Caracterização das DNA topoisomerases II de Trypanosoma rangeli

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia, Florianópolis, 2010DNA topoisomerases são enzimas que participam de diversos processos celulares tais como: replicação, transcrição, recombinação e segregação dos cromossomos. Atuando na clivagem transitória de uma fita (tipo I) ou ambas as fitas (tipo II) da molécula de DNA, as topoisomerases são alvos de agentes bactericidas e de drogas antitumorais e podem ser um importante alvo para quimioterapia de doenças causadas por parasitos. Neste estudo, descrevemos e caracterizamos os genes que codificam as topoisomerases tipo II de Trypanosoma rangeli (TrTop2). Os genes TrTop2 apresentaram um quadro aberto de leitura com 3.696 (TrTop2mt) e 4.368 pares de bases (TrTop2?), codificando polipeptídios preditos de 1.232 (138,8 kDa) e 1.456 (164,1 kDa) aminoácidos, respectivamente. Ambos os genes apresentaram uma alta similaridade da sequência protéica com topoisomerases ortólogas de outros tripanosomatídeos, sobretudo com T. cruzi (96% e 85%). Entre os dois genes a similaridade a nível de aminoácidos foi de 56%, sendo ambos de cópia única no genoma do T. rangeli. Anticorpos dirigidos a fragmentos protéicos de ambas as proteínas foram utilizados em ensaios de western blot e revelaram bandas de aproximadamente 130 kDa em extratos protéicos totais de T. rangeli. Embora com tamanho similar, foram identificadas proteínas distintas quando avaliadas por eletroforese 2D (pI 6,4 e 7,6). Através de géis de poliacrilamida em condições não desnaturantes foi possível determinar que ambas as proteínas nativas formam um complexo protéico de alto peso molecular indicando uma possível interação entre proteínas ou subunidades. Ensaios de imunolocalização com os distintos anticorpos contra DNA topoisomerases II apontaram diferentes padrões de localização celular, com reconhecimento no núcleo ou no cinetoplasto em formas epimastigotas e em alguns casos dispersa no citoplasma de formas tripomastigotas. A novobiocina, inibidor de topoisomerases tipo II procarióticas, foi ativo in vitro contra o T. rangeli. Acima de 300 ?g/ml observa-se uma redução no crescimento dos parasitos com alterações morfológicas e estruturais a nível nuclear e do cinetoplasto. Acima de 150 ?g/ml observa-se completa inibição da diferenciação celular in vitro. Utilizando as proteínas mtHSP70, DHLADH e a DNA topoisomerase II mitocondrial (TrTop2mt) como marcadores biológicos, foi observado redução da expressão das mesmas durante a diferenciação do T. rangeli, assim como nos tratamentos com novobiocina. Conclui-se que eventos relacionados as DNA topoisomerases II podem ser essenciais na redução do crescimento e da diferenciação celular do T. rangeli

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

The Genome of Anopheles darlingi, the main neotropical malaria vector

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vectorhuman and vectorparasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles- darlingi. © 2013 The Author(s)

TRENDS ON TRYPANOSOMA (HERPETOSOMA) RANGELI RESEARCH

Trypanosoma rangeli is a hemoflagellate protozoan parasite presenting an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, in a wide geographical area in Latin America. Despite considered as non-pathogenic for man, T. rangeli shares several characteristics with T. cruzi such as vertebrate and invertebrate reservoirs, vectors and approximately half of the soluble antigenic determinants. Despite the importance of specific detection, little is know about T. rangeli in comparison to T. cruzi; several questions lack proper answers, including the controversies concerning T. rangeli´s taxonomic position. In this context, this short review attempted to congregate current aspects on the research of this parasite, approaching several subjects as life cycle, vector suscepbility, specific genes studies and genomic data

Molecular epidemiology of HIV-1 in Santa Catarina State confirms increases of subtype C in Southern Brazil.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2015-02-11T16:22:38Z No. of bitstreams: 1 Locateli D. Molecular....pdf: 182648 bytes, checksum: 93e90c3cf5e279ef1db4c007c1ef8cbb (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-02-11T16:22:47Z (GMT) No. of bitstreams: 1 Locateli D. Molecular....pdf: 182648 bytes, checksum: 93e90c3cf5e279ef1db4c007c1ef8cbb (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-02-11T16:35:14Z (GMT) No. of bitstreams: 1 Locateli D. Molecular....pdf: 182648 bytes, checksum: 93e90c3cf5e279ef1db4c007c1ef8cbb (MD5)Made available in DSpace on 2015-02-11T16:35:14Z (GMT). No. of bitstreams: 1 Locateli D. Molecular....pdf: 182648 bytes, checksum: 93e90c3cf5e279ef1db4c007c1ef8cbb (MD5) Previous issue date: 2007UFSC. Laboratório de Imunologia Aplicada. Departamento de Microbiologia e Parasitologia. Florianópolis, SC, BrasilUFSC. Laboratórios de Protozoologia e de Bioinformática. Departamento de Microbiologia e Parasitologia. Florianópolis, SC, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório Avançado de Saúde Pública. Salvador, BA, Brasil / Escola Bahiana de Medicina e Saúde Pública. FBDC. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório Avançado de Saúde Pública. Salvador, BA, Brasil / Escola Bahiana de Medicina e Saúde Pública. FBDC. Salvador, BA, BrasilHospital Regional Homero de Miranda Gomes. São José, SC, BrasilUFSC. Laboratório de Imunologia Aplicada. Departamento de Microbiologia e Parasitologia. Florianópolis, SC, BrasilInstituto Adolfo Lutz. Laboratório de Retrovírus. São Paulo, SP, BrasilUFSC. Laboratórios de Protozoologia e de Bioinformática. Departamento de Microbiologia e Parasitologia. Florianópolis, SC, BrasilUFSC. Laboratório de Imunologia Aplicada. Departamento de Microbiologia e Parasitologia. Florianópolis, SC, BrasilRecent studies have demonstrated an increased prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C in southern Brazil. Although Santa Catarina State (SC) is located in this area and presents one of the country's highest incidences of HIV/AIDS, knowledge on the molecular epidemiology of HIV-1 in such State is lacking. The aim of this study was to investigate the HIV-1 molecular diversity and epidemiological profile of HIV-1-infected patients from SC. DNA samples were PCR amplified and HIV-1 subtypes were determined using both env and gag genes by direct sequencing. Phylogenetic analyses revealed that 48% were subtype C and 23% were subtype B. Possible recombinant forms were observed for both B/C (23%) and B/F (6%) subtypes. Our results, for the first time, identifies HIV-1 subtype C as a major clade circulating in SC and contributes to the understanding of HIV epidemics in the country by confirming the epidemic spread of the HIV-1 subtype C in southern Brazil

Swab pooling: A new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution.

To minimize sample dilution effect on SARS-CoV-2 pool testing, we assessed analytical and diagnostic performance of a new methodology, namely swab pooling. In this method, swabs are pooled at the time of collection, as opposed to pooling of equal volumes from individually collected samples. Paired analysis of pooled and individual samples from 613 patients revealed 94 positive individuals. Having individual testing as reference, no false-positives or false-negatives were observed for swab pooling. In additional 18,922 patients screened with swab pooling (1,344 pools), mean Cq differences between individual and pool samples ranged from 0.1 (Cr.I. -0.98 to 1.17) to 2.09 (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic patients were screened using 4,400 RT-qPCR assays. This corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests. Therefore, swab pooling represents a major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations

Number of gene clusters shared by the <i>T. rangeli</i>, <i>T. cruzi</i>, <i>T. brucei</i> and <i>L. major</i> genomes.

<p>Analyzes were performed using the following genome versions and gene numbers retrieved from the TriTrypDB: <i>Leishmania major</i> Friedlin (V. 7.0/8,400 genes), <i>Trypanosoma brucei</i> TREU927 (V. 5.0/10,574 genes), <i>Trypanosoma cruzi</i> CL Brener Esmeraldo (V. 7.0/10,342 genes) and Non-Esmeraldo (V. 7.0/10,834 genes). A total of 7,613 <i>T. rangeli</i> genes were used. BBH analysis used a cut-off value of 1e-05, positive similarity type and similarity value of 40% following manual trimming for comparison with COG analysis in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003176#pntd.0003176-ElSayed1" target="_blank">[55]</a> generating the numbers in the rectangles.</p