functional genomics of mycobacteria: genomics x proteomics x diagnosis simpósio imunopatogenia e...

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


Mycobacterial genomes:

19981998

20012001

20032003

20072007


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


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


BCG Moreau Genome Sequencing

Contig distribution (vs. M. bovis)


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


Assembled genome: 4.320.000 bp% G+C = 65.64

Assembled genome: 4.320.000 bp% G+C = 65.64

Jacenir R.S.Malle



37.555.553 bp included in the assembly

23.429.107 bp high quality

High quality coverage = 6x

Overall coverage = 9x

RD16

RD01

RD02



DNA-based tools for quality control


Patente de invenção, nºPI0604958-3 Depósito de patente internacional, PCT/BR 2007/000322


BCG Moreau Genome Annotation:

stop


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



pI pI=7

1D 2D

MW

pI

2D-Electrophoresis


Proteomics

sup

cap

s

cap

s

sup

- Tw + Tw

Extracellular proteins are available sooner during infection for

modulation of host’s response


Mycobacterial Proteomics

Mendonça-Lima & Barker

cytoplasm

Cellmembrane Cell wall

‘capsule’ orsurface

Extracellularmedium Abdallah et al., Nat Rev Microbiol 2007


Mycobacterial Fractionation

Culture filtrate proteins

Remove culture medium

0.2um filter

Bacterial veilsSurface-associated

proteinsMechanical agitation

Centrifugation

15 day cultures in protein-free

medium


Mycobacterial Fractionation

More

au

Past

eur

Sauton IVM

1

2

3 4


Proteomics of the BCG Moreau vaccine


Proteomics of the BCG Moreau vaccine

Moreau Pasteur


Culture filtrate proteins

Moreau Pasteur


Surface-associated proteins


Web interface

http://bioinfo.pdtis.fiocruz.br/cgi-bin/2d/2d.cgi

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


Immunoproteomics

http://images.google.com.br/imgres?imgurl=http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpg&imgrefurl=http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/page_protein.html&h=715&w=800&sz=592&tbnid=jYxBaZ9eaRoJ:&tbnh=126&tbnw=141&hl=pt-BR&start=2&prev=/images?q=sds-page&hl=pt-BR&lr=

http://images.google.com.br/imgres?imgurl=http://www.mrc-dunn.cam.ac.uk/pictures/2d-gel-b.jpg&imgrefurl=http://www.mrc-dunn.cam.ac.uk/research/proteomes.html&h=400&w=394&sz=18&tbnid=JY-VaM4nNCQJ:&tbnh=120&tbnw=118&hl=pt-BR&start=12&prev=/images?q=proteome+and+gel&hl=pt-BR&lr=

http://images.google.com.br/imgres?imgurl=http://www-smbh.univ-paris13.fr/lbtp/Biochemistry/biochimie/Amultimedia/proteanXL.gif&imgrefurl=http://www-smbh.univ-paris13.fr/lbtp/Biochemistry/biochimie/technSDSPAGE.htm&h=242&w=255&sz=27&tbnid=EFU3L0TCpLkJ:&tbnh=100&tbnw=105&hl=pt-BR&start=32&prev=/images?q=bio+rad&start=20&hl=pt-BR&lr=&sa=N

http://images.google.com.br/imgres?imgurl=http://www.technochemical.com/pierce/supers/westpico/supersignal_kit.jpg&imgrefurl=http://www.technochemical.com/pierce/supers/westpico/westpico.htm&h=161&w=250&sz=16&hl=pt-BR&start=9&tbnid=Dm11R9imgKcgKM:&tbnh=71&tbnw=111&prev=/images?q=supersignal+%2B+pierce&gbv=2&svnum=10&hl=pt-BR&sa=X

Culture filtrate Surface-associated

3,0 4,6 5,4 6,2 7,0 10,0



Immunoproteomics



Immunoproteomics

CPqGM/FIOCRUZ: Theolis Barbosa

Revaccinated Controls

IFN alto IFN baixo

T 0 mo

T 2 mo

T 12 mo



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

functional genomics of mycobacteria: genomics x proteomics x diagnosis simpósio imunopatogenia e...

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