DNA metabolism in Trypanosoma cruzi: mitochondrial repair pathways and hybrid formation

Submitted by Bruno Repoles ([email protected]) on 2020-02-10T18:13:02Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: cfd6801dba008cb6adbd9838b81582ab (MD5) Tese Final Bruno Repoles.pdf: 8337600 bytes, checksum: 3bdf339b1e4fbae650cd0ba18cdb36b1 (MD5)Approved for entry into archive by Camila Silva ([email protected]) on 2020-02-27T18:16:50Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: cfd6801dba008cb6adbd9838b81582ab (MD5) Tese Final Bruno Repoles.pdf: 8337600 bytes, checksum: 3bdf339b1e4fbae650cd0ba18cdb36b1 (MD5)Made available in DSpace on 2020-03-04T19:27:17Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: cfd6801dba008cb6adbd9838b81582ab (MD5) Tese Final Bruno Repoles.pdf: 8337600 bytes, checksum: 3bdf339b1e4fbae650cd0ba18cdb36b1 (MD5) Previous issue date: 2019-09-27CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas GeraisCAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorTrypanosoma cruzi é um membro da ordem Kinetoplastida e, como tal, possui uma mitocôndria única e modificada, chamada de cinetoplasto. O cinetoplasto possui diversas características únicas em comparação com a mitocôndria de outros eucariotos. Apesar de diversas proteínas de sistemas de reparo de DNA já terem sido descritas como presentes na mitocôndria destes parasitos, não existem muitas informações sobre as vias de reparo que estão envolvidas na manutenção do DNA do cinetoplasto (kDNA). Neste trabalho investigamos possíveis vias de reparo de DNA que podem estar envolvidas com o metabolismo do kDNA de T. cruzi. Estudamos o fenótipo de uma cepa superexpressora da proteína do reparo por excisão de bases TcMYH. Foi visto, por PCR quantitativa, que este superexpressor possui mais danos no kDNA no primeiro momento, em comparação com a cepa selvagem. Foi visto também, utilizando um reagente desenhado para causar danos especificamente no DNA mitocondrial, que este mutante é mais sensível que a célula selvagem e que esta superexpressão gera mais sítios AP no DNA total do parasito. Estes resultados sugerem que o parasita é capaz de lidar com dano oxidativo em seu kDNA. Uma das características únicas do cinetoplasto é a presença de proteínas específicas chamadas de proteínas associadas ao cinetoplasto (KAP). Apesar de alguns trabalhos descreverem a localização destas proteínas nos sítios antipodais, regiões na qual vários processos do metabolismo do kDNA ocorrem, suas funções não são claras ainda. Neste trabalho também estudamos a função da proteína TcKAP7, que possui homologia com o fator de transcrição mitocondrial A (TFAM). Nossos resultados demonstraram que a falta de TcKAP7 está relacionada com a sensibilidade à radiação UV e cisplatina, além de resistência a altos níveis de estresse oxidativo no parasita. Este mesmo fenótipo de sensibilidade foi observado em heminocautes de um homólogo de TcKAP7 em outro tripanossomatídeo, o parasita de insetos Angomonas deanei. Nossos resultados demonstram que TcKAP7 está envolvida na resposta ao dano de DNA mitocondrial. Além de investigar os mecanismos de reparo do kDNA, neste trabalho também investigamos a troca de material genético em diferentes cepas de T. cruzi. Nossos resultados demonstram que T. cruzi é capaz de realizar troca de material genético dentro de uma população e que, as diversas cepas distintas, são capazes de trocar material genético entre elas. Esta troca também parece estar diretamente ligada ao nível de TcRad51 nestas cepas. Estes resultados mostram que este parasita é capaz de trocar material genético e que a recombinação homóloga pode ter papel fundamental neste processo.Trypanosoma cruzi is a member of the Kinetoplastida and as so, present a single and unique mitochondria, called kinetoplast, which possesses several distinct features in comparison with other eukaryotes mitochondria. Although several proteins of the DNA repair systems have already being described as present on the parasite mitochondria, there’s not much information about the DNA repair pathways involved on the kinetoplast DNA (kDNA) maintenance. In this work we investigate possible DNA repair pathways that could be involved on T. cruzi kDNA metabolism. To first investigate the repair proteins related to the kDNA metabolism we analyzed the effect of overexpressing the protein TcMYH, which is involved in base excision DNA repair in T. cruzi. As shown by qPCR, the overexpressor strain of TcMYH has more damages on the kDNA at the first moment, when compared to the wild type strain. Also, when we used a reagent designed to specifically cause damage on the mitochondrial DNA, we could observe that this mutant is more sensitive than the WT control strain, and that the overexpression of the protein generates more AP sites on the parasite organelle. These results suggest that the parasite is able to deal with oxidative damage that attacks the kDNA. One of the unique features of the kinetoplast is the presence of specific proteins called kinetoplast associated proteins (KAP). Although some works described the location of those proteins on the antipodal sites, regions of DNA metabolism on the kinetoplast, their function are not yet clear. In this work we study the function of the proteinTcKAP7, which has sequence homology with the Mitochondrial transcription factor A (TFAM). Our results demonstrate that the absence of TcKAP7 is related to a long-term sensitivity to UV radiation and cisplatin; and a resistance to high levels oxidative stress on the parasite. The same phenotype has been observed for Angomonas deanei mutants, another trypanossomatid and an insect parasite. In this work we also investigate the genetic exchange events in different T. cruzi strains. Our results demonstrate that T. cruzi is able to perform genetic exchange among individuals in a same population. Also, the variety of T. cruzi strains are able to perform genetic exchange among them. This exchange is also linked to the levels of TcRad51 on these strains. Together, these results demonstrate that this parasite is able to perform genetic exchange and that the homologous recombination could be linked to this process

Mitochondrial DNA Instability in Mammalian Cells

Significance: The small, multicopy mitochondrial genome (mitochondrial DNA [mtDNA]) is essential for efficient energy production, as alterations in its coding information or a decrease in its copy number disrupt mitochondrial ATP synthesis. However, the mitochondrial replication machinery encounters numerous challenges that may limit its ability to duplicate this important genome and that jeopardize mtDNA stability, including various lesions in the DNA template, topological stress, and an insufficient nucleotide supply. Recent Advances: An ever-growing array of DNA repair or maintenance factors are being reported to localize to the mitochondria. We review current knowledge regarding the mitochondrial factors that may contribute to the tolerance or repair of various types of changes in the mitochondrial genome, such as base damage, incorporated ribonucleotides, and strand breaks. We also discuss the newly discovered link between mtDNA instability and activation of the innate immune response. Critical Issues: By which mechanisms do mitochondria respond to challenges that threaten mtDNA maintenance? What types of mtDNA damage are repaired, and when are the affected molecules degraded instead? And, finally, which forms of mtDNA instability trigger an immune response, and how? Future Directions: Further work is required to understand the contribution of the DNA repair and damage-tolerance factors present in the mitochondrial compartment, as well as the balance between mtDNA repair and degradation. Finally, efforts to understand the events underlying mtDNA release into the cytosol are warranted. Pursuing these and many related avenues can improve our understanding of what goes wrong in mitochondrial disease

The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method

The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues

Assessment of genetic mutation frequency induced by oxidative stress in Trypanosoma cruzi

Abstract Trypanosoma cruzi is the etiological agent of Chagas disease, a public health challenge due to its morbidity and mortality rates, which affects around 6-7 million people worldwide. Symptoms, response to chemotherapy, and the course of Chagas disease are greatly influenced by T. cruzi‘s intra-specific variability. Thus, DNA mutations in this parasite possibly play a key role in the wide range of clinical manifestations and in drug sensitivity. Indeed, the environmental conditions of oxidative stress faced by T. cruzi during its life cycle can generate genetic mutations. However, the lack of an established experimental design to assess mutation rates in T. cruzi precludes the study of conditions and mechanisms that potentially produce genomic variability in this parasite. We developed an assay that employs a reporter gene that, once mutated in specific positions, convert G418-sensitive into G418-insenstitive T. cruzi. We were able to determine the frequency of DNA mutations in T. cruzi exposed and non-exposed to oxidative insults assessing the number of colony-forming units in solid selective media after plating a defined number of cells. We verified that T. cruzi‘s spontaneous mutation frequency was comparable to those found in other eukaryotes, and that exposure to hydrogen peroxide promoted a two-fold increase in T. cruzi‘s mutation frequency. We hypothesize that genetic mutations in T. cruzi can arise from oxidative insults faced by this parasite during its life cycle

Assessment of genetic mutation frequency induced by oxidative stress in Trypanosoma cruzi

<div><p>Abstract Trypanosoma cruzi is the etiological agent of Chagas disease, a public health challenge due to its morbidity and mortality rates, which affects around 6-7 million people worldwide. Symptoms, response to chemotherapy, and the course of Chagas disease are greatly influenced by T. cruzi‘s intra-specific variability. Thus, DNA mutations in this parasite possibly play a key role in the wide range of clinical manifestations and in drug sensitivity. Indeed, the environmental conditions of oxidative stress faced by T. cruzi during its life cycle can generate genetic mutations. However, the lack of an established experimental design to assess mutation rates in T. cruzi precludes the study of conditions and mechanisms that potentially produce genomic variability in this parasite. We developed an assay that employs a reporter gene that, once mutated in specific positions, convert G418-sensitive into G418-insenstitive T. cruzi. We were able to determine the frequency of DNA mutations in T. cruzi exposed and non-exposed to oxidative insults assessing the number of colony-forming units in solid selective media after plating a defined number of cells. We verified that T. cruzi‘s spontaneous mutation frequency was comparable to those found in other eukaryotes, and that exposure to hydrogen peroxide promoted a two-fold increase in T. cruzi‘s mutation frequency. We hypothesize that genetic mutations in T. cruzi can arise from oxidative insults faced by this parasite during its life cycle.</p></div

Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response

Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas disease. Like most living organisms, it is susceptible to oxidative stress, and must adapt to distinct environments. Hence, DNA repair is essential for its survival and the persistence of infection. Therefore, we studied whether T. cruzi has a homolog counterpart of the MutY enzyme (TcMYH), important in the DNA Base Excision Repair (BER) mechanism. Analysis of T. cruzi genome database showed that this parasite has a putative MutY DNA glycosylase sequence. We performed heterologous complementation assays using this genomic sequence. TcMYH complemented the Escherichia coli MutY-strain, reducing the mutation rate to a level similar to wild type. In in vitro assays, TcMYH was able to remove an adenine that was opposite to 8-oxoguanine. We have also constructed a T. cruzi lineage that over-expresses MYH. Although in standard conditions this lineage has similar growth to control cells, the over-expressor is more sensitive to hydrogen peroxide and glucose oxidase than the control, probably due to accumulation of AP sites in its DNA. Localization experiments with GFP-fused TcMYH showed this enzyme is present in both nucleus and mitochondrion. QPCR and MtOX results reinforce the presence and function of TcMYH in these two organelles. Our data suggest T. cruzi has a functional MYH DNA glycosylase, which participates in nuclear and mitochondrial DNA Base Excision Repair.FONDECYT (Chile) 1130113 ERANET-LAC ELAC2014/HID-0328 CNPq (Brazil) 444334/2014-9 FAPEMIG (Brazil) APQ-00827-15 APQ-02308-1

The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi

Submitted by Nuzia Santos ([email protected]) on 2019-06-24T16:52:14Z No. of bitstreams: 1 The recombinase Rad51.pdf: 7462652 bytes, checksum: d2c0b2a73c5ee203c2681bcf6f90618e (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2019-06-24T16:57:35Z (GMT) No. of bitstreams: 1 The recombinase Rad51.pdf: 7462652 bytes, checksum: d2c0b2a73c5ee203c2681bcf6f90618e (MD5)Made available in DSpace on 2019-06-24T16:57:35Z (GMT). No. of bitstreams: 1 The recombinase Rad51.pdf: 7462652 bytes, checksum: d2c0b2a73c5ee203c2681bcf6f90618e (MD5) Previous issue date: 2018Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Instituto Butantan. Centro de Toxinas, Resposta Imune e Sinalização Celular. Laboratório Especial de Ciclo Celular. São Paulo, SP, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Instituto Butantan. Centro de Toxinas, Resposta Imune e Sinalização Celular. Laboratório Especial de Ciclo Celular. São Paulo, SP, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG Brazil.Universidade Federal de Minas Gerais. Departamento de Morfologia. Belo Horizonte, MG, Brazil.Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Instituto Butantan. Centro de Toxinas, Resposta Imune e Sinalização Celular. Laboratório Especial de Ciclo Celular. São Paulo, SP, Brazil.Universidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, Brazil.Detection of genetic exchange has been a limiting factor to deepen the knowledge on the mechanisms by which Trypanosoma cruzi is able to generate progeny and genetic diversity. Here we show that incorporation of halogenated thymidine analogues, followed by immunostaining, is a reliable method not only to detect T. cruzi fused-cell hybrids, but also to quantify their percentage in populations of this parasite. Through this approach, we were able to detect and quantify fused-cell hybrids of T. cruzi clones CL Brener and Y. Given the increased detection of fused-cell hybrids in naturally-occurring hybrid CL Brener strain, which displays increased levels of RAD51 and BRCA2 transcripts, we further investigated the role of Rad51 – a recombinase involved in homologous recombination – in the process of genetic exchange. We also verified that the detection of fused-cell hybrids in T. cruzi overexpressing RAD51 is increased when compared to wild-type cells, suggesting a key role for Rad51 either in the formation or in the stabilization of fused-cell hybrids in this organism

The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions.

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite