Publications by authors named "Gloria Regina Franco"

40 Publications

Differential Modulation of Mouse Heart Gene Expression by Infection With Two Strains: A Transcriptome Analysis.

Front Genet 2020 3;11:1031. Epub 2020 Sep 3.

Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, UFMG, Belo Horizonte, Brazil.

The protozoan () is a well-adapted parasite to mammalian hosts and the pathogen of Chagas disease in humans. As both host and 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 (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 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 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.
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http://dx.doi.org/10.3389/fgene.2020.01031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495023PMC
September 2020

Drug repositioning for psychiatric and neurological disorders through a network medicine approach.

Transl Psychiatry 2020 05 12;10(1):141. Epub 2020 May 12.

Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.

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.
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http://dx.doi.org/10.1038/s41398-020-0827-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217930PMC
May 2020

Whole-genome sequencing of the endemic Antarctic fungus Antarctomyces pellizariae reveals an ice-binding protein, a scarce set of secondary metabolites gene clusters and provides insights on Thelebolales phylogeny.

Genomics 2020 09 7;112(5):2915-2921. Epub 2020 May 7.

Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, 31.270-901, Brazil.

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.
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http://dx.doi.org/10.1016/j.ygeno.2020.05.004DOI Listing
September 2020

The Influence of Recombinational Processes to Induce Dormancy in .

Front Cell Infect Microbiol 2020 28;10. Epub 2020 Jan 28.

Laboratory of Biochemistry Genetics, Department of Biochemistry and Immunology, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

The protozoan 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 amastigotes has been shown. Here, we quantify the percentage of dormant parasites from different DTUs during their replicative epimastigote and amastigote stages. For this study, cells of 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 , 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 strains present higher transcription of RAD51-a key gene in recombination process -we also measured the percentage of dormant cells from clone CL Brener harboring single knockout for RAD51. Our results showed a significative reduction of dormant cells in this 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 strains to generate dormant cells, and that homologous recombination may be important for dormancy in this parasite.
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http://dx.doi.org/10.3389/fcimb.2020.00005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025536PMC
January 2020

Trypanosoma cruzi RNA-binding protein ALBA30 aggregates into cytoplasmic foci under nutritional stress.

Parasitol Res 2020 Feb 3;119(2):749-753. Epub 2020 Jan 3.

Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

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.
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http://dx.doi.org/10.1007/s00436-019-06554-wDOI Listing
February 2020

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

PLoS Negl Trop Dis 2018 11 13;12(11):e0006875. Epub 2018 Nov 13.

Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

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.
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http://dx.doi.org/10.1371/journal.pntd.0006875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258567PMC
November 2018

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

Sci Rep 2018 09 6;8(1):13335. Epub 2018 Sep 6.

Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, 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.
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http://dx.doi.org/10.1038/s41598-018-31541-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127316PMC
September 2018

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

Genet Mol Biol 2018 Apr./Jun;41(2):466-474. Epub 2018 Jun 11.

Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

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.
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http://dx.doi.org/10.1590/1678-4685-GMB-2017-0281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082238PMC
June 2018

Genome sequence and effectorome of Moniliophthora perniciosa and Moniliophthora roreri subpopulations.

BMC Genomics 2018 Jul 3;19(1):509. Epub 2018 Jul 3.

Departamento de Ciências Biológicas (DCB), Centro de Biotecnologia e Genética (CBG), Universidade Estadual de Santa Cruz (UESC), Rodovia Ilhéus-Itabuna, km 16, Ilhéus, 45662-900, Bahia, Brazil.

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.
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http://dx.doi.org/10.1186/s12864-018-4875-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029071PMC
July 2018

Landscape of the spliced leader trans-splicing mechanism in Schistosoma mansoni.

Sci Rep 2018 03 1;8(1):3877. Epub 2018 Mar 1.

Laboratório de Genética Bioquímica, Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901, Brazil.

Spliced leader dependent trans-splicing (SLTS) has been described as an important RNA regulatory process that occurs in different organisms, including the trematode Schistosoma mansoni. We identified more than seven thousand putative SLTS sites in the parasite, comprising genes with a wide spectrum of functional classes, which underlines the SLTS as a ubiquitous mechanism in the parasite. Also, SLTS gene expression levels span several orders of magnitude, showing that SLTS frequency is not determined by the expression level of the target gene, but by the presence of particular gene features facilitating or hindering the trans-splicing mechanism. Our in-depth investigation of SLTS events demonstrates widespread alternative trans-splicing (ATS) acceptor sites occurring in different regions along the entire gene body, highlighting another important role of SLTS generating alternative RNA isoforms in the parasite, besides the polycistron resolution. Particularly for introns where SLTS directly competes for the same acceptor substrate with cis-splicing, we identified for the first time additional and important features that might determine the type of splicing. Our study substantially extends the current knowledge of RNA processing by SLTS in S. mansoni, and provide basis for future studies on the trans-splicing mechanism in other eukaryotes.
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http://dx.doi.org/10.1038/s41598-018-22093-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832876PMC
March 2018

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

Infect Genet Evol 2017 11 29;55:332-342. Epub 2017 Sep 29.

Departamento de Bioquímica e Imunologia, ICB, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Caixa Postal 486, Belo Horizonte 30161-970, MG, Brazil. Electronic address:

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 overexpresses MYH. Although in standard conditions this lineage has similar growth to control cells, the overexpressor 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.
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http://dx.doi.org/10.1016/j.meegid.2017.09.030DOI Listing
November 2017

Catalase expression impairs oxidative stress-mediated signalling in Trypanosoma cruzi.

Parasitology 2017 Sep 27;144(11):1498-1510. Epub 2017 Jun 27.

Departamento de Bioquímica e Imunologia,Universidade Federal de Minas Gerais,Belo Horizonte, MG,Brazil.

Trypanosoma cruzi is exposed to oxidative stresses during its life cycle, and amongst the strategies employed by this parasite to deal with these situations sits a peculiar trypanothione-dependent antioxidant system. Remarkably, T. cruzi's antioxidant repertoire does not include catalase. In an attempt to shed light on what are the reasons by which this parasite lacks this enzyme, a T. cruzi cell line stably expressing catalase showed an increased resistance to hydrogen peroxide (H2O2) when compared with wild-type cells. Interestingly, preconditioning carried out with low concentrations of H2O2 led untransfected parasites to be as much resistant to this oxidant as cells expressing catalase, but did not induce the same level of increased resistance in the latter ones. Also, presence of catalase decreased trypanothione reductase and increased superoxide dismutase levels in T. cruzi, resulting in higher levels of residual H2O2 after challenge with this oxidant. Although expression of catalase contributed to elevated proliferation rates of T. cruzi in Rhodnius prolixus, it failed to induce a significant increase of parasite virulence in mice. Altogether, these results indicate that the absence of a gene encoding catalase in T. cruzi has played an important role in allowing this parasite to develop a shrill capacity to sense and overcome oxidative stress.
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http://dx.doi.org/10.1017/S0031182017001044DOI Listing
September 2017

The polymorphism rs17782313 near MC4R gene is related with anthropometric changes in women submitted to bariatric surgery over 60 months.

Clin Nutr 2018 08 22;37(4):1286-1292. Epub 2017 May 22.

Departamento de Bioquimica e Imunologia - ICB, Universidade Federal de Minas, Belo Horizonte, MG, Brazil.

Objective: Evaluate whether the polymorphism rs17782313 near MC4R gene influences long-term outcomes after bariatric surgery.

Methods: The rs16782313 polymorphism was genotyped in 217 individuals undergoing bariatric surgery and analyzed in detail in 141 women. Data for comorbidities, BMI, excess weight loss (EWL), and body composition were obtained before and during 60 months after surgery.

Results: The risk allele was found in 65 (47%) of the 141 women. Pre-surgical body weight and BMI were higher in carriers of the rs17782313 polymorphism (CC + CT group) than in non-carriers (TT group) (p = 0.039 and 0.047, respectively). The number of women who acquired surgical success (EWL > 50%), was lower in CC + CT group compared to TT group (p = 0.015). The minimum BMI seen during the 60 months of follow-up was higher in CC + CT group compared to TT group (p = 0.028). The number of women who presented BMI < 30 kg/m (no longer classified as obesity) after 24 months of surgery was inferior in CC + CT group (6 out 35 patients - 17%) than in TT group (19 out 49 patients - 37%, p = 0.043). Moreover, the number of patients maintaining BMI > 35 kg/m were higher carriers (18 out 35 patients - 51%) compare to non-carriers (16 out 49 patients - 32%, p = 0.045).

Conclusion: Women with extreme obesity carrying rs17782313 MC4R polymorphism present a higher pre-surgical BMI, are more unlikely to reach non-obesity BMI (<30 kg/m) and tend to maintain a BMI > 35 kg/m that characterize treatment failure.
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http://dx.doi.org/10.1016/j.clnu.2017.05.018DOI Listing
August 2018

Early polymerase chain reaction detection of Chagas disease reactivation in heart transplant patients.

J Heart Lung Transplant 2017 Jul 24;36(7):797-805. Epub 2017 Feb 24.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Background: Heart transplantation is a valuable therapeutic option for Chagas disease patients with severe cardiomyopathy. During patient follow-up, the differential diagnosis between cardiac transplant rejection and Chagas disease infection reactivation remains a challenging task, which hinders rapid implementation of the appropriate treatment. Herein we investigate whether polymerase chain reaction (PCR) strategies could facilitate early detection of Trypanosoma cruzi (T cruzi) in transplanted endomyocardial biopsies (EMBs).

Methods: In this study we analyzed 500 EMB specimens obtained from 58 chagasic cardiac transplant patients, using PCR approaches targeted to nuclear (rDNA 24Sα) and kinetoplastid (kDNA) markers, and compared the efficiency of these approaches with that of other tests routinely used.

Results: T cruzi DNA was detected in 112 EMB specimens derived from 39 patients (67.2%). The first positive result occurred at a median 1.0 month post-transplant. Conventional histopathologic, blood smear and hemoculture analyses showed lower sensitivity and higher median time to the first positive result. Patient follow-up revealed that 31 of 39 PCR-positive cases presented clinical reactivation of Chagas disease at different time-points after transplantation. PCR techniques showed considerable sensitivity (0.82) and specificity (0.60), with area under the receiver operating characteristic (ROC) curves of 0.708 (p = 0.001). Moreover, PCR techniques anticipated the clinical signs of Chagas disease reactivation by up to 36 months, with a median time of 6 months and an average of 9.1 months.

Conclusions: We found a good association between the PCR diagnosis and the clinical signs of the disease, indicating that the PCR approaches used herein are suitable for early diagnosis of Chagas disease reactivation, with high potential to assist physicians in treatment decisions. For this purpose, an algorithm is proposed for surveillance based on the molecular tests.
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http://dx.doi.org/10.1016/j.healun.2017.02.018DOI Listing
July 2017

Computer aided identification of a Hevein-like antimicrobial peptide of bell pepper leaves for biotechnological use.

BMC Genomics 2016 12 15;17(Suppl 12):999. Epub 2016 Dec 15.

Department of Biochemistry and Molecular Biology, Universidade Federal de Viçosa, Viçosa, MG, 36570-900, Brazil.

Background: Antimicrobial peptides from plants present mechanisms of action that are different from those of conventional defense agents. They are under-explored but have a potential as commercial antimicrobials. Bell pepper leaves ('Magali R') are discarded after harvesting the fruit and are sources of bioactive peptides. This work reports the isolation by peptidomics tools, and the identification and partially characterization by computational tools of an antimicrobial peptide from bell pepper leaves, and evidences the usefulness of records and the in silico analysis for the study of plant peptides aiming biotechnological uses.

Results: Aqueous extracts from leaves were enriched in peptide by salt fractionation and ultrafiltration. An antimicrobial peptide was isolated by tandem chromatographic procedures. Mass spectrometry, automated peptide sequencing and bioinformatics tools were used alternately for identification and partial characterization of the Hevein-like peptide, named HEV-CANN. The computational tools that assisted to the identification of the peptide included BlastP, PSI-Blast, ClustalOmega, PeptideCutter, and ProtParam; conventional protein databases (DB) as Mascot, Protein-DB, GenBank-DB, RefSeq, Swiss-Prot, and UniProtKB; specific for peptides DB as Amper, APD2, CAMP, LAMPs, and PhytAMP; other tools included in ExPASy for Proteomics; The Bioactive Peptide Databases, and The Pepper Genome Database. The HEV-CANN sequence presented 40 amino acid residues, 4258.8 Da, theoretical pI-value of 8.78, and four disulfide bonds. It was stable, and it has inhibited the growth of phytopathogenic bacteria and a fungus. HEV-CANN presented a chitin-binding domain in their sequence. There was a high identity and a positive alignment of HEV-CANN sequence in various databases, but there was not a complete identity, suggesting that HEV-CANN may be produced by ribosomal synthesis, which is in accordance with its constitutive nature.

Conclusions: Computational tools for proteomics and databases are not adjusted for short sequences, which hampered HEV-CANN identification. The adjustment of statistical tests in large databases for proteins is an alternative to promote the significant identification of peptides. The development of specific DB for plant antimicrobial peptides, with information about peptide sequences, functional genomic data, structural motifs and domains of molecules, functional domains, and peptide-biomolecule interactions are valuable and necessary.
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http://dx.doi.org/10.1186/s12864-016-3332-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5249031PMC
December 2016

Adenine Glycosylase MutY of Corynebacterium pseudotuberculosis presents the antimutator phenotype and evidences of glycosylase/AP lyase activity in vitro.

Infect Genet Evol 2016 10 22;44:318-329. Epub 2016 Jul 22.

Laboratory of Molecular Biology, Federal University of São João Del-Rei (CCO), Av. Sebastião Gonçalves Coelho, 400, Divinópolis, MG 35501-296, Brazil. Electronic address:

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis, a disease that predominantly affects small ruminants, causing significant economic losses worldwide. As a facultative intracellular pathogen, this bacterium is exposed to an environment rich in reactive oxygen species (ROS) within macrophages. To ensure its genetic stability, C. pseudotuberculosis relies on efficient DNA repair pathways for excision of oxidative damage such as 8-oxoguanine, a highly mutagenic lesion. MutY is an adenine glycosylase involved in adenine excision from 8-oxoG:A mismatches avoiding genome mutation incorporation. The purpose of this study was to characterize MutY protein from C. pseudotuberculosis and determine its involvement with DNA repair. In vivo functional complementation assay employing mutY gene deficient Escherichia coli transformed with CpmutY showed a 13.5-fold reduction in the rate of spontaneous mutation, compared to cells transformed with empty vector. Also, under oxidative stress conditions, CpMutY protein favored the growth of mutY deficient E. coli, relative to the same strain in the absence of CpMutY. To demonstrate the involvement of this enzyme in recognition and excision of 8-oxoguanine lesion, an in vitro assay was performed. CpMutY protein was capable of recognizing and excising 8-oxoG:A but not 8-oxoG:C presenting evidences of glycosylase/AP lyase activity in vitro. In silico structural characterization revealed the presence of preserved motifs related to the MutY activity on DNA repair, such as catalytic residues involved in glycosylase/AP lyase activity and structural DNA-binding elements, such as the HhH motif and the [4Fe-4S] cluster. The three-dimensional structure of CpMutY, generated by comparative modeling, exhibits a catalytic domain very similar to that of E. coli MutY. Taken together, these results indicate that the CpmutY encodes a functional protein homologous to MutY from E. coli and is involved in the prevention of mutations and the repair of oxidative DNA lesions.
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http://dx.doi.org/10.1016/j.meegid.2016.07.028DOI Listing
October 2016

Structural and evolutionary insights into endogenous alpha-phospholipase A2 inhibitors of Latin American pit vipers.

Toxicon 2016 Mar 21;112:35-44. Epub 2016 Jan 21.

Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG, Brazil. Electronic address:

Phospholipases A2 are major components of snake venoms (svPLA2s) and are able to induce multiple local and systemic deleterious effects upon envenomation. Several snake species are provided with svPLA2 inhibitors (sbPLIs) in their circulating blood, which confer a natural resistance against the toxic components of homologous and heterologous venoms. The sbPLIs belong to any of three structural classes named α, β and γ. In the present study, we identified, characterized and performed structural and evolutionary analyses of sbαPLIs transcripts and the encoded proteins, in the most common Latin American pit vipers belonging to Crotalus, Bothrops and Lachesis genera. Mutation data indicated that sbαPLIs from Latin American snakes might have evolved in an accelerated manner, similarly to that reported for sbαPLIs from Asian snakes, and possibly co-evoluted with svPLA2s in response to the diversity of target enzymes. The importance of sbαPLI trimerization for the effective binding and inhibition of acidic svPLA2s is discussed and conserved cationic residues located at the central pore of the inhibitor trimer are suggested to be a significant part of the binding site of sbαPLIs to acidic svPLA2s. Our data contribute to the current body of knowledge on the structural and evolutionary characteristics of sbPLIs, in general, and may assist in the future development of selective inhibitors for secretory PLA2 from several sources.
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http://dx.doi.org/10.1016/j.toxicon.2016.01.058DOI Listing
March 2016

A Basic Protein Comparative Three-Dimensional Modeling Methodological Workflow Theory and Practice.

IEEE/ACM Trans Comput Biol Bioinform 2014 Nov-Dec;11(6):1052-65

When working with proteins and studying its properties, it is crucial to have access to the three-dimensional structure of the molecule. If experimentally solved structures are not available, comparative modeling techniques can be used to generate useful protein models to subsidize structure-based research projects. In recent years, with Bioinformatics becoming the basis for the study of protein structures, there is a crescent need for the exposure of details about the algorithms behind the softwares and servers, as well as a need for protocols to guide in silico predictive experiments. In this article, we explore different steps of the comparative modeling technique, such as template identification, sequence alignment, generation of candidate structures and quality assessment, its peculiarities and theoretical description. We then present a practical step-by-step workflow, to support the Biologist on the in silico generation of protein structures. Finally, we explore further steps on comparative modeling, presenting perspectives to the study of protein structures through Bioinformatics. We trust that this is a thorough guide for beginners that wish to work on the comparative modeling of proteins.
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http://dx.doi.org/10.1109/TCBB.2014.2325018DOI Listing
March 2016

Homology-independent metrics for comparative genomics.

Comput Struct Biotechnol J 2015 4;13:352-7. Epub 2015 May 4.

Laboratório Multiusuário de Bioinformática, Embrapa Informática Agropecuária, André Tosello Avenue, 209, Barão Geraldo, Campinas, São Paulo CEP 13083-886, Brazil.

A mainstream procedure to analyze the wealth of genomic data available nowadays is the detection of homologous regions shared across genomes, followed by the extraction of biological information from the patterns of conservation and variation observed in such regions. Although of pivotal importance, comparative genomic procedures that rely on homology inference are obviously not applicable if no homologous regions are detectable. This fact excludes a considerable portion of "genomic dark matter" with no significant similarity - and, consequently, no inferred homology to any other known sequence - from several downstream comparative genomic methods. In this review we compile several sequence metrics that do not rely on homology inference and can be used to compare nucleotide sequences and extract biologically meaningful information from them. These metrics comprise several compositional parameters calculated from sequence data alone, such as GC content, dinucleotide odds ratio, and several codon bias metrics. They also share other interesting properties, such as pervasiveness (patterns persist on smaller scales) and phylogenetic signal. We also cite examples where these homology-independent metrics have been successfully applied to support several bioinformatics challenges, such as taxonomic classification of biological sequences without homology inference. They where also used to detect higher-order patterns of interactions in biological systems, ranging from detecting coevolutionary trends between the genomes of viruses and their hosts to characterization of gene pools of entire microbial communities. We argue that, if correctly understood and applied, homology-independent metrics can add important layers of biological information in comparative genomic studies without prior homology inference.
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http://dx.doi.org/10.1016/j.csbj.2015.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4446528PMC
June 2015

Proteomic analysis of Trypanosoma cruzi response to ionizing radiation stress.

PLoS One 2014 19;9(5):e97526. Epub 2014 May 19.

Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097526PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026238PMC
December 2014

Unveiling benznidazole's mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi.

Environ Mol Mutagen 2014 May 18;55(4):309-21. Epub 2013 Dec 18.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, Minas Gerais.

Benznidazole (BZ) is the most commonly used drug for the treatment of Chagas disease. Although BZ is known to induce the formation of free radicals and electrophilic metabolites within the parasite Trypanosoma cruzi, its precise mechanisms of action are still elusive. Here, we analyzed the survival of T. cruzi exposed to BZ using genetically modified parasites overexpressing different DNA repair proteins. Our results indicate that BZ induces oxidation mainly in the nucleotide pool, as heterologous expression of the nucleotide pyrophosphohydrolase MutT (but not overexpression of the glycosylase TcOgg1) increased drug resistance in the parasite. In addition, electron microscopy indicated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomic DNA undergoes extensive heterochromatin unpacking following exposure to the drug. Furthermore, the overexpression of proteins involved in the recombination-mediated DNA repair increased resistance to BZ, reinforcing the idea that the drug causes double-stranded breaks. Our results also show that the overexpression of mitochondrial DNA repair proteins increase parasite survival upon BZ exposure, indicating that the drug induces lesions in the mitochondrial DNA as well. These findings suggest that BZ preferentially oxidizes the nucleotide pool, and the extensive incorporation of oxidized nucleotides during DNA replication leads to potentially lethal double-stranded DNA breaks in T. cruzi DNA.
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http://dx.doi.org/10.1002/em.21839DOI Listing
May 2014

Evidence of substantial recombination among Trypanosoma cruzi II strains from Minas Gerais.

Infect Genet Evol 2014 Mar 1;22:183-91. Epub 2013 Dec 1.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. Electronic address:

Due to the scarcity of evidence of sexuality in Trypanosoma cruzi, the causative agent of Chagas disease, it has been general accepted that the parasite reproduction is essentially clonal with infrequent genetic recombination. This assumption is mainly supported by indirect evidence, such as Hardy-Weinberg imbalances, linkage disequilibrium and a strong correlation between independent sets of genetic markers of T. cruzi populations. However, because the analyzed populations are usually isolated from different geographic regions, the possibility of population substructuring as generating these genetic marker imbalances cannot be eliminated. To investigate this possibility, we firstly compared the allele frequencies and haplotype networks using seven different polymorphic loci (two from mitochondrial and five from different nuclear chromosomes) in two groups of TcII strains: one including isolates obtained from different regions in Latin America and the other including isolates obtained only from patients of the Minas Gerais State in Brazil. Our hypothesis was that if the population structure is essentially clonal, Hardy-Weinberg disequilibrium and a sharp association between the clusters generated by analyzing independent markers should be observed in both strain groups, independent of the geographic origin of the samples. The results demonstrated that the number of microsatellite loci in linkage disequilibrium decreased from 4 to 1 when only strains from Minas Gerais were analyzed. Moreover, we did not observed any correlation between the clusters when analyzing the nuclear and mitochondrial loci, suggesting independent inheritance of these markers among the Minas Gerais strains. Besides, using a second subset of five physically linked microsatellite loci and the Minas Gerais strains, we could also demonstrate evidence of homologous recombination roughly proportional to the relative distance among them. Taken together, our results do not support a clonal population structure for T. cruzi, particularly in TcII, which coexists in the same geographical area, suggesting that genetic exchanges among these strains may occur more frequently than initially expected.
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http://dx.doi.org/10.1016/j.meegid.2013.11.021DOI Listing
March 2014

A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni.

Mem Inst Oswaldo Cruz 2013 Sep;108(6):707-17

Grupo de Genômica e Biologia Computacional, Centro de Pesquisas René Rachou, Fiocruz, Belo HorizonteMG, Brasil.

Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).
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http://dx.doi.org/10.1590/0074-0276108062013006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970683PMC
September 2013

LSSP-PCR of Trypanosoma cruzi: how the single primer sequence affects the kDNA signature.

BMC Res Notes 2013 May 2;6:174. Epub 2013 May 2.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Background: Low-stringency single specific primer PCR (LSSP-PCR) is a highly sensitive and discriminating technique that has been extensively used to genetically characterize Trypanosoma cruzi populations in the presence of large amounts of host DNA. To ensure high sensitivity, in most T. cruzi studies, the variable regions of the naturally amplified kinetoplast DNA (kDNA) minicircles were targeted, and this method translated the intraspecific polymorphisms of these molecules into specific and reproducible kDNA signatures. Although the LSSP-PCR technique is reproducible under strict assay conditions, the complex banding pattern generated can be significantly altered by even a single-base change in the target DNA. Our survey of the literature identified eight different primers with similar, if not identical, names that have been used for kDNA amplification and LSSP-PCR of T. cruzi. Although different primer sequences were used in these studies, many of the authors cited the same reference report to justify their primer choice. We wondered whether these changes in the primer sequence could affect also the parasite LSSP-PCR profiles.

Findings: To answer this question we compared the kDNA signatures obtained from three different and extensively studied T. cruzi populations with the eight primers found in the literature. Our results clearly demonstrate that even minimal modifications in the oligonucleotide sequences, especially in the 3' or 5' end, can significantly change the kDNA signature of a T. cruzi strain.

Conclusions: These results highlight the necessity of careful preservation of primer nomenclature and sequence when reproducing an LSSP-PCR work to avoid confusion and allow comparison of results among different laboratories.
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http://dx.doi.org/10.1186/1756-0500-6-174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653686PMC
May 2013

Modeling the zing finger protein SmZF1 from Schistosoma mansoni: Insights into DNA binding and gene regulation.

J Mol Graph Model 2013 Feb 23;39:29-38. Epub 2012 Oct 23.

Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Zinc finger proteins are widely found in eukaryotes, representing an important class of DNA-binding proteins frequently involved in transcriptional regulation. Zinc finger motifs are composed by two antiparallel β-strands and one α-helix, stabilized by a zinc ion coordinated by conserved histidine and cysteine residues. In Schistosoma mansoni, these regulatory proteins are known to modulate morphological and physiological changes, having crucial roles in parasite development. A previously described C(2)H(2) zinc finger protein, SmZF1, was shown to be present in cell nuclei of different life stages of S. mansoni and to activate gene transcription in a heterologous system. A high-quality SmZF1 tridimensional structure was generated using comparative modeling. Molecular dynamics simulations of the obtained structure revealed stability of the zinc fingers motifs and high flexibility on the terminals, comparable to the profile observed on the template X-ray structure based on thermal b-factors. Based on the protein tridimensional features and amino acid composition, we were able to characterize four C(2)H(2) zinc finger motifs, the first involved in protein-protein interactions while the three others involved in DNA binding. We defined a consensus DNA binding sequence using three distinct algorithms and further carried out docking calculations, which revealed the interaction of fingers 2-4 with the predicted DNA. A search for S. mansoni genes presenting putative SmZF1 binding sites revealed 415 genes hypothetically under SmZF1 control. Using an automatic annotation and GO assignment approach, we found that the majority of those genes code for proteins involved in developmental processes. Taken together, these results present a consistent base to the structural and functional characterization of SmZF1.
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http://dx.doi.org/10.1016/j.jmgm.2012.10.004DOI Listing
February 2013

Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi.

PLoS One 2012 2;7(8):e42484. Epub 2012 Aug 2.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-/-) (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2)O(2)). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2)O(2) of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042484PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411635PMC
January 2013

Unequivocal identification of subpopulations in putative multiclonal Trypanosoma cruzi strains by FACs single cell sorting and genotyping.

PLoS Negl Trop Dis 2012 10;6(7):e1722. Epub 2012 Jul 10.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Trypanosoma cruzi, the etiological agent of Chagas disease, is a polymorphic species. Evidence suggests that the majority of the T. cruzi populations isolated from afflicted humans, reservoir animals, or vectors are multiclonal. However, the extent and the complexity of multiclonality remain to be established, since aneuploidy cannot be excluded and current conventional cloning methods cannot identify all the representative clones in an infection. To answer this question, we adapted a methodology originally described for analyzing single spermatozoids, to isolate and study single T. cruzi parasites. Accordingly, the cloning apparatus of a Fluorescence-Activated Cell Sorter (FACS) was used to sort single T. cruzi cells directly into 96-wells microplates. Cells were then genotyped using two polymorphic genomic markers and four microsatellite loci. We validated this methodology by testing four T. cruzi populations: one control artificial mixture composed of two monoclonal populations--Silvio X10 cl1 (TcI) and Esmeraldo cl3 (TcII)--and three naturally occurring strains, one isolated from a vector (A316A R7) and two others derived from the first reported human case of Chagas disease. Using this innovative approach, we were able to successfully describe the whole complexity of these natural strains, revealing their multiclonal status. In addition, our results demonstrate that these T. cruzi populations are formed of more clones than originally expected. The method also permitted estimating of the proportion of each subpopulation of the tested strains. The single-cell genotyping approach allowed analysis of intrapopulation diversity at a level of detail not achieved previously, and may thus improve our comprehension of population structure and dynamics of T. cruzi. Finally, this methodology is capable to settle once and for all controversies on the issue of multiclonality.
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http://dx.doi.org/10.1371/journal.pntd.0001722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393670PMC
November 2012

DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions.

Mol Biochem Parasitol 2012 Jun 24;183(2):122-31. Epub 2012 Feb 24.

Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil.

Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polβ) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolβ) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolβ showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolβ overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolβ was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolβ during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle.
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http://dx.doi.org/10.1016/j.molbiopara.2012.02.007DOI Listing
June 2012

Trypanosoma cruzi gene expression in response to gamma radiation.

PLoS One 2012 11;7(1):e29596. Epub 2012 Jan 11.

Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Trypanosoma cruzi is an organism highly resistant to ionizing radiation. Following a dose of 500 Gy of gamma radiation, the fragmented genomic DNA is gradually reconstructed and the pattern of chromosomal bands is restored in less than 48 hours. Cell growth arrests after irradiation but, while DNA is completely fragmented, RNA maintains its integrity. In this work we compared the transcriptional profiles of irradiated and non-irradiated epimastigotes at different time points after irradiation using microarray. In total, 273 genes were differentially expressed; from these, 160 were up-regulated and 113 down-regulated. We found that genes with predicted functions are the most prevalent in the down-regulated gene category. Translation and protein metabolic processes, as well as generation of precursor of metabolites and energy pathways were affected. In contrast, the up-regulated category was mainly composed of obsolete sequences (which included some genes of the kinetoplast DNA), genes coding for hypothetical proteins, and Retrotransposon Hot Spot genes. Finally, the tyrosyl-DNA phosphodiesterase 1, a gene involved in double-strand DNA break repair process, was up-regulated. Our study demonstrated the peculiar response to ionizing radiation, raising questions about how this organism changes its gene expression to manage such a harmful stress.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029596PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256153PMC
May 2012

Coinfection with different Trypanosoma cruzi strains interferes with the host immune response to infection.

PLoS Negl Trop Dis 2010 Oct 12;4(10):e846. Epub 2010 Oct 12.

Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

A century after the discovery of Trypanosoma cruzi in a child living in Lassance, Minas Gerais, Brazil in 1909, many uncertainties remain with respect to factors determining the pathogenesis of Chagas disease (CD). Herein, we simultaneously investigate the contribution of both host and parasite factors during acute phase of infection in BALB/c mice infected with the JG and/or CL Brener T. cruzi strains. JG single infected mice presented reduced parasitemia and heart parasitism, no mortality, levels of pro-inflammatory mediators (TNF-α, CCL2, IL-6 and IFN-γ) similar to those found among naïve animals and no clinical manifestations of disease. On the other hand, CL Brener single infected mice presented higher parasitemia and heart parasitism, as well as an increased systemic release of pro-inflammatory mediators and higher mortality probably due to a toxic shock-like systemic inflammatory response. Interestingly, coinfection with JG and CL Brener strains resulted in intermediate parasitemia, heart parasitism and mortality. This was accompanied by an increase in the systemic release of IL-10 with a parallel increase in the number of MAC-3(+) and CD4(+) T spleen cells expressing IL-10. Therefore, the endogenous production of IL-10 elicited by coinfection seems to be crucial to counterregulate the potentially lethal effects triggered by systemic release of pro-inflammatory mediators induced by CL Brener single infection. In conclusion, our results suggest that the composition of the infecting parasite population plays a role in the host response to T. cruzi in determining the severity of the disease in experimentally infected BALB/c mice. The combination of JG and CL Brener was able to trigger both protective inflammatory immunity and regulatory immune mechanisms that attenuate damage caused by inflammation and disease severity in BALB/c mice.
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http://dx.doi.org/10.1371/journal.pntd.0000846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953483PMC
October 2010