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Functional Genomics of Mycobacteria: Genomics x Proteomics x Diagnosis Simpósio Imunopatogenia e Pesquisa Básica III Encontro Nacional de Tuberculose Salvador.

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Presentation on theme: "Functional Genomics of Mycobacteria: Genomics x Proteomics x Diagnosis Simpósio Imunopatogenia e Pesquisa Básica III Encontro Nacional de Tuberculose Salvador."— Presentation transcript:

1 Functional Genomics of Mycobacteria: Genomics x Proteomics x Diagnosis Simpósio Imunopatogenia e Pesquisa Básica III Encontro Nacional de Tuberculose Salvador - Junho 2008 Leila Mendonça-Lima Lab. Genômica Funcional e Bioinformática Instituto Oswaldo Cruz - FIOCRUZ FIOCRUZ

2 Functional Genomics: Schwann cells infected with live M. leprae Correlating genomic information to the organisms behavior in the situation under study. How genomic information translates into the pattern of protein expression. FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute How this pattern of protein expression affects host-pathogen interaction. Mendonça-Lima & Barker

3 Jan FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

4 Mycobacterial genomes: FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

5

6 Jean-Marie Camille Guérin (1872 – 1961) Léon Charles Albert Calmette ( ) B acille de C almette et G uérin

7 BCG Vaccine Over 3 billion people vaccinated with BCG Safe, inexpensive, can be administered soon after birth Immunization with dead BCG does not protect Efficacy varies (0 - 80% in adults) Globally, BCG prevents only 5% of all potencially avoidable deaths due to TB In Brazil: BCG Moreau In use since 1930 Fundação Ataulpho de Paiva (FAP) produces > 17million doses/year >90% coverage for BCG vaccination In Brazil: BCG Moreau In use since 1930 Fundação Ataulpho de Paiva (FAP) produces > 17million doses/year >90% coverage for BCG vaccination FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

8 M. bovis (1908) Russia (1924) Moreau (1925) Japan (1925) Sweden (1926) Birkhaug (1927) Prague (1947) Glaxo (1954) Danish (1931) Tice (1934) Connaught (1948) Frappier (1937) Phipps (1938) Pasteur (Liofilizada 1961) RD2 RD1 BCG (1921) RD14 RD16 RD8 sigK mma3 IS serial passages in potato/glycerin/bile medium CRP (HTH) CRP (cNMP) 1927 Adapted from Behr et al, Science 1999 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

9 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute BCG Moreau Genome Sequencing

10 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute BCG Moreau Genome Sequencing

11 Contig distribution (vs. M. bovis) FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute BCG Moreau Genome Sequencing: Shotgun

12 Design of specific oligonucleotides for each region Chromosome walking PCR > DNA sequencing Specific sequencing protocols for each region BCG Moreau Genome Sequencing: Finishing FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

13 Assembled genome: bp % G+C = Assembled genome: bp % G+C = Jacenir R.S.Malle FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute BCG Moreau Genome Sequencing bp included in the assembly bp high quality High quality coverage = 6x Overall coverage = 9x

14 RD16 RD01 RD02 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute BCG Moreau Genome Sequencing

15 DNA-based tools for quality control FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Patente de invenção, nºPI Depósito de patente internacional, PCT/BR 2007/000322

16 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute BCG Moreau Genome Annotation:

17 stop FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute BCG Moreau Functional Genomics BCG evolution = loss of genomic information Loss of genomic information sometimes = functional gain InDels:

18 Proteomics Plataforma de Eletroforese 2D PDTIS-FIOCRUZ Lab. Genômica Funcional e Bioinformática IOC - FIOCRUZ FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

19 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute pI =7 1D 2D MW pI 2D-Electrophoresis

20 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Proteomics

21 sup caps sup - Tw+ Tw Extracellular proteins are available sooner during infection for modulation of hosts response FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Mycobacterial Proteomics Mendonça-Lima & Barker

22 cytoplasm Cell membrane Cell wall capsule or surface Extracellular medium Abdallah et al., Nat Rev Microbiol 2007 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Mycobacterial Fractionation

23 Culture filtrate proteins Remove culture medium 0.2um filter Bacterial veils Surface-associated proteins Mechanical agitation Centrifugation 15 day cultures in protein-free medium FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Mycobacterial Fractionation

24 Moreau Pasteur SautonIVM FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Proteomics of the BCG Moreau vaccine

25 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Proteomics of the BCG Moreau vaccine

26 MoreauPasteur FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Culture filtrate proteins

27 MoreauPasteur FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Surface-associated proteins

28 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Web interface

29 IEF 7cm pH 4 -7 SDS-PAGE (2ºD) 12% Coomassie G-250 Transfer of proteins to nitrocellulose membrane Blocking: Anti-human-IgG peroxidase conjugate Reversible protein stain Pooled sera (IgG) Chemiluminescence substrate Super Signal West Pico Image detecting GS-800 Matching signals for immunoblots and proteins spots Sample Fractionation - ZOOM®IEF fractionator Fractions pI 3 – 4,6 4,6 – 5,4 5,4 – 6,2 6, Mass Spectrometry Pronnex/FAPERJ; TDR/WHO FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Immunoproteomics

30 Culture filtrateSurface-associated 3,04,65,46,27,010,0 Pronnex/FAPERJ; TDR/WHO FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Immunoproteomics

31 Pronnex/FAPERJ; TDR/WHO FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Immunoproteomics CPqGM/FIOCRUZ: Theolis Barbosa

32 RevaccinatedControls IFN altoIFN baixo T 0 mo T 2 mo T 12 mo Pronnex/FAPERJ; TDR/WHO FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute Immunoproteomics CPqGM/FIOCRUZ: Theolis Barbosa

33 Leila de Mendonça Lima Wim M. Degrave Thomas Otto Marcia Berredo Pinho Marcelo Alves Ferreira Antônio B. de Miranda Teca Calcagno Galvão Leonardo Ferreira Gomes Melissa Pontes Pereira Patrícia M. Ferrão Talita Duarte Pagani Aline dos Santos Moreira Renata Ferreira da Silva Mariana Reis da Cunha Luis Roberto Castello Branco Renata Monteiro Maia Érico Albuquerque Carolina Zavarese PDTIS / FIOCRUZ REDE PROTEOMA & GENOMA ESTRUTURAL Lab. Genômica Funcional e Bioinformática (IOC - FIOCRUZ) Fundação Ataulpho de Paiva – FAP Functional Genomics and Proteomics FAPERJ FIOCRUZ CNPq PDTIS/FIOCRUZ Technological Platforms: DNA Sequencing 2D-Electrophoresis Mass Spectrometry Bioinformatics


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