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Universidade Estadual De Campinas Instituto De Biologia UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE BIOLOGIA MARINA AIELLO PADILLA ATIVIDADE ANTIVIRAL DE EXTRATOS PRODUZIDOS DE BACTÉRIAS ISOLADAS E COLETADAS EM CUPINZEIROS FRENTE A VÍRUS DE IMPORTÂNCIA HUMANA E ANIMAL ANTIVIRAL ACTIVITY OF EXTRACTS PRODUCED BY ISOLATED BACTERIA COLLECTED IN TERMITE MOUNDS AGAINST IMPORTANT HUMAN AND ANIMAL VIRUSES CAMPINAS 2015 MARINA AIELLO PADILLA ATIVIDADE ANTIVIRAL DE EXTRATOS PRODUZIDOS DE BACTÉRIAS ISOLADAS E COLETADAS EM CUPINZEIROS FRENTE A VÍRUS DE IMPORTÂNCIA HUMANA E ANIMAL ANTIVIRAL ACTIVITY OF EXTRACTS PRODUCED BY ISOLATED BACTERIA COLLECTED IN TERMITE MOUNDS AGAINST HUMAN AND ANIMAL VIRUSES Tese apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do Título de Doutora em Genética e Biologia Molecular na área de Microbiologia Thesis presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Genetics and Molecular Biology in Microbiology area ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA TESE PELA ALUNA MARINA AIELLO PADILLA E ORIENTADA PELA PROFA. DRA. CLARICE WEIS ARNS CAMPINAS 2015 Campinas, 04 de dezembro de 2015. COMISSÃO EXAMINADORA Profa. Dra. Clarice Weis Arns Profa. Dra.Carmen Lucia Queiroga Prof. Dr. José Luiz Proença Modena Profa. Dra. Isabela Cristina Simoni Prof. Dr. Eduardo Furtado Flores Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica do aluno. DEDICATÓRIA À todos os meus amores. AGRADECIMENTOS À Deus por toda a força que me deu nessa jornada e por ter colocado pessoas tão especiais na minha vida. Meus pais, Telma e Miguel, agradeço simplesmente por ter o privilégio de ter vocês como pais. Nada seria possível, ou pelo menos não teria a mesma satisfação, se não pudesse ser compartilhado com vocês. “ Amo tanto e de tanto amar...” À toda a minha família: meus irmãos, cunhadas, minha avó Terezinha que acreditaram e me apoiaram nessa jornada, como em todas as outras. Vocês são essenciais! Aos meus sobrinhos lindos: Miguel, Alice e Bebel. Vocês me ensinam, me ajudam (mesmo sem perceber) e me mostraram um novo jeito de amar: o amor de tia coruja! Amo mais que demais. Ao Marco, principalmente pela “paciência com a Mari” (potássio), mas também pelos momentos de risadas, conselhos, companheirismo, cumplicidade........Obrigada! Te quiero. À minha orientadora, profa. Dra. Clarice, pela oportunidade, confiança, incentivo, ensinamentos, conversas e convivência. Aprendi muito durante todo esse período que estamos trabalhando juntas. Muito obrigada! Aos meus amigos mais que especiais Matheus, Fernanda que sempre estiveram do meu lado e me ajudam em todos os sentidos para que as coisas dêem certo. Vocês são incríveis! Aos meus amigos lindos que eu amo tanto: Flora, Buchalla, Sônia, Henrique, Rodrigo, Priscila, Eduardo, Lara, Diego, Michele, Juliana. Obrigada pelo amor, pela amizade e cumplicidade. Aos meus amigos queridos e grande equipe do LVA. Não só companheiros de trabalho, mas também de boas risadas! Matheus, Juliana, Ana Paula, Ana Caroline, Raíssa, Paulo Simas, Leonardo, Paula, Luciana, Priscila e todos outros que passaram por lá. Aos meus pequenos: Wood e Erick, que apesar do trabalho que as vezes me dão, são meus companheiros de casa. A vida é melhor sabendo que quando estou em casa estarei na companhia de vocês. Aos meus anjinhos que olham e cuidam de mim: meus avôs Cadorna e Miguel, minha avó Catharina e o Kidinho. Aos amigos e colaboradores que ganhei com as etapas realizadas no CPQBA: toda a equipe do Dr. Rodney na DQPN e a equipe da Dra. Fabiana na DRM. Obrigada por me receberem tão bem, pela paciência, pelos ensinamentos, pelas ajudas, pelas conversas, risadas e cafés. Agradeço também aos professores que gentilmente se disponibilizaram a participar da avaliação da minha tese. Tenho grande admiração por vocês e os tenho como exemplos de profissionais. À coordenação do curso de pós graduação em Genética e Biologia Molecular À FASPESP e CNPq pelo suporte financeiro. À UNICAMP pela formação científica. À todos que de alguma forma estão do meu lado e me ajudam a ser uma pessoa melhor, MUITO OBRIGADA!!!!! “A frase mais excitante que se pode ouvir na ciência, a que anuncia novos descobrimentos, não é Eureka! (o achei!) senão “é estranho”...” Issac Asimov RESUMO As infecções virais acometem tanto humanos quanto animais. Problemas associados a falta de vacinas e o desenvolvimento de resistência aos medicamentos estimulam a pesquisa por compostos antivirais. As bactérias são responsáveis pela produção de inúmeros compostos com atividades biológicas importantes, portanto, esse trabalho teve como objetivo a busca por compostos antivirais produzidos por bactérias coletadas em cupinzeiros. Extratos provenientes de diferentes isolados de bactérias associadas ao cupim foram avaliados quanto a atividade antiviral in vitro frente a três vírus de importância para a saúde humana e animal: herpes humano tipo 1 (HHV-1), calicivírus felino (FCV)- modelo para o norovírus humano- e o vírus da diarréia viral bovina (BVDV) – modelo para o vírus da hepatite C. Dos 90 extratos analisados, 10% apresentaram porcentagem de inibição (PI%) ≥ 98% contra pelo menos um dos vírus: cinco foram ativos frente ao HHV-1, três apresentaram atividade contra o FCV e dois foram ativos frente ao BVDV. Das espécies ativas, seis pertencem ao gênero Streptomyces , com uma delas identificada como S. chartreusis e quatro identificadas como sendo do gênero Achromobacter. Três extratos apresentaram índice de seletividade (IS) acima de 3,5 contra o HHV-1 com destaque ao CDPA10 ( Streptomyces sp) com IS=17,10. Frente ao FCV e o BVDV dois extratos foram considerados promissores: o LC22 (IS=11,00), identificado como Streptomyces sp e o CDPA27 ( S. chartreusis ) com IS=3,5, respectivamente. Os extratos que destacaram-se quanto aos valores e IS foram também avaliados em uma triagem de mecanismo de ação. Dos três extratos com IS acima de 3,5 frente ao HHV-1, um apresentou atividade antiviral no pós tratamento. Os outros,incluindo o CDPA10 agem como inativadores virais. O extrato LC22, ativo contra o FCV apresentou atividade no pós tratamento e também como inativador viral. Por fim, o extrato proveniente do S. chartreusis (CDPA27), agiu no pós tratamento frente ao BVDV. Esses três extratos, CDPA10, LC22 e CDPA27 foram selecionados para o fracionamento em coluna C 18 fase reversa em ordem crescente de metanol, originando seis frações. Todas as frações foram avaliadas contra seus respectivos vírus e como resultado foi possível observar que a fração 6 (100% metanol) foi a mais promissora dos três extratos. Essa fração do CDPA10 e do CDPA27, ativos contra o HHV-1 e BVDV respectivamente, apresentaram valor de PI=99%, enquanto a fração 6 do extrato LC22 (ativo contra o FCV) apresentou PI=90%. Nas avaliações quanto ao IS dessas frações promissoras, observou-se que os valores aumentaram consideravelmente tanto para o CDPA10 como para o CDPA27, já a fração do LC22 teve o valor de IS reduzido para 3,24. Finalmente, todos os extratos e suas frações promissoras foram analisados por cromatografia gasosa acoplada a espectrometria de massa (CG-MS). Esta análise, quando comparada aos compostos já descritos na biblioteca NIST/2005, não permitiu a identificação dos compostos devido a baixa similaridade. Estudos mais detalhados devem ser realizados a fim de identificar as espécies bacterianas e seus respectivos compostos ativos. Os resultados obtidos revelam novas perspectivas para a pesquisa antiviral a partir de produtos naturais e demonstra o potencial antiviral de compostos produzidos por bacterias associadas ao cupim. Palavras-chave: atividade antiviral, cupinzeiro, bactérias, HHV-1, BVDV, FCV, Streptomyces ABSTRACT Viral infections affect both humans and animals. Problems associated with a lack of vaccines and development of drug resistance stimulates the research for antiviral compounds. Bacteria are responsible for producing many compounds with important biological activities, so this study aimed to research for antiviral compounds produced by isolated associated bacteria. Extracts from different isolated termite associated bacteria were evaluated for in vitro antiviral activity against three important viruses to human and animal health: human herpesvirus type 1 (HHV-1), feline calicivirus (FCV) - surrogate model for human norovirus -and bovine viral diarrhea virus (BVDV) - surrogate model for hepatitis C virus. Of 90 extracts analyzed, 10% had percentage of inhibition (PI%) ≥ 98% against at least one virus: five were active against HHV-1, three showed activity against FCV and two were active against BVDV. Of the active species, six belong to Streptomyces genus, with one of them identified as S. chartreusis and four were identified as Achromobacter genus. Three extracts showed selectivity index (SI) above 3.5 against HHV-1, highlighting CDPA10 (Streptomyces sp) with SI = 17.10. Against the FCV and BVDV only two extracts were considered promising: LC22 (SI = 11.00), identified as Streptomyces sp and CDPA27 ( S. chartreusis ) with SI = 3.5, respectively. The extracts that stood out about the values of SI were also evaluated in a screening of mechanism of action. Of the three extracts with SI above 3.5 against HHV-1, only one showed antiviral activity in post-treatment, the others, including CDPA10 act as virus inactivation. The LC22, extract active against FCV, showed activity in the post-treatment as well as virus inactivation.
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