Download - Zebrafish embryo screen to identify mycobacterial genes

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


09/27/10

Entry into macrophages

Survival & replication in macrophages

Formation of stable granuloma

Recruitment of immune cells & dissemination

neutrophil

lymphocyte

Granuloma formation


09/27/10

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


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:


09/27/10



Formation of stable granuloma

Recruitment of immune cells & dissemination

neutrophil

lymphocyte

Where in the process are mutants attenuated?


09/27/10

Step &: Macrophage infection



&


Step : Recruitment of immune cells

Wildtype infection

Mutant 223 infection (glycolipid mutant)

neutrophils

Transgenic zebrafish expressing GFP in macrophages

neutrophils


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


Thanks to…• Gunny van den Brink• Theo Verboom• Astrid van der Sar• Wilbert Bitter

• Organizers• Lectures• Sponsors• Participants

09/27/10
