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Functional Genomics of Mycobacteria: Genomics x Proteomics x Diagnosis

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Presentation on theme: "Functional Genomics of Mycobacteria: Genomics x Proteomics x Diagnosis"— 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: Correlating genomic information to the organism’s “behavior” in the situation under study. How genomic information translates into the pattern of protein expression. How this pattern of protein expression affects host-pathogen interaction. Mendonça-Lima & Barker Schwann cells infected with live M. leprae FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

3 Jan. 2007 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

4 Mycobacterial genomes:
1998 2001 2003 2007 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

5

6 Léon Charles Albert Calmette Jean-Marie Camille Guérin
( ) Jean-Marie Camille Guérin (1872 – 1961) Bacille de Calmette et Guérin Bacille de Calmette et Guérin

7 Over 3 billion people vaccinated with BCG
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 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

8 230 serial passages in potato/glycerin/bile medium
M. bovis (1908) 230 serial passages in potato/glycerin/bile medium RD1 CRP (HTH) “ BCG ” (1921) Russia (1924) RD16 Moreau (1925) Japan (1925) IS6110 Sweden (1926) Birkhaug (1927) 1927 CRP (cNMP) RD2 Prague (1947) Danish (1931) mma3 Glaxo (1954) sigK Tice (1934) RD8 Frappier (1937) Connaught (1948) Phipps (1938) RD14 Pasteur (Liofilizada 1961) FIOCRUZ Adapted from Behr et al, Science 1999 Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

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

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

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

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

13 Assembled genome: 4.320.000 bp % G+C = 65.64
BCG Moreau Genome Sequencing bp included in the assembly bp high quality High quality coverage = 6x Overall coverage = 9x Assembled genome: bp % G+C = 65.64 Jacenir R.S.Malle FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

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

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

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

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

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

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

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

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

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

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

24 Proteomics of the BCG Moreau vaccine
Sauton IVM Moreau 1 Pasteur 2 3 4 FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

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

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

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

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

29 Immunoproteomics Pronnex/FAPERJ; TDR/WHO Sample pI
Transfer of proteins to nitrocellulose membrane Reversible protein stain 3 – 4,6 Fractionation - ZOOM®IEF fractionator 4,6 – 5,4 IEF 7cm pH 4 -7 5,4 – 6,2 Fractions 6,2 - 7 7 - 10 Blocking: SDS-PAGE (2ºD) 12% Coomassie G-250 Image detecting GS-800 Pooled sera (IgG) Anti-human-IgG peroxidase conjugate Matching signals for immunoblots and proteins spots Chemiluminescence substrate Super Signal West Pico Mass Spectrometry Pronnex/FAPERJ; TDR/WHO FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

30 Pronnex/FAPERJ; TDR/WHO
Immunoproteomics 3,0 4,6 5,4 6,2 7,0 10,0 Culture filtrate Surface-associated Pronnex/FAPERJ; TDR/WHO FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

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

32 Pronnex/FAPERJ; TDR/WHO
Immunoproteomics Revaccinated Controls IFN alto IFN baixo T 0 mo T 2 mo T 12 mo WB- 2D Differential profile CPqGM/FIOCRUZ: Theolis Barbosa Pronnex/FAPERJ; TDR/WHO FIOCRUZ Lab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute

33 PDTIS / FIOCRUZ REDE PROTEOMA & GENOMA ESTRUTURAL Lab. Genômica Funcional e Bioinformática (IOC - FIOCRUZ) 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 PDTIS/FIOCRUZ Technological Platforms: DNA Sequencing 2D-Electrophoresis Mass Spectrometry Bioinformatics FIOCRUZ Functional Genomics and Proteomics Fundação Ataulpho de Paiva – FAP Luis Roberto Castello Branco Renata Monteiro Maia Érico Albuquerque Carolina Zavarese FAPERJ CNPq


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