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Esther Stoop
Department of Medical Microbiology & Infection Control,VU University Medical CenterAmsterdam, the Netherlands
Zebrafish embryo screen to identify mycobacterial genes
involved in granuloma formation
lundi 27 septembre 2010
Mycobacteria
• Mycobacterium tuberculosis– TB
• Mycobacterium marinum– TB in cold blooded animals
• Hallmark: granuloma formation
• Host versus microbe
• Aim: identify mycobacterial factors involved in granuloma formation
Granuloma in Danio rerio
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Entry into macrophages
Survival & replication in macrophages
Formation of stable granuloma
Recruitment of immune cells & dissemination
neutrophil
lymphocyte
Granuloma formation
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M. marinum
5 days post infection
Mutant screen
….1000
Zebrafish embryo28 hours post fertilization
Injection site: caudal vein
Innate immunitylundi 27 septembre 2010
Vaccine strain• Live attenuated M. bovis BCG strain
– Delayed granuloma formation– Deletion in ESX-1 (T7SS)– ESX-1 involved in initial survival in macrophages
Rv3866Rv3867
Rv3868Rv3869
Rv3870 Rv3871PE35
PPE68
CFP10ESAT6
Rv3876Rv3877
Rv3878Rv3879c
Rv3880cRv3881c
ESX-1 region
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20 out of 1000 mutants form less granulomas
Mutant 58 ~128 CFU
Wildtype ~137 CFU
ESX-1 (T7SS) screen = reliable
PE/PPE protein family putative virulence factors ~10% genome
SecA2 secretion system associated with virulence ROS
Synthesis of glycolipids surface molecules interaction with host
Synthesis of polysaccharides capsule interaction with host
Metabolism
Unknown
Functions of mutated genes:
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Entry into macrophages
Survival & replication in macrophages
Formation of stable granuloma
Recruitment of immune cells & dissemination
neutrophil
lymphocyte
Where in the process are mutants attenuated?
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Step &: Macrophage infection
Entry into macrophages
Survival & replication in macrophages
&
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Step : Recruitment of immune cells
Wildtype infection
Mutant 223 infection (glycolipid mutant)
neutrophils
Transgenic zebrafish expressing GFP in macrophages
neutrophils
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Conclusions
• Granuloma screen works– Identification of known virulence factors confirms reliability of screen– New genes involved in granuloma formation
• Dissect the process of granuloma formation
• In vitro studies• Transgenic zebrafish lines• RNA interference
• Adult zebrafish experiments– Embryos: innate immunity only
lundi 27 septembre 2010
Thanks to…• Gunny van den Brink• Theo Verboom• Astrid van der Sar• Wilbert Bitter
• Organizers• Lectures• Sponsors• Participants
09/27/10
lundi 27 septembre 2010
