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DAIANI CRISTINA SAVI.Pdf 1 UNIVERSIDADE FEDERAL DO PARANÁ DAIANI CRISTINA SAVI GÊNERO Microbispora: RECLASSIFICAÇÃO FILOGENÉTICA, BIOPROSPECÇÃO E IDENTIFICAÇÃO DE METABÓLITOS SECUNDÁRIOS CURITIBA 2015 2 UNIVERSIDADE FEDERAL DO PARANÁ DAIANI CRISTINA SAVI GÊNERO Microbispora: RECLASSIFICAÇÃO FILOGENÉTICA, BIOPROSPECÇÃO E IDENTIFICAÇÃO DE METABÓLITOS SECUNDÁRIOS Tese apresentada ao Programa de Pós- Graduação em Microbiologia, Parasitologia e Patologia, Setor de Ciências Biológicas, Universidade Federal do Paraná como requisito parcial à obtenção de título de Doutora em Microbiologia. Orientadora: Profª Drª Chirlei Glienke Co-Orientadores: Drª Josiane Gomes Figueiredo e Dr Jurgen Rohr CURITIBA 2015 3 4 AGRADECIMENTOS A Deus pela oportunidade e ensinamentos; Aos meus pais e irmã pelo apoio, compreensão e incentivo; Ao Programa de Pós-graduação em Microbiologia, Parasitologia e Patologia pela oportunidade; À Profª Drª Chirlei Glienke pela confiança, respeito, paciência e ensinamentos que ajudaram a me moldar tanto quando pesquisadora como pessoa, gratidão eterna; À Profa Dra Lygia Vitória Galli Terasawa e à Profa Dra Vanessa Kava-Cordeiro por todo o ensinamento, paciência e disponibilidade dentro e fora do laboratório; Ao Prof Dr Jurgen Rohr, pela oportunidade, por me receber tão bem, paciência e orientação na parte química; À Drª Josiane Gomes Figueiredo, pela co-orientação, auxílio, ensinamentos e pela amizade; Ao Dr Khaled Shaaban por todo auxílio e ensinamentos no desenvolvimento da parte de identificação de compostos; Ao Rodrigo Aluízio pela amizade e auxílio nas analises filogenéticas; Ao Prof Dr Charles Windson Isidoro Haminiuk e a Profa Dra Sheila Winnischofer pelo auxílio com os testes de atividade antioxidante e antitumoral; À Profª Drª Andréa E. M. Stinghen pelo auxílio e orientação na prática de docência; Aos Professores do Programa de Pós-graduação em Microbiologia, Parasitologia e Patologia, que contribuíram para minha formação; 5 À Luciana Marques, secretária do Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia, pelas orientações e informações; À Ana Paula Chiaverini pelo auxílio técnico; Ao Prof Dr Emanuel Maltempi de Souza, pela facilidade do uso do sequenciador; Ao Valter Antonio Baura pelo auxílio técnico e científico na parte do sequenciamento; A todos os membros (atuais e de um passado remoto) do Laboratório de Genética de Microrganismos/UFPR pelo auxílio, paciência e momentos de descontração; A CAPES pelo auxílio financeiro; A todos que colaboraram com o desenvolvimento do trabalho e me incentivaram, meus sinceros agradecimentos. 6 “No problem can be solved from the same level of consciousness that created it” Albert Einstein 7 RESUMO A ioprospecção de microrganismos hoje é a fonte mais promissora para obtenção de novos compostos bioativos. Actinomicetos do solo pertencentes ao gênero Streptomyces, produzem uma vasta gama de compostos amplamente utilizados pela industría farmacêutica e agronômica. Porém, devido ao grande número de pesquisas envolvendo estes microrganismos a probablidade da descoberta de novos compostos a partir de linhagens isoladas do solo torna-se cada vez menos provável, levando a exploração de actinomicetos de outras fontes naturais, como por exemplo, isolados de plantas medicinais. Neste contexto, o presente trabalho objetivou o isolamento, bioprospecção, e a identificação de actinomicetos endofíticos da planta medicinal Vochysia divergens. O trabalho foi dividido em 3 capítulos voltados a bioprospecção e avaliação da atividade biológica dos isolados endofíticos - principalmente pertencentes ao gênero Microbispora - seguido de identificação dos compostos produzidos pela linhagem mais promissora e posterior estudo filogenético dos isolados pertencentes ao gênero Microbispora. O primeiro capítulo, intitulado “Antitumor, antioxidant and antibacterial activities of secondary metabolites extracted by endophytic actinomycetes isolated from Vochysia divergens”, explorou o isolamento e biodiversidade de actinomicetos isolados da planta Vochysia divergens, bem como a capacidade de seis isolados em produzir compostos com atividade antibacteriana, antioxidante e antitumoral. O segundo capítulo, “Microbispora sp. LGMB259 Endophytic Actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing β- Carbolines and Indoles with Biological Activity” objetivou o isolamento e identificação de compostos produzidos pela linhagem de Microbispora sp. LGMB259. No trabalho foram identificados sete compostos, quatro β-carbolinas e três indóis, sendo que o composto 1-vinil- β-carbolina-3-ácido carboxílico foi responsável pela atividade biológica desta linhagem, o qual apresentou atividade antibacteriana, antifúngica e citotóxica. O último capítulo, intitulado “Multilocus Sequence Analysis of the Genus Microbispora” baseou-se em análise multigênica utilizando os genes 16S rRNA, 23S rRNA, gyrB e rpoB para o estudo filogenético dentro do gênero Microbispora. As análises realizadas sugerem que as espécies M. amethystogenes, M. chromogenes, M. karnatakensis, M. parva, M. aerata, M. thermodiastatica e M. thermorosea são espécies distintas da espécie M. rosea; diferentemente do proposto na literatura por Miyadoh et al. (1990). M. aerata e M. thermodiastica compartilharam elevada similaridade genética e provavelmente pertencem a mesma espécie, assim como M. indica e M. rosea. As linhagens endofíticas pertencentes aos clusters Microbispora sp. 1, Microbispora sp. 2 e Microbispora sp. 3 são diferentes das espécies de Microbispora previamente descritas, sendo que mais estudos são necessários para a descrição das mesmas como novas espécies. Foi proposto também a análise concatenada dos genes gyrB-rpoB como uma alternativa à técnica de hibridização de DNA para a identificação de espécies dentro do gênero Microbispora, e o valor de 98,0% como o valor de homologia mínimo para classificar os isolados de Microbispora como pertencentes a mesma espécie. Palavras-chave: Vochysia divergens – Gênero Microbispora – metabólitos secundários – β- carbolinas – reclassificação filogenética 8 ABSTRACT The Bioprospecting of Microorganisms is the most promising source for new bioactive compounds. Actinomycetes - mainly that belonging to the genus Streptomyces - isolated from soil produced a wide range of bioactive compounds widely used by the pharmaceutical and agronomic industries. However, due to the large number of research involving these microorganisms the probablidade of discovery novel compounds isolated from soil strains is increasingly less, leading to exploration of actinomycetes isolated from others natural sources such as, endophytic isolated by medicial plants. In this context, our objectives were the isolation, bioprospecting, and the identification of endophytic actinomycetes from medicinal plant Vochysia divergens. The work was divided into three chapters and includes bioprospecting and avaliation of biological activity of endophytic isolates - mainly belonging to the genus Microbispora - followed by identification of compounds produced by the most promising strain and subsequent phylogenetic study of isolates belonging to Microbispora genus. The first chapter, entitled "Antitumor, antioxidant and antibacterial activities of secondary metabolites extracted by endophytic actinomycetes isolated from Vochysia divergens" is about the isolation and biodiversity of actinomycetes from the medicinal plant Vochysia divergens, and the ability of six isolates to produce compounds with antibacterial, antioxidant and antitumor activities. The second chapter, "Microbispora sp. LGMB259 Endophytic actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing β-Carbolines and Indoles with Biological Activity" aimed to isolate and identify compounds produced by the strain Microbispora sp. LGMB259. We identified seven compounds produced by this strain - four β-carbolines and three indoles- wherein the compound 1-vinyl-β-carboline-3-carboxylic acid was responsible for the biological activities of this strain, which showed antibacterial, antifungal and cytotoxic activities. The last chapter, entitled "Multilocus Sequence Analysis of the Genus Microbispora" was based on multigene analysis using the 16S rRNA, 23S rRNA, gyrB and rpoB for phylogenetic studies in the Microbispora genus. The analyzes suggest that the species M. amethystogenes, M. chromogenes, M. karnatakensis, M. parva, M. aerata, M. thermodiastatica and M. thermorosea are distinct from M. rosea; different by proposed for Miyadoh et al. (1990). However M. aerata and M. thermodiastatica probably are the same species, as well M. indica and M. rosea. Endophytic isolates belongs to the clusters Microbispora sp. 1, Microbispora sp. 2 and Microbispora sp. 3 are different from the Microbispora species previous described and futures studies are required to describe these species. It is also proposed the concatenated analyses of gyrB-rpoB genes as a useful alternative to DNA–DNA hybridization for the identification and phylogenetic analysis in the Microbispora genus, and values less than 98,0% to characterizate and determine relationship at species level. Key-words: Vochysia divergens – Microbispora Genus – secondary metabolites – β- carbolines – phylogenetic reclassification 9 SUMÁRIO I. JUSTIFICATIVA 10 II. OBJETIVOS 11 III. METAS 11 IV. INTRODUÇÃO 12 V. REVISÃO DA LITERATURA 15 Vochysia divergens e o Pantanal Sul Mato-Grossense 15 Microrganismos endofíticos 17 Bioprospecção 18 Metabólitos secundários 20 Gênero Microbispora 21 VI. CAPÍTULO 1 “Antitumor, antioxidant and
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