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BARBARA MORIEL.Pdf UNIVERSIDADE FEDERAL DO PARANÁ BÁRBARA MORIEL SEQUENCIAMENTO E COMPARAÇÃO DO GENOMA DE Aeromonas caviae 8LM CURITIBA 2017 BÁRBARA MORIEL SEQUENCIAMENTO E COMPARAÇÃO DO GENOMA DE Aeromonas caviae 8LM Tese apresentada como requisito parcial à obtenção do grau de Doutor em Ciências Farmacêuticas, no Curso de Pós-Graduação em Ciências Farmacêuticas, Setor de Ciências da Saúde, da Universidade Federal do Paraná. Orientadores: Profa. Dra. Cyntia Maria Telles Fadel-Picheth Prof. Dr. Leonardo Magalhães Cruz CURITIBA 2017 Moriel, Bárbara Sequenciamento e comparação do genoma de Aeromonas caviae 8LM / Bárbara Moriel – Curitiba, 2017. 148 f. ; il. (algumas color.) ; 30 cm Orientadora: Professora Dra. Cyntia Maria Telles Fadel-Picheth Coorientador: Professor Dr. Leonardo Magalhães Cruz Tese (doutorado) – Programa de Pós-Graduação em Ciências Farmacêuticas, Setor de Ciências da Saúde. Universidade Federal do Paraná. Inclui bibliografia 1. Aeromonas. 2. Genoma. 3. Genômica. I. Fadel-Picheth, Cyntia Maria Telles. II. Cruz, Leonardo Magalhães. III. Universidade Federal do Paraná. IV. Título. CDD 616.014 AGRADECIMENTOS Gostaria de agradecer os meus orientadores, Profª. Cynthia e Prof. Leonardo da Universidade Federal do Paraná, pela oportunidade de trabalhar com esse projeto tão desafiador. Ao Prof. Rupert Mutzel da Freie Universität Berlin e Prof. Torsten Semmler do Intituto Robert Koch em Berlin pela oportunidade de realizar o estágio de doutorado sanduíche. Agradeço também o Programa de Pós-Gradução em Ciências Farmacêuticas e o Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Paraná, onde foram realizadas as análises desse projeto. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Capes, pelo aporte financeiro durante o doutoramento. Às minhas colegas de laboratório pela ajuda com as análises e as agradáveis conversas durante todos esses anos. Aos Professores Dieval, Rodrigo e Helisson, do Departamento de Bioquímica e Biologia Molecular da Universidade Federal do Paraná, pela orientação e grande auxílio nas análises de bioinformática. Ao Douglas por toda ajuda e paciência principalmente em explicar como lidar com as ferramentas de bioinformática. À minha família e amigos pelo suporte durante todos esses anos. “A ciência nunca resolve um problema sem criar pelo menos outros dez." (George Bernard Shaw) RESUMO Aeromonas são bacilos gram-negativos, encontrados em ambientes aquáticos, capazes de causar diversas infecções em humanos destacando-se a diarréia. Os objetivos deste trabalho foram sequenciar o genoma da estirpe A. caviae 8LM, isolada a partir de fezes diarreicas, e realizar análises de genômica comparativa. O sequenciamento foi realizado empregando os sistemas 454 GS Junior, Illumina MiSeq e PacBio. Nas análises de genômica comparativa foram empregadas diversas ferramentas de bioinformática para análise estrutural e do conteúdo gênico de estirpes de Aeromonas. O genoma de A. caviae 8LM apresenta 4,6Mb, conteúdo GC de 61,7%, dispondo de 4.101 CDS e 10 operons de rRNA distribuídos próximos da origem de replicação. Foram identificados diversos genes relacionados com virulência incluindo motilidade e adesão, toxinas, sistemas de secreção tipo II e VI, entre outros. As análises de genômica comparativa mostram a similaridade do genoma de A. caviae 8LM com outras estirpes da espécie. As análises realizadas indicaram divergências na identificação de algumas Aeromonas e outras, identificadas apenas ao nível de gênero puderam ser identificadas ao nível de espécie. O pan-genoma de Aeromonas é formado por 9.785 genes e o core genoma 701 genes. Foram detectadas regiões do genoma que podem conter possíveis marcadores moleculares para a espécie A. caviae. Os resultados deste trabalho trazem informações sobre o genoma e virulência de A. caviae 8LM, e sugerem a presença de possíveis marcadores moleculares com potencial para o desenvolvimento de testes diagnósticos. Palavras-chave: Aeromonas. Genoma completo. Genômica comparativa. ABSTRACT Aeromonas are gram-negative bacilli, found in aquatic environments, are capable of causing several human infections, highlighting diarrhea. The proposes of this work were to sequence the genome of strain A. caviae 8LM, isolated from diarrheal feces, and carry out comparative genomics analyzes. Sequencing was performed applying the 454 GS Junior, Illumina MiSeq and PacBio systems. In comparative genomics analyzes, several bioinformatics tools were applied for structural and gene content analysis of Aeromonas strains. The genome of A. caviae 8LM presents 4.6Mb, GC content of 61.7%, 4,101 CDS and 10 distributed rRNA operons close to the origin of replication. Several genes related to virulence including motility and adhesion, toxins, type II and VI secretion systems, among others were identified. Comparative genomics analyzes exhibited the similarity of the genome of A. caviae 8LM with other strains of the species. The analyzes indicated differences in identification of some Aeromonas and others that were only identified at genus level could be identified at species level. The pan-genome of Aeromonas consists of 9,785 genes and the core genome 701 genes. Genomic regions containing possible molecular markers for A. caviae species were detected. The results of this work provide information about the genome and virulence of A. caviae 8LM, and suggest the presence of possible molecular markers with potential for the development of diagnostic tests. Key-words: Aeromonas. Complete genome. Comparative genomics. LISTA DE FIGURAS FIGURA 1 – SISTEMAS DE SECREÇÃO EM BACTÉRIAS ..................................... 27 FIGURA 2 – SEQUENCIA DE dnaA box EM A. caviae 8LM .................................... 49 FIGURA 3 – DISTRIBUIÇÃO DOS OPERONS DE rRNA EM A. caviae 8LM .......... 52 FIGURA 4 – BLAST-ATLAS DE Aeromonas COM GENOMAS COMPLETOS ........ 61 FIGURA 5 – PRESENÇA/AUSÊNCIA DE SEQUÊNCIAS NO PAN-GENOMA DE Aeromonas COM GENOMA COMPLETO................................................................. 66 FIGURA 6 – NÚMERO DE SEQUENCIAS ÚNICAS NAS ESPÉCIES DE Aeromonas ................................................................................................................................... 67 FIGURA 7 – PRESENÇA/AUSÊNCIA DE GENES NO PAN-GENOMA DE Aeromonas ................................................................................................................ 71 FIGURA 8 – ÁRVORE FILOGENÉTICA DOS GENES HOUSEKEEPING ............... 74 FIGURA 9 – ÁRVORE FILOGENÉTICA DOS GENES DO CORE GENOMA .......... 76 FIGURA 10 – ORGANIZAÇÃO GÊNICA DO FLAGELO LATERAL ......................... 81 FIGURA 11 – ORGANIZAÇÃO GÊNICA DO FLAGELO POLAR ............................. 82 FIGURA 12 – ORGANIZAÇÃO GÊNICA DO T6SS .................................................. 83 LISTA DE GRÁFICOS GRÁFICO 1 – NÚMERO DE GENOMAS DE Aeromonas DEPOSITADOS NO NCBI ................................................................................................................................... 46 GRÁFICO 2 – ANOTAÇÃO DO GENOMA DE A. caviae 8LM PELO RAST ............ 48 GRÁFICO 3 – CURVAS Z PARA O GENOMA DE A. caviae 8LM ........................... 49 GRÁFICO 4 – DOTPLOT ENTRE A. caviae E A. caviae 8LM .................................. 57 GRÁFICO 5 – DOTPLOT ENTRE A. hydrophila E A. caviae 8LM ............................ 58 GRÁFICO 6 – DOTPLOT ENTRE AS DEMAIS Aeromonas E A. caviae 8LM .......... 59 GRÁFICO 7 – NÚMERO DE GENES NO PAN-GENOMA DE Aeromonas COM GENOMAS COMPLETOS ......................................................................................... 65 GRÁFICO 8 – COMPRIMENTO DAS SEQUENCIAS DE AMINOÁCIDOS .............. 73 LISTA DE TABELAS TABELA 1 – NOMENCLATURA DOS GENES DO T6SS ......................................... 29 TABELA 2 – TESTES BIOQUÍMICOS PARA IDENTIFICAÇÃO DE A. caviae 8LM . 31 TABELA 3 – GENES HOUSEKEEPING UTILIZADOS PARA A RECONSTRUÇÃO FILOGENÉTICA ........................................................................................................ 41 TABELA 4 – DADOS DO SEQUENCIAMENTO DE A. caviae 8LM ......................... 44 TABELA 5 – GENOMAS DE Aeromonas spp. SEQUENCIADOS ............................ 47 TABELA 6 – OPERONS DE RNA RIBOSSOMAL EM A. caviae 8LM ...................... 50 TABELA 7 – MAIORES VALORES DE GGDC–DDH ENTRE Aeromonas ............... 54 TABELA 8 – MAIORES VALORES DE ANI ENTRE Aeromonas ............................. 55 TABELA 9 – ILHAS GENÔMICAS DE A. caviae 8LM .............................................. 62 TABELA 10 – NOVA IDENTIFICAÇÃO DAS ESTIRPES DE Aeromonas ................ 77 TABELA 11 – FATORES DE VIRULÊNCIA EM A. caviae 8LM ................................ 79 LISTA DE ABREVIATURAS E SIGLAS ANI - Identidade média de nucleotídeos ATP - Adenosina trifosfato BLAST - Basic Local Alignment Search Tool BLASTp - Protein Basic Local Alignment Search Tool cAMP - Adenosina monofostato cíclico CDS - Sequência codificadora de ácido desoxirribonucleico DDH - Hibridização ácido desoxirribonucleico – ácido desoxirribonucleico DNA - Ácido desoxirribonucleico GGDC - Genome-to-Genome Distance Calculator HSPs - Segmentos de alta pontuação MCL - Markov Cluster Algorithm MUMmer - Maximal Unique Matches NCBI - National Center for Biotechnology Information RAST - Rapid Annotation using Subsystem Technology RNA - Ácido ribonucleico rRNA - Ácido ribonucleico ribossômico T2SS - Sistema de secreção tipo II T3SS - Sistema de secreção tipo III T6SS - Sistema de secreção tipo VI tRNA - Ácido ribonucleico transportador SUMÁRIO 1 INTRODUÇÃO ............................................................................................
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