Post on 28-Mar-2015
Functional Genomics of Mycobacteria:Genomics x Proteomics x Diagnosis
Simpósio Imunopatogenia e Pesquisa BásicaIII Encontro Nacional de Tuberculose
Salvador - Junho 2008
Leila Mendonça-LimaLab. Genômica Funcional e Bioinformática
Instituto Oswaldo Cruz - FIOCRUZ FIOCRUZ
Functional Genomics:
Schwann cells infected with live M. leprae
Correlating genomic information to the organism’s “behavior” in the situation under study.
How genomic information translates into the pattern of protein expression.
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
How this pattern of protein expression affects host-pathogen interaction.
Mendonça-Lima & Barker
http://www.ncbi.nlm.nih.gov/Genbank/genbankstats.html
Jan. 200771.292.211.453
Jan. 200771.292.211.453
67.218.344 67.218.344
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Mycobacterial genomes:
19981998
20012001
20032003
20072007
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Jean-Marie Camille Guérin(1872 – 1961)
Léon Charles Albert Calmette (1863-1933)
Bacille de Calmette et GuérinBacille de Calmette et Guérin
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
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
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
IS6110
230 serial passages in potato/glycerin/bile
mediumCRP (HTH)
CRP (cNMP)
1927
Adapted from Behr et al, Science 1999 FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
BCG Moreau Genome Sequencing
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
BCG Moreau Genome Sequencing
Contig distribution (vs. M. bovis)
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
BCG Moreau Genome Sequencing: Shotgun
Design of specific oligonucleotides for each region
Chromosome walking
PCR > DNA sequencing
Specific sequencing protocols for each region
BCG Moreau Genome Sequencing: Finishing
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Assembled genome: 4.320.000 bp% G+C = 65.64
Assembled genome: 4.320.000 bp% G+C = 65.64
Jacenir R.S.Malle
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
BCG Moreau Genome Sequencing
37.555.553 bp included in the assembly
23.429.107 bp high quality
High quality coverage = 6x
Overall coverage = 9x
RD16
RD01
RD02
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
BCG Moreau Genome Sequencing
DNA-based tools for quality control
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Patente de invenção, nºPI0604958-3 Depósito de patente internacional, PCT/BR 2007/000322
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
BCG Moreau Genome Annotation:
stop
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
BCG Moreau Functional Genomics
BCG evolution = loss of genomic informationBCG evolution = loss of genomic information
Loss of genomic information sometimes = functional gainLoss of genomic information sometimes = functional gain
InDels:InDels:
Proteomics
Plataforma de Eletroforese 2DPDTIS-FIOCRUZLab. Genômica Funcional e BioinformáticaIOC - FIOCRUZ
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
pI pI=7
1D 2D
MW
pI
2D-Electrophoresis
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Proteomics
sup
cap
s
cap
s
sup
- Tw + Tw
Extracellular proteins are available sooner during infection for
modulation of host’s response
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Mycobacterial Proteomics
Mendonça-Lima & Barker
cytoplasm
Cellmembrane Cell wall
‘capsule’ orsurface
Extracellularmedium Abdallah et al., Nat Rev Microbiol 2007
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Mycobacterial Fractionation
Culture filtrate proteins
Remove culture medium
0.2um filter
Bacterial veilsSurface-associated
proteinsMechanical agitation
Centrifugation
15 day cultures in protein-free
medium
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Mycobacterial Fractionation
More
au
Past
eur
Sauton IVM
1
2
3 4
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Proteomics of the BCG Moreau vaccine
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Proteomics of the BCG Moreau vaccine
Moreau Pasteur
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Culture filtrate proteins
Moreau Pasteur
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Surface-associated proteins
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Web interface
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
6,2 - 7
7 - 10
Mass Spectrometry
Pronnex/FAPERJ; TDR/WHOPronnex/FAPERJ; TDR/WHO
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Immunoproteomics
Culture filtrate Surface-associated
3,0 4,6 5,4 6,2 7,0 10,0
Pronnex/FAPERJ; TDR/WHOPronnex/FAPERJ; TDR/WHO
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Immunoproteomics
Pronnex/FAPERJ; TDR/WHOPronnex/FAPERJ; TDR/WHO
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Immunoproteomics
CPqGM/FIOCRUZ: Theolis Barbosa
Revaccinated Controls
IFN alto IFN baixo
T 0 mo
T 2 mo
T 12 mo
Pronnex/FAPERJ; TDR/WHOPronnex/FAPERJ; TDR/WHO
FIOCRUZLab. Functional Genomics & Bioinformatics, Oswaldo Cruz Institute
Immunoproteomics
CPqGM/FIOCRUZ: Theolis Barbosa
Leila de Mendonça LimaWim M. DegraveThomas OttoMarcia Berredo PinhoMarcelo Alves FerreiraAntônio B. de MirandaTeca Calcagno GalvãoLeonardo Ferreira GomesMelissa Pontes PereiraPatrícia M. FerrãoTalita Duarte PaganiAline dos Santos MoreiraRenata Ferreira da SilvaMariana Reis da Cunha
Luis Roberto Castello BrancoRenata Monteiro Maia Érico Albuquerque Carolina Zavarese
PDTIS / FIOCRUZREDE PROTEOMA & GENOMA ESTRUTURAL
Lab. Genômica Funcional e Bioinformática (IOC - FIOCRUZ)
Fundação Ataulpho de Paiva – FAP 2006 -2008Functional Genomics
and Proteomics
FAPERJFAPERJ
FIOCRUZ
CNPqCNPq
PDTIS/FIOCRUZ Technological Platforms:
• DNA Sequencing
• 2D-Electrophoresis
• Mass Spectrometry
• Bioinformatics