SCIENCE VOL MAY 2011 Objective: To evaluate the role of Gbps in macrophages infected with intracellular bacteria sensitive to IFN- mediated killing RAW264.7 macrophages + siRNA duplexes 4h IFN treatement for 16-20h Infection with Listeria monocytogenes or Mycobacterium bovis Gbp1 + Gbp7 Gbp 7 + Gbp10 Gbp10 + Gbp1 Gbp1 S52N Gbp7 S52N Gbp1 +/+ Gbp1 -/- L. monocytogenes or M. bovis IFN- Gbp1 +/+ or Gbp1 -/- Oral infected L. monocytogenes Quantified CFU in liver and spleen and 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 2h 30 min Protective Gbps target bacteria-containing vacuoles Infection with L. monocytogenes or M. bovis IFN treatementmicroscopy GST-Gbp7 (Cterminal domain) + IFN-induced macrophage lysate copreciptation GFP-Gbp7 (full-length) + IFN-induced macrophage lysate copreciptation HA-p67 phox + IFN-induced macrophage lysate copreciptation (P67 phox allows robust copreciptation of endogenous gp91 phox ) Infection with M. bovis IFN treatement microscopy siRNA or doxycycline Gbp7 is important for INF--induced oxidant protection aganist intracellular bacteria Infection with L. monocytogenes or M. bovis IFN treatement 16 hours O 2 - production P62 delivers ubiquitinated cargo to autolysosomes eYFP-GBp1/ eYFP-GBp7/ eYFP-GBp10 + IFN-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 + IFN-induced macrophage lysate copreciptation IFN treatement scRNAs, Gbp7 siRNAs or stably expressing eGFPGbp7 microscopy Conclusion