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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