SCIENCE VOL 332 6 MAY 2011
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Transcript of SCIENCE VOL 332 6 MAY 2011
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