RESUMO
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.
Assuntos
Galinhas , Ensaio de Imunoadsorção Enzimática , Orthoreovirus Aviário , Doenças das Aves Domésticas , Proteínas Recombinantes , Infecções por Reoviridae , Animais , Orthoreovirus Aviário/imunologia , Orthoreovirus Aviário/genética , Orthoreovirus Aviário/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Reoviridae/veterinária , Infecções por Reoviridae/diagnóstico , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/diagnóstico , Proteínas Recombinantes/imunologia , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Proteínas Virais/imunologia , Proteínas Virais/genéticaRESUMO
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.
Assuntos
Proteínas de Fluorescência Verde , Tripanossomicidas , Trypanosoma cruzi , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Proteínas de Fluorescência Verde/genética , Tripanossomicidas/farmacologia , Nitroimidazóis/farmacologia , Testes de Sensibilidade Parasitária , Animais , Concentração Inibidora 50 , Avaliação Pré-Clínica de Medicamentos , Sobrevivência Celular/efeitos dos fármacosRESUMO
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.
RESUMO
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.
Assuntos
Leishmania mexicana , Leishmania , Leishmaniose Cutânea , Humanos , Animais , Camundongos , Epitopos , Antígenos de Histocompatibilidade Classe I , Antígenos HLA , Leishmania/metabolismo , Peptídeos/química , Vacinas de Subunidades Antigênicas , Complexo Principal de HistocompatibilidadeRESUMO
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.
Assuntos
Parasitos , Trypanosoma cruzi , Animais , Cromatina , Histonas/genética , Histonas/metabolismo , Mamíferos , Nucleossomos , Parasitos/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMO
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.
Assuntos
Antígenos Virais/imunologia , Infecções por Birnaviridae/veterinária , Galinhas , Vírus da Doença Infecciosa da Bursa/genética , Doenças das Aves Domésticas/virologia , Proteínas Estruturais Virais/imunologia , Animais , Infecções por Birnaviridae/epidemiologia , Infecções por Birnaviridae/virologia , Brasil/epidemiologia , Regulação Viral da Expressão Gênica , Doenças das Aves Domésticas/epidemiologiaRESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Recombinação Homóloga , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologia , Animais , Linhagem Celular , Macaca mulatta , Mutação , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , Rad51 Recombinase/genética , Especificidade da Espécie , Trypanosoma cruzi/citologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
In the protozoan parasite Trypanosoma cruzi - the causative agent of Chagas disease - gene expression control is mainly post-transcriptional, where RNA-binding proteins (RBPs) play a central role, by controlling mRNA stability, distribution and translation. A large variety of RBPs are encoded in the T. cruzi genome, including the CCCH-type zinc finger (CCCH ZnF) protein family, which is characterized by the presence of the C-X7/8-C-X5-C-X3-H (CCCH) motif. In the related parasite T. brucei, CCCH ZnF proteins have been shown to control key differentiation steps in the parasite's life cycle. However, little is known about the CCCH ZnF proteins in T. cruzi. We have worked on the generation of T. cruzi mutants for CCCH ZnF proteins in an effort to shed light on the functions of these proteins in this parasite. Here, we characterize the expression and function of the CCCH ZnF protein TcZC3H31 of T. cruzi. TcZC3H31 is almost exclusively expressed in epimastigotes and metacyclic trypomastigotes, the parasite forms found in the invertebrate host. Importantly, we show that the epimastigote form of the T. cruzi knockout for the TcZC3H31 gene (TcZC3H31 KO) is incapable, both in vitro and in vivo (in infected triatomine insects), to differentiate into the metacyclic trypomastigote form, which is responsible for infection transmission from vectors to humans. The epimastigote forms recovered from the excreta of insects infected with TcZC3H31 KO parasites do not have the typical epimastigote morphology, suggesting that parasites are arrested in a mid-differentiation step. Also, epimastigotes overexpressing TcZC3H31 differentiate into metacyclics more efficiently than wild-type epimastigotes, in vitro. These data suggest that TcZC3H31 is an essential positive regulator of T. cruzi differentiation into the human-infective metacyclic form.
Assuntos
Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trypanosoma cruzi/citologia , Trypanosoma cruzi/crescimento & desenvolvimento , Dedos de Zinco , Animais , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Insetos , Proteínas de Protozoários/genética , Proteínas de Ligação a RNA/genética , Trypanosoma cruzi/genéticaRESUMO
BACKGROUND: Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. RESULTS: A wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli. For immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2, fagA, fagB, NlpC/P60 protein family and LPxTG putative protein family. CONCLUSION: Proteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.
Assuntos
Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/veterinária , Animais , Antígenos de Bactérias/sangue , Bacteriófagos/genética , Sequência de Bases , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/patogenicidade , Bases de Dados de Ácidos Nucleicos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Genes Bacterianos/genética , Genoma Bacteriano , Doenças das Cabras/sangue , Doenças das Cabras/imunologia , Doenças das Cabras/microbiologia , Cabras , Linfadenite/imunologia , Linfadenite/microbiologia , Reação em Cadeia da Polimerase/veterinária , Ovinos , Doenças dos Ovinos/sangue , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Vacinas de DNA/uso terapêuticoRESUMO
The AP-1 Adaptor Complex assists clathrin-coated vesicle assembly in the trans-Golgi network (TGN) of eukaryotic cells. However, the role of AP-1 in the protozoan Trypanosoma cruzi-the Chagas disease parasite-has not been addressed. Here, we studied the function and localization of AP-1 in different T. cruzi life cycle forms, by generating a gene knockout of the large AP-1 subunit gamma adaptin (TcAP1-γ), and raising a monoclonal antibody against TcAP1-γ. Co-localization with a Golgi marker and with the clathrin light chain showed that TcAP1-γ is located in the Golgi, and it may interact with clathrin in vivo, at the TGN. Epimastigote (insect form) parasites lacking TcAP1-γ (TcγKO) have reduced proliferation and differentiation into infective metacyclic trypomastigotes (compared with wild-type parasites). TcγKO parasites have also displayed significantly reduced infectivity towards mammalian cells. Importantly, TcAP1-γ knockout impaired maturation and transport to lysosome-related organelles (reservosomes) of a key cargo-the major cysteine protease cruzipain, which is important for parasite nutrition, differentiation and infection. In conclusion, the defective processing and transport of cruzipain upon AP-1 ablation may underlie the phenotype of TcγKO parasites.
Assuntos
Doença de Chagas/parasitologia , Cisteína Endopeptidases/química , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/fisiologia , Trypanosoma cruzi/genética , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/química , Vesículas Revestidas por Clatrina , Endocitose , Teste de Complementação Genética , Complexo de Golgi/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Organelas , Plasmídeos/metabolismo , Proteínas de Protozoários , Proteínas Recombinantes/química , Rede trans-Golgi/metabolismoRESUMO
BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.
Assuntos
Vírus de RNA/genética , Hepatite C/diagnóstico , Hepacivirus/genética , Escherichia coli/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Levivirus/genética , Modelos BiológicosRESUMO
BACKGROUND: Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES: The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS: The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS: We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.
Assuntos
Hepatite C/diagnóstico , Levivirus/genética , Vírus de RNA/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Escherichia coli/genética , Hepacivirus/genética , Modelos Biológicos , Padrões de ReferênciaRESUMO
Replication Protein A (RPA), the major single stranded DNA binding protein in eukaryotes, is composed of three subunits and is a fundamental player in DNA metabolism, participating in replication, transcription, repair, and the DNA damage response. In human pathogenic trypanosomatids, only limited studies have been performed on RPA-1 from Leishmania. Here, we performed in silico, in vitro and in vivo analysis of Trypanosoma cruzi RPA-1 and RPA-2 subunits. Although computational analysis suggests similarities in DNA binding and Ob-fold structures of RPA from T. cruzi compared with mammalian and fungi RPA, the predicted tridimensional structures of T. cruzi RPA-1 and RPA-2 indicated that these molecules present a more flexible tertiary structure, suggesting that T. cruzi RPA could be involved in additional responses. Here, we demonstrate experimentally that the T. cruzi RPA complex interacts with DNA via RPA-1 and is directly related to canonical functions, such as DNA replication and DNA damage response. Accordingly, a reduction of TcRPA-2 expression by generating heterozygous knockout cells impaired cell growth, slowing down S-phase progression. Moreover, heterozygous knockout cells presented a better efficiency in differentiation from epimastigote to metacyclic trypomastigote forms and metacyclic trypomastigote infection. Taken together, these findings indicate the involvement of TcRPA in the metacyclogenesis process and suggest that a delay in cell cycle progression could be linked with differentiation in T. cruzi.
Assuntos
Diferenciação Celular , DNA de Protozoário/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteína de Replicação A/química , Proteína de Replicação A/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Doença de Chagas , Simulação por Computador , DNA de Cadeia Simples/metabolismo , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteína de Replicação A/genética , Proteína de Replicação A/isolamento & purificação , Trypanosoma cruzi/genéticaRESUMO
The widespread occurrence of anthelmintic-resistant gastrointestinal nematodes (GINs), particularly Haemonchus contortus, in sheep production systems has magnified the need to identify and develop alternative control strategies. Strategies include the selection of genetically GIN-resistant sheep and the implementation of biological parasite control to reduce dependence on anthelmintic drugs. In this study, we aimed to establish the molecular identity of bacterial communities present in the abomasum of sheep classified as resistant or susceptible to H. contortus. Thirty-eight sheep were experimentally infected with L3 Haemonchus contortus and analyzed for fecal egg count (FEC), and hematocrit (Ht) to establish haemonchosis resistance or susceptibility. Four resistant sheep (RS) and four susceptible sheep (SS) were selected for microbial sampling and subsequent phylogenetic analysis. Molecular identification of the bacteria was based on amplification of the bacterial 16S rRNA gene, construction of a 16S rDNA clone library, and subsequent gene sequencing. Significant differences (p = 0.05) were observed in the occurrence of different phyla identified in RS and SS libraries: Firmicutes (61.4% and 37.2%, respectively), Proteobacteria (10.2% and 37.2%, respectively), Bacteroidetes (12.8% and 5.8%, respectively), and unclassified bacteria (12.8% and 17%, respectively). Differences between the proportions of bacterial communities present in the RS and SS pool samples were observed, contributing as a first step toward the assessment of the association between the gastrointestinal tract microbiota and nematode resistance in sheep.(AU)
Na produção de ovinos, a disseminação de nematódeos gastrintestinais (NGI) resistentes aos antihelmínticos, em especial Haemonchus contortus, tem levado à busca de estratégias de controle alternativo, como a seleção de animais geneticamente resistentes aos NGI e o controle biológico. Neste trabalho, buscou-se identificar molecularmente as comunidades bacterianas presentes no abomaso de animais classificados como resistentes ou susceptíveis ao H. contortus. Foram utilizados 38 ovinos para classificação em resistentes ou susceptíveis à hemoncose, por meio de infecções experimentais com L3 de H. contortus e posterior análise de variações na contagem de ovos por grama de fezes (ΔOPG) e hematócrito (ΔHt). Destes, foram selecionados para colheita de material e posterior análise filogenética, quatro ovinos resistentes (OR) e quatro susceptíveis (OS). A identificação molecular de bactérias foi realizada por técnicas moleculares a partir da amplificação do gene RNAr 16S bacteriano, construção de bibliotecas de clones de RNAr 16S e posterior sequenciamento gênico. O trabalho mostrou diferença significativa (p=0,05) na porcentagem dos filos predominantes para as bibliotecas OR e OS, respectivamente: Firmicutes (61,4% e 37,2%), Proteobacterias (10,2% e 37,2%), Bacteroidetes (12,8% e 5,8%) e Bactérias não classificadas (12,8% e 17%). Diferenças entre os pools OR e OS com relação à proporção de comunidades bacterianas presentes podem ser observadas, sendo o primeiro passo para que se possa avaliar a relação entre a microbiota do trato gastrointestinal e a resistência a parasitos.(AU)
Assuntos
Animais , Ovinos/parasitologia , Gastroenteropatias/parasitologia , Haemonchus , Bactérias/genética , RNA Ribossômico , Bacillus thuringiensis , Trichostrongylus , Controle Biológico de VetoresRESUMO
The widespread occurrence of anthelmintic-resistant gastrointestinal nematodes (GINs), particularly Haemonchus contortus, in sheep production systems has magnified the need to identify and develop alternative control strategies. Strategies include the selection of genetically GIN-resistant sheep and the implementation of biological parasite control to reduce dependence on anthelmintic drugs. In this study, we aimed to establish the molecular identity of bacterial communities present in the abomasum of sheep classified as resistant or susceptible to H. contortus. Thirty-eight sheep were experimentally infected with L3 Haemonchus contortus and analyzed for fecal egg count (FEC), and hematocrit (Ht) to establish haemonchosis resistance or susceptibility. Four resistant sheep (RS) and four susceptible sheep (SS) were selected for microbial sampling and subsequent phylogenetic analysis. Molecular identification of the bacteria was based on amplification of the bacterial 16S rRNA gene, construction of a 16S rDNA clone library, and subsequent gene sequencing. Significant differences (p = 0.05) were observed in the occurrence of different phyla identified in RS and SS libraries: Firmicutes (61.4% and 37.2%, respectively), Proteobacteria (10.2% and 37.2%, respectively), Bacteroidetes (12.8% and 5.8%, respectively), and unclassified bacteria (12.8% and 17%, respectively). Differences between the proportions of bacterial communities present in the RS and SS pool samples were observed, contributing as a first step toward the assessment of the association between the gastrointestinal tract microbiota and nematode resistance in sheep.
Na produção de ovinos, a disseminação de nematódeos gastrintestinais (NGI) resistentes aos antihelmínticos, em especial Haemonchus contortus, tem levado à busca de estratégias de controle alternativo, como a seleção de animais geneticamente resistentes aos NGI e o controle biológico. Neste trabalho, buscou-se identificar molecularmente as comunidades bacterianas presentes no abomaso de animais classificados como resistentes ou susceptíveis ao H. contortus. Foram utilizados 38 ovinos para classificação em resistentes ou susceptíveis à hemoncose, por meio de infecções experimentais com L3 de H. contortus e posterior análise de variações na contagem de ovos por grama de fezes (ΔOPG) e hematócrito (ΔHt). Destes, foram selecionados para colheita de material e posterior análise filogenética, quatro ovinos resistentes (OR) e quatro susceptíveis (OS). A identificação molecular de bactérias foi realizada por técnicas moleculares a partir da amplificação do gene RNAr 16S bacteriano, construção de bibliotecas de clones de RNAr 16S e posterior sequenciamento gênico. O trabalho mostrou diferença significativa (p=0,05) na porcentagem dos filos predominantes para as bibliotecas OR e OS, respectivamente: Firmicutes (61,4% e 37,2%), Proteobacterias (10,2% e 37,2%), Bacteroidetes (12,8% e 5,8%) e Bactérias não classificadas (12,8% e 17%). Diferenças entre os pools OR e OS com relação à proporção de comunidades bacterianas presentes podem ser observadas, sendo o primeiro passo para que se possa avaliar a relação entre a microbiota do trato gastrointestinal e a resistência a parasitos.
Assuntos
Animais , Bacillus thuringiensis , Bactérias/genética , Gastroenteropatias/parasitologia , Haemonchus , Ovinos/parasitologia , RNA Ribossômico , Trichostrongylus , Controle Biológico de VetoresRESUMO
The Trypanosoma cruzi adenylyl cyclase (AC) multigene family encodes different isoforms (around 15) sharing a variable large N-terminal domain, which is extracellular and receptor-like, followed by a transmembrane helix and a conserved C-terminal catalytic domain. It was proposed that these key enzymes in the cAMP signalling pathway allow the parasite to sense its changing extracellular milieu in order to rapidly adapt to its new environment, which is generally achieved through a differentiation process. One of the critical differentiation events the parasitic protozoan T. cruzi undergoes during its life cycle, known as metacyclogenesis, occurs in the digestive tract of the insect and corresponds to the differentiation from noninfective epimastigotes to infective metacyclic trypomastigote forms. By in vitro monitoring the activity of AC during metacyclogenesis, we showed that both the activity of AC and the intracellular cAMP content follow a similar pattern of transient stimulation in a two-step process, with a first activation peak occurring during the first hours of nutritional stress and a second peak between 6 and 48 h, corresponding to the cellular adhesion. During this differentiation process, a general mechanism of upregulation of AC expression of both mRNA and protein is triggered and in particular for a major subclass of these enzymes that are present in various gene copies commonly associated to the THT gene clusters. Although the scattered genome distribution of these gene copies is rather unusual in trypanosomatids and seems to be a recent acquisition in the evolution of the T. cruzi clade, their encoded product redistributed on the flagellum of the parasite upon differentiation could be important to sense the extracellular milieu.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Adenilil Ciclases/química , Adenilil Ciclases/genética , Sequência de Aminoácidos , Animais , Doença de Chagas/parasitologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Família Multigênica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Regulação para CimaRESUMO
Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the “culture PCR” approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.