/Cdc6 and Pol theta-helicase directly interacted, and Pol theta-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Pol theta-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Pol theta-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins.