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1 Universidade De São Paulo Faculdade De Medicina De 1 UNIVERSIDADE DE SÃO PAULO FACULDADE DE MEDICINA DE RIBEIRÃO PRETO PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA CELULAR E MOLECULAR MARISTELA PREVIATO MELLO Caracterização funcional de fatores de transcrição da família MarR de Chromobacterium violaceum Ribeirão Preto 2018 2 UNIVERSIDADE DE SÃO PAULO FACULDADE DE MEDICINA DE RIBEIRÃO PRETO PROGRAMA DE PÓS-GRADUAÇÃO EM BIOLOGIA CELULAR E MOLECULAR MARISTELA PREVIATO MELLO Caracterização funcional de fatores de transcrição da família MarR de Chromobacterium violaceum Orientador: Prof. Dr. José Freire da Silva Neto Ribeirão Preto 2018 1 MARISTELA PREVIATO MELLO Caracterização funcional de fatores de transcrição da família MarR de Chromobacterium violaceum Versão Original Tese apresentada à Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo para obtenção do título de Doutor em Ciências. Área de concentração: Biologia Celular e Molecular Orientador: Prof. Dr. José Freire da Silva Neto Ribeirão Preto 2018 2 Autorizo a reprodução e divulgação total ou parcial deste trabalho, por qualquer meio convencional ou eletrônico, para fins de estudo e pesquisa, desde que citada a fonte. Previato-Mello, Maristela Caracterização funcional de fatores de transcrição da família MarR de Chromobacterium violaceum. Ribeirão Preto, 2018. 151 p. : il. ; 30 cm Tese de Doutorado, apresentada à Faculdade de Medicina de Ribeirão Preto/USP. Área de concentração: Biologia Celular e Molecular. Orientador: da Silva Neto, José Freire. 1. Biologia Celular. 2. Biologia Molecular. 3. Microbiologia. 4. Regulação gênica. 5. Reguladores transcricionais da família MarR. 3 Candidata: PREVIATO-MELLO, Maristela. Título da Tese: Caracterização funcional de fatores de transcrição da família MarR de Chromobacterium violaceum. Tese apresentada à Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo para obtenção do título de Doutor em Ciências. Área de concentração: Biologia Celular e Molecular Avaliada em: Banca Examinadora Prof. Dr. ____________________________________________________________ Instituição: __________________________________________________________ Julgamento: _________________________________________________________ Prof. Dr. ____________________________________________________________ Instituição: __________________________________________________________ Julgamento: _________________________________________________________ Prof. Dr. ____________________________________________________________ Instituição: __________________________________________________________ Julgamento: _________________________________________________________ Prof. Dr. ____________________________________________________________ Instituição: __________________________________________________________ Julgamento: _________________________________________________________ Prof. Dr. ____________________________________________________________ Instituição: __________________________________________________________ Julgamento: _________________________________________________________ 4 5 Dedico este trabalho às pessoas que amo: meus pais Lourdes e Laerte, minhas irmãs Mariângela e Mariana e meu marido Adriano. 6 AGRADECIMENTOS Ao Prof. Dr. José Freire da Silva Neto pela excelente orientação, dedicação, ensinamentos, confiança, compreensão, companhia e principalmente pelas longas e quase diárias conversas sobre nosso trabalho. Aos amigos de laboratório Kelly, Juliana, Greicy, Bianca, Júlia, Renato, Carlos e Waldir e amigos de corredor Relber, Flávia e Lívia pela feliz convivência diária, risadas, celebrações e aprendizado. À técnica Claudia R. M. Faggion por todo auxílio, preocupação, risadas e conversas gostosas. A todos os docentes, funcionários e alunos da USP-Ribeirão Preto que compartilharam conhecimento comigo. A todos os alunos que passaram pelo laboratório e me deram a oportunidade de ensinar e aprender durante os cursos de verão, iniciação científica ou matéria do curso de Ciências Biomédicas. Às secretarias Gabriela B. Zamoner, Lúcia H. P. Raphael e Camila Fabris, pela competência e atenção ao me ajudarem nos processos burocráticos. Ao Prof. Dr. Luis E. S. Netto e ao pós-doutorando Dr. Diogo de A. Meireles do Instituto de Biociênicas (IB-USP) pela colaboração em meu primeiro artigo. Aos professores Dr. Paulo S. R. Coelho, Dr. Dario S. Zamboni, Dr.ª Angela K. Cruz, Dr.ª Maria Cristina R. A. Barreira, Dr. Luis Lamberti P. da Silva, Dr.ª Maria Celia Jamur, Dr.ª Maria Luisa Paçó-Larson, Dr.ª Munira M. A. Baqui, Dr.ª Mariana K. Osako, Dr. Rafael S. Rocha e Dr. Marcelo D. Gomes por disponibilizarem seus equipamentos, reagentes ou espaço físico. Ao professor Dr. Geraldo A. Passos e as alunas Ma. Nicole Pezzi e Dr.ª Amanda F. Assis por realizarem as leituras das lâminas do ensaio de Microarranjo de DNA e extraírem os dados. Aos professores Dr. Luiz R. O. Tosi e Dr. Rafael S. Rocha e ao pós-doutorando Dr. Warrison A. C. de Andrade por participarem da minha banca de qualificação e auxiliarem com sugestões. Ao Sr. Domingos S. de Souza Filho por manter o biotério de camundongos e sempre ajudar, e à Dr.ª Katiuchia U. Sales por adequar as regras para uso do biotério. Ao serviço de esterilização, representado pelas técnicas Maria Helena da S. Martins e Joana S. de Souza, por toda ajuda. 7 Às técnicas Ma. Tânia P. A. Defina, Viviane Ambrósio e Silvia R. A. Nascimento por todo auxílio durante os experimentos com material radioativo. Aos técnicos Vani M. Alves e Antonio R. Meirelles por toda ajuda e ensinamentos durante o experimento de histologia. À técnica Dr. Roberta Ribeiro Costa Rosales pelo treinamento e auxílio para o uso do microscópio IVIS Spectrum. Às técnicas Maira C. Nakamura e Sandra M. Oliveira Thomaz por sempre permitirem o uso dos equipamentos e reagentes dos laboratórios onde são responsáveis. À Faculdade de Medicina de Ribeirão Preto, ao Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos e ao Programa de Pós-Graduação em Biologia Celular e Molecular pela infraestrutura para realização da minha pesquisa. Aos amigos da faculdade Naila, Laís, Renata e Tiago pela torcida e amizade. Aos alunos e à Prof. Dr.ª Marilis do Valle Marques do Laboratório de Fisiologia e Genética Bacteriana, por tudo que me ensinaram durante o mestrado. Aos amigos Vânia e José por me acolherem durante o período das provas para o ingresso no departamento, pelas caronas e almoços compartilhados. Às pessoas da minha família e amigos que sempre torcem por mim. Aos familiares queridos que partiram durante essa caminhada. Às minhas queridas irmãs Mariângela e Mariana por todo carinho, incentivo, preocupação e amor. Aos meus pais Lourdes e Laerte por todo empenho, dedicação e amor para que todas nós estudássemos. Ao meu querido esposo Adriano Mello pela confiança, desprendimento, força, cumplicidade, companheirismo e amor. 8 AGRADECIMENTOS PELO APOIO FINANCEIRO À Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), pelo auxílio financeiro (processo nº 2012/20435-9) para o desenvolvimento desta pesquisa e pela concessão da bolsa de doutorado (processo nº 2013/25745-9). À Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da FMRP-USP (FAEPA) pelo auxílio financeiro destinado ao Laboratório de Regulação da Expressão Gênica e Patogenicidade Bacteriana. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), fundação do Ministério da Educação (MEC), pelo apoio institucional ao Programa de Pós-Graduação em Biologia Celular e Molecular e ao Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos da Faculdade de Medicina de Ribeirão Preto. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pelo apoio institucional ao Programa de Pós-Graduação em Biologia Celular e Molecular e ao Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos da Faculdade de Medicina de Ribeirão Preto. 9 RESUMO PREVIATO-MELLO, Maristela. Caracterização funcional de fatores de transcrição da família MarR de Chromobacterium violaceum. 2018. 151 f. Tese (Doutorado em Biologia Celular e Molecular) - Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, 2018. Os fatores de transcrição da família MarR atuam como sensores diretos de sinais intracelulares e regulam vários processos em bactérias, incluindo virulência e degradação de compostos aromáticos. Neste trabalho, identificamos de modo global os fatores de transcrição da família MarR envolvidos na virulência do patógeno oportunista de humanos Chromobacterium violaceum. Usando mutagênese por troca alélica, geramos mutantes nulos não polares para doze dos quinze reguladores da família MarR encontrados no genoma de C. violaceum. Em ensaios de virulência, quando injetados por via intraperitoneal em camundongos BALB/c, os mutantes ΔCV_0210 (ΔohrR), ΔCV_0577 e ΔCV_2726 foram menos virulentos, enquanto o mutante ΔCV_1776 foi mais virulento, quando comparados à linhagem selvagem. Para os demais nove mutantes MarR não houve diferença na virulência. Para definir o regulon de alguns destes reguladores da família MarR, os perfis de expressão gênica foram determinados por ensaios de microarranjo de DNA e Northern blot para as linhagens mutantes ΔCV_0210 (ΔohrR), ΔCV_1776, ΔCV_1810 e ΔCV_2726, para a linhagem selvagem superexpressando CV_2726 e para a linhagem selvagem em estresse oxidativo com hidroperóxido de cumeno (CHP). O regulon do repressor CV_1810 compreendeu dois operons divergentes, que codificam enzimas que possivelmente metabolizam compostos aromáticos, mas produtos do catabolismo
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