SCIENCE VOL 332 6 MAY 2011

Objective:To evaluate the role of Gbps in macrophages

infected with intracellular bacteria sensitive to IFN-γ mediated killing

RAW264.7 macrophages

+ siRNAduplexes 4h

IFNγ treatementfor 16-20h

Infection with Listeria monocytogenes or Mycobacterium bovis

Gbp1 + Gbp7Gbp 7 + Gbp10Gbp10 + Gbp1

Gbp1 S52N Gbp7 S52N Gbp1 +/+ Gbp1 -/-

± L. monocytogenes or

M. bovis

±IFN-γ

Gbp1+/+ or Gbp1-/-

Oral infected L. monocytogenes

Quantified CFU in liver and spleenand

Weight loss

Gbp1+/+ or Gbp1-/-

i.V Mycobacterium bovis Quantified CFU in lung,

liver and spleen

The 65-Gbps are a class of bacterial host defense proteins that operate in vitro and in vivo

2h30 min

Protective Gbps target bacteria-containing vacuoles

Infection with L. monocytogenes or M. bovis

IFNγ treatement microscopy

GST-Gbp7 (Cterminal domain) + IFNγ-induced macrophage lysate

copreciptation

GFP-Gbp7 (full-length) + IFNγ-induced macrophage lysate

copreciptation

HA-p67phox + IFNγ-induced macrophage lysate

copreciptation

(P67phox allows robust copreciptation of endogenous gp91phox)

Infection with M. bovis

IFNγ treatement

microscopy± siRNA or ±doxycycline

Gbp7 is important for INF-γ-induced oxidant protection aganist intracellular bacteria

Infection with L. monocytogenes or M. bovis

IFNγ treatement16 hours

O2- production

P62 delivers ubiquitinated cargo to autolysosomes

eYFP-GBp1/ eYFP-GBp7/ eYFP-GBp10 + IFNγ-induced macrophage lysate

copreciptation

Gbp1 binds to p62 and delivery the autophagossome to the lysosome.

After a Gbp-interaction screen…

eYFP-GBp1/ eYFP-GBp7/ eYFP-GBp10 + IFNγ-induced macrophage lysate

copreciptation

IFNγ treatement scRNAs, Gbp7 siRNAs or stably expressing eGFPGbp7

microscopy

Conclusion