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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
+ siRNA
duplexes
IFNγ treatement
4h
for 16-20h
RAW264.7 macrophages
Gbp1 + Gbp7
Gbp 7 + Gbp10
Gbp10 + Gbp1
Infection with Listeria
monocytogenes or
Mycobacterium bovis
±
Gbp1 S52N
Gbp7 S52N
Gbp1 +/+
Gbp1 -/-
IFN-γ
±
L. monocytogenes
or
M. bovis
L. monocytogenes
Oral infected
Gbp1+/+ or Gbp1-/-
Quantified CFU in liver and spleen
and
Weight loss
Mycobacterium bovis
i.V
Gbp1+/+
or
Quantified CFU in lung,
liver and spleen
Gbp1-/-
The 65-Gbps are a class of bacterial host defense proteins that
operate in vitro and in vivo
IFNγ treatement
30 min
Infection with L. monocytogenes
or M. bovis
microscopy
2h
Protective Gbps target bacteria-containing vacuoles
copreciptation
GST-Gbp7 (Cterminal domain) +
IFNγ-induced macrophage lysate
copreciptation
GFP-Gbp7 (full-length) +
IFNγ-induced macrophage lysate
copreciptation
HA-p67phox +
IFNγ-induced macrophage lysate
(P67phox allows robust copreciptation of endogenous gp91phox)
IFNγ
treatement
± siRNA or ±
doxycycline
Infection with
M. bovis
microscopy
IFNγ treatement
16 hours
Infection with L. monocytogenes
or M. bovis
O2- production
Gbp7 is important for INF-γ-induced oxidant protection aganist
intracellular bacteria
After a Gbp-interaction screen…
copreciptation
eYFP-GBp1/ eYFP-GBp7/ eYFP-GBp10 +
IFNγ-induced macrophage lysate
P62 delivers ubiquitinated cargo to autolysosomes
Gbp1 binds to p62 and delivery the autophagossome to the
lysosome.
copreciptation
eYFP-GBp1/ eYFP-GBp7/ eYFP-GBp10 +
IFNγ-induced macrophage lysate
IFNγ treatement
scRNAs, Gbp7 siRNAs or stably
expressing eGFPGbp7
microscopy
Conclusion