ABSTRACT
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.
Subject(s)
Ataxia Telangiectasia Mutated Proteins , DNA Damage , DNA Repair , R-Loop Structures , Transcription, Genetic , Trypanosoma cruzi , Ultraviolet Rays , Trypanosoma cruzi/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/genetics , Protozoan Proteins/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , Cell Death , Apoptosis , HumansABSTRACT
In hominids, including Homo sapiens, uric acid is the end product of purine catabolism. In contrast, other placental mammals further degrade uric acid to (S)-allantoin by enzymes such as urate oxidase (uricase), HIU hydrolase (HIUase), and OHCU decarboxylase. Some organisms, such as frogs and fish, hydrolyze (S)-allantoin to allantoate and eventually to (S)-ureidoglycolate and urea, while marine invertebrates convert urea to ammonium. In H. sapiens, mutations in the uricase gene led to a reduction in the selective pressure for maintaining the integrity of the genes encoding the other enzymes of the purine catabolism pathway, resulting in an accumulation of uric acid. The hyperuricemia resulting from this accumulation is associated with gout, cardiovascular disease, diabetes, and preeclampsia. Many commonly used drugs, such as aspirin, can also increase uric acid levels. Despite the apparent absence of these enzymes in H. sapiens, there appears to be production of transcripts for uricase (UOX), HIUase (URAHP), OHCU decarboxylase (URAD), and allantoicase (ALLC). While some URAHP transcripts are classified as long non-coding RNAs (lncRNAs), URAD and ALLC produce protein-coding transcripts. Given the presence of these transcripts in various tissues, we hypothesized that they may play a role in the regulation of purine catabolism and the pathogenesis of diseases associated with hyperuricemia. Here, we specifically investigate the unique aspects of purine catabolism in H. sapiens, the effects mutations of the uricase gene, and the potential regulatory role of the corresponding transcripts. These findings open new avenues for research and therapeutic approaches for the treatment of hyperuricemia and related diseases.
ABSTRACT
RNA processing is a highly conserved mechanism that serves as a pivotal regulator of gene expression. Alternative processing generates transcripts that can still be translated but lead to potentially nonfunctional proteins. A plethora of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), strategically manipulate the host's RNA processing machinery to circumvent antiviral responses. We integrated publicly available omics datasets to systematically analyze isoform-level expression and delineate the nascent peptide landscape of SARS-CoV-2-infected human cells. Our findings explore a suggested but uncharacterized mechanism, whereby SARS-CoV-2 infection induces the predominant expression of unproductive splicing isoforms in key IFN signaling, interferon-stimulated (ISGs), class I MHC, and splicing machinery genes, including IRF7, HLA-B, and HNRNPH1. In stark contrast, cytokine and chemokine genes, such as IL6 and TNF, predominantly express productive (protein-coding) splicing isoforms in response to SARS-CoV-2 infection. We postulate that SARS-CoV-2 employs an unreported tactic of exploiting the host splicing machinery to bolster viral replication and subvert the immune response by selectively upregulating unproductive splicing isoforms from antigen presentation and antiviral response genes. Our study sheds new light on the molecular interplay between SARS-CoV-2 and the host immune system, offering a foundation for the development of novel therapeutic strategies to combat COVID-19.
Subject(s)
Alternative Splicing , COVID-19 , Interferons , Protein Isoforms , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , COVID-19/virology , COVID-19/genetics , COVID-19/immunology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Interferons/metabolism , Interferons/genetics , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolismABSTRACT
The pathophysiology of post-traumatic brain injury (TBI) behavioral and cognitive changes is not fully understood, especially in its mild presentation. We designed a weight drop TBI model in mice to investigate the role of neuroinflammation in behavioral and cognitive sequelae following mild TBI. C57BL/6 mice displayed depressive-like behavior at 72 h after mild TBI compared with controls, as indicated by a decrease in the latency to first immobility and climbing time in the forced swim test. Additionally, anxiety-like behavior and hippocampal-associated spatial learning and memory impairment were found in the elevated plus maze and in the Barnes maze, respectively. Levels of a set of inflammatory mediators and neurotrophic factors were analyzed at 6 h, 24 h, 72 h, and 30 days after injury in ipsilateral and contralateral hemispheres of the prefrontal cortex and hippocampus. Principal components analysis revealed two principal components (PC), which represented 59.1% of data variability. PC1 (cytokines and chemokines) expression varied between both hemispheres, while PC2 (neurotrophic factors) expression varied only across the investigated brain areas. Our model reproduces mild TBI-associated clinical signs and pathological features and might be a valuable tool to broaden the knowledge regarding mild TBI pathophysiology as well as to test potential therapeutic targets.
Subject(s)
Brain Concussion , Brain Injuries, Traumatic , Mice , Animals , Brain Concussion/complications , Mice, Inbred C57BL , Brain/pathology , Brain Injuries, Traumatic/complications , Nerve Growth Factors , Cognition , Maze Learning/physiology , Disease Models, AnimalABSTRACT
Acute rejection (AR) is a process triggered via the recognition of grafted organ-derived antigens by the immune system, which could present as a life-threatening condition. In the context of a kidney transplant, despite improvement with immunosuppressive therapies, AR maintains a significant incidence of 10%, and currently available drugs generally act in similar and canonical pathways of lymphocyte activation. This prompted the research for different approaches to identify potential novel targets that could improve therapeutic interventions. Here, we conducted a transcriptome analysis comparing groups of acute rejection (including T cell-mediated rejection and antibody-mediated rejection) to stable grafts that included differentially expressed genes, transcription factor and kinase enrichment, and Gene Set Enrichment Analysis. These analyses revealed inflammasome enhancement in rejected grafts and AIM2 as a potential component linked to acute rejection, presenting a positive correlation to T-cell activation and a negative correlation to oxidative phosphorylation metabolism. Also, the AIM2 expression showed a global accuracy in discerning acute rejection grafts (area under the curve (AUC) = 0.755 and 0.894, p < 0.0001), and meta-analysis comprising different studies indicated a considerable enhancement of AIM2 in rejection (standardized mean difference (SMD) = 1.45, [CI 95%, 1.18 to 1.71]), especially for T cell-mediated rejection (TCMR) (SMD = 2.01, [CI 95%, 1.58 to 2.45]). These findings could guide future studies of AIM2 as either an adjuvant target for immunosuppression or a potential biomarker for acute rejection and graft survival.
Subject(s)
Graft Rejection , Inflammasomes , Biomarkers , Kidney , Transcription FactorsABSTRACT
Thousands of biomedical scientific articles, including those describing genes associated with human diseases, are published every week. Computational methods such as text mining and machine learning algorithms are now able to automatically detect these associations. In this study, we used a cognitive computing text-mining application to construct a knowledge network comprising 3,723 genes and 99 diseases. We then tracked the yearly changes on these networks to analyze how our knowledge has evolved in the past 30 years. Our systems approach helped to unravel the molecular bases of diseases and detect shared mechanisms between clinically distinct diseases. It also revealed that multi-purpose therapeutic drugs target genes that are commonly associated with several psychiatric, inflammatory, or infectious disorders. By navigating this knowledge tsunami, we were able to extract relevant biological information and insights about human diseases.
ABSTRACT
The growth of sequenced bacterial genomes has revolutionized the assessment of microbial diversity. Pseudomonas is a widely diverse genus, containing more than 254 species. Although type strains have been employed to estimate Pseudomonas diversity, they represent a small fraction of the genomic diversity at a genus level. We used 10,035 available Pseudomonas genomes, including 210 type strains, to build a genomic distance network to estimate the number of species through community identification. We identified taxonomic inconsistencies with several type strains and found that 25.65 % of the Pseudomonas genomes deposited on Genbank are misclassified. The phylogenetic tree using single-copy genes from representative genomes in each species cluster in the distance network revealed at least 14 Pseudomonas groups, including the P. alcaligenes group proposed here. We show that Pseudomonas is likely an admixture of different genera and should be further divided. This study provides an overview of Pseudomonas diversity from a network and phylogenomic perspective that may help reduce the propagation of mislabeled Pseudomonas genomes.
Subject(s)
Genetic Variation , Genome, Bacterial , Pseudomonas , Genome, Bacterial/genetics , Genomics , Phylogeny , Pseudomonas/classification , Pseudomonas/geneticsABSTRACT
BACKGROUND: PTEN is the most frequently lost tumor suppressor in primary prostate cancer (PCa) and its loss is associated with aggressive disease. However, the transcriptional changes associated with PTEN loss in PCa have not been described in detail. In this study, we highlight the transcriptional changes associated with PTEN loss in PCa. METHODS: Using a meta-analysis approach, we leveraged two large PCa cohorts with experimentally validated PTEN and ERG status by Immunohistochemistry (IHC), to derive a transcriptomic signature of PTEN loss, while also accounting for potential confounders due to ERG rearrangements. This signature was expanded to lncRNAs using the TCGA quantifications from the FC-R2 expression atlas. RESULTS: The signatures indicate a strong activation of both innate and adaptive immune systems upon PTEN loss, as well as an expected activation of cell-cycle genes. Moreover, we made use of our recently developed FC-R2 expression atlas to expand this signature to include many non-coding RNAs recently annotated by the FANTOM consortium. Highlighting potential novel lncRNAs associated with PTEN loss and PCa progression. CONCLUSION: We created a PCa specific signature of the transcriptional landscape of PTEN loss that comprises both the coding and an extensive non-coding counterpart, highlighting potential new players in PCa progression. We also show that contrary to what is observed in other cancers, PTEN loss in PCa leads to increased activation of the immune system. These findings can help the development of new biomarkers and help guide therapy choices.
Subject(s)
Gene Expression Regulation, Neoplastic , PTEN Phosphohydrolase/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Transcriptome , Adaptive Immunity , Biomarkers, Tumor , Gene Expression Profiling , Humans , Immunity, Innate , Immunohistochemistry , Male , PTEN Phosphohydrolase/genetics , Prostatic Neoplasms/pathology , Transcriptional Regulator ERG/metabolismABSTRACT
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.
ABSTRACT
Bioinformatics is a fast-evolving research field, requiring effective educational initiatives to bring computational knowledge to Life Sciences. Since 2017, an organizing committee composed of graduate students and postdoctoral researchers from the Universidade Federal de Minas Gerais (Brazil) promotes a week-long event named Summer Course in Bioinformatics (CVBioinfo). This event aims to diffuse bioinformatic principles, news, and methods mainly focused on audiences of undergraduate students. Furthermore, as the advent of the COVID-19 global pandemic has precluded in-person events, we offered the event in online mode, using free video transmission platforms. Herein, we present and discuss the insights obtained from promoting the Online Workshop in Bioinformatics (WOB) organized in November 2020, comparing it to our experience in previous in-person editions of the same event.
ABSTRACT
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.
ABSTRACT
The protozoan Trypanosoma cruzi (T. cruzi) is a well-adapted parasite to mammalian hosts and the pathogen of Chagas disease in humans. As both host and T. cruzi are highly genetically diverse, many variables come into play during infection, making disease outcomes difficult to predict. One important challenge in the field of Chagas disease research is determining the main factors leading to parasite establishment in the chronic stage in some organs, mainly the heart and/or digestive system. Our group previously showed that distinct strains of T. cruzi (JG and Col1.7G2) acquired differential tissue distribution in the chronic stage in dually infected BALB/c mice. To investigate changes in the host triggered by the two distinct T. cruzi strains, we assessed the gene expression profiles of BALB/c mouse hearts infected with either JG, Col1.7G2 or an equivalent mixture of both parasites during the initial phase of infection. This study demonstrates the clear differences in modulation of host gene expression by both parasites. Col1.7G2 strongly activated Th1-polarized immune signature genes, whereas JG caused only minor activation of the host immune response. Moreover, JG strongly reduced the expression of genes encoding ribosomal proteins and mitochondrial proteins related to the electron transport chain. Interestingly, the evaluation of gene expression in mice inoculated with a mixture of the parasites produced expression profiles with both up- and downregulated genes, indicating the coexistence of both parasite strains in the heart during the acute phase. This study suggests that different strains of T. cruzi may be distinguished by their efficiency in activating the immune system, modulating host energy metabolism and reactive oxygen species production and decreasing protein synthesis during early infection, which may be crucial for parasite persistence in specific organs.
ABSTRACT
Psychiatric and neurological disorders (PNDs) affect millions worldwide and only a few drugs achieve complete therapeutic success in the treatment of these disorders. Due to the high cost of developing novel drugs, drug repositioning represents a promising alternative method of treatment. In this manuscript, we used a network medicine approach to investigate the molecular characteristics of PNDs and identify novel drug candidates for repositioning. Using IBM Watson for Drug Discovery, a powerful machine learning text-mining application, we built knowledge networks containing connections between PNDs and genes or drugs mentioned in the scientific literature published in the past 50 years. This approach revealed several drugs that target key PND-related genes, which have never been used to treat these disorders to date. We validate our framework by detecting drugs that have been undergoing clinical trial for treating some of the PNDs, but have no published results in their support. Our data provides comprehensive insights into the molecular pathology of PNDs and offers promising drug repositioning candidates for follow-up trials.
Subject(s)
Drug Repositioning , Nervous System Diseases , Computational Biology , Data Mining , Humans , Machine Learning , Nervous System Diseases/drug therapyABSTRACT
The snow-covered surfaces of Antarctica comprise an extreme environment that favors the development of life forms with adaptations to adverse low-temperature habitats. The ability to survive and such temperatures might involve the production of antifreeze proteins and ice-binding proteins that attenuate the effects of intense cold temperatures. He, we sequenced and reconstructed the nuclear and mitochondrial genomes of the endemic Antarctic fungus Antarctomyces pellizariae UFMGCB 12416. We then have identified a putative ice-binding protein-coding gene, mapped the presence of secondary metabolite gene clusters, and reconstructed the phylogenetic relationships of a A. pellizariae with others Leotiomycetes from the alignment of hundreds of orthologous single-copy proteins. Our results will deepen the understanding of microbial ice-binding proteins and the genomic aspects of psychrophilic fungi. DATASET: The GenBank/EMBL/DDBJ accession number for the gene sequence of ice-binding protein from A. pellizariae determined in this study is MN867686. The Whole Genome Shotgun project of strain A. pellizariae UFMGCB 12416 has been deposited at DDBJ/ENA/GenBank under accession WCAA00000000. The version described in this paper is version WCAA01000000. The mitochondrial genome has been deposited under accession MT197497.
Subject(s)
Ascomycota/genetics , Carrier Proteins/genetics , Fungal Proteins/genetics , Amino Acid Sequence , Antarctic Regions , Ascomycota/classification , Ascomycota/metabolism , Carrier Proteins/chemistry , Fungal Proteins/chemistry , Genome, Bacterial , Genome, Mitochondrial , Ice , Phylogeny , Secondary Metabolism/genetics , Sequence Alignment , Whole Genome SequencingABSTRACT
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.
Subject(s)
Homologous Recombination , Trypanosoma cruzi/genetics , Trypanosoma cruzi/physiology , Animals , Cell Line , Macaca mulatta , Mutation , Protozoan Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Protozoan/genetics , Rad51 Recombinase/genetics , Species Specificity , Trypanosoma cruzi/cytology , Trypanosoma cruzi/growth & developmentABSTRACT
Trypanosoma cruzi, the causative agent of Chagas disease, has a complex life cycle that requires the adaptation to different environments. In the absence of traditional mechanisms for regulation of gene expression, this parasite relies on posttranscriptional control events, which allow the progression of its life cycle in different hosts and stress conditions. In this context, different stress conditions trigger the aggregation of RNA-binding proteins and their target mRNAs into cytoplasmic foci known as RNA granules, which act as RNA-sorting centers. In this study, we have characterized the T. cruzi RNA-binding protein ALBA30 during nutritional stress conditions. Using a recombinant form of TcALBA30 to facilitate its detection (rTcALBA30), we showed that this protein resides in the cytoplasm in normal growth conditions but is recruited into cytoplasmic foci after starvation. Moreover, evaluation of rTcALBA30 in parasites that reached the stationary phase of growth also showed the recruitment of this protein into cytoplasmic foci. Our results indicate that, similar to TbALBA3, TcALBA30 aggregates into stress granules in parasites submitted to nutritional stress.
Subject(s)
Gene Expression Regulation/genetics , Protozoan Proteins/genetics , RNA-Binding Proteins/genetics , Stress, Physiological/physiology , Trypanosoma cruzi/genetics , Animals , Cell Cycle , Chagas Disease/parasitology , Cytoplasm/metabolism , Life Cycle Stages/physiology , RNA, Messenger/metabolism , StarvationABSTRACT
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.
Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , Protozoan Proteins/metabolism , Rad51 Recombinase/metabolism , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/genetics , Animals , Chagas Disease/parasitology , DNA Breaks, Double-Stranded/radiation effects , DNA Repair/radiation effects , Humans , Male , Mice , Oxidative Stress , Protozoan Proteins/genetics , Rad51 Recombinase/genetics , Trypanosoma cruzi/metabolism , Trypanosoma cruzi/radiation effects , Ultraviolet RaysABSTRACT
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.
Subject(s)
Homologous Recombination/physiology , Protozoan Proteins/metabolism , Rad51 Recombinase/metabolism , Trypanosoma cruzi/enzymology , Protozoan Proteins/genetics , Rad51 Recombinase/genetics , Trypanosoma cruzi/geneticsABSTRACT
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.
ABSTRACT
BACKGROUND: The hemibiotrophic pathogens Moniliophthora perniciosa (witches' broom disease) and Moniliophthora roreri (frosty pod rot disease) are among the most important pathogens of cacao. Moniliophthora perniciosa has a broad host range and infects a variety of meristematic tissues in cacao plants, whereas M. roreri infects only pods of Theobroma and Herrania genera. Comparative pathogenomics of these fungi is essential to understand Moniliophthora infection strategies, therefore the detection and in silico functional characterization of effector candidates are important steps to gain insight on their pathogenicity. RESULTS: Candidate secreted effector proteins repertoire were predicted using the genomes of five representative isolates of M. perniciosa subpopulations (three from cacao and two from solanaceous hosts), and one representative isolate of M. roreri from Peru. Many putative effectors candidates were identified in M. perniciosa: 157 and 134 in cacao isolates from Bahia, Brazil; 109 in cacao isolate from Ecuador, 92 and 80 in wild solanaceous isolates from Minas Gerais (Lobeira) and Bahia (Caiçara), Brazil; respectively. Moniliophthora roreri showed the highest number of effector candidates, a total of 243. A set of eight core effectors were shared among all Moniliophthora isolates, while others were shared either between the wild solanaceous isolates or among cacao isolates. Mostly, candidate effectors of M. perniciosa were shared among the isolates, whereas in M. roreri nearly 50% were exclusive to the specie. In addition, a large number of cell wall-degrading enzymes characteristic of hemibiotrophic fungi were found. From these, we highlighted the proteins involved in cell wall modification, an enzymatic arsenal that allows the plant pathogens to inhabit environments with oxidative stress, which promotes degradation of plant compounds and facilitates infection. CONCLUSIONS: The present work reports six genomes and provides a database of the putative effectorome of Moniliophthora, a first step towards the understanding of the functional basis of fungal pathogenicity.