1. 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

2. RAW264.7 macrophages + siRNA duplexes 4h IFNγ treatement for 16-20h Infection with Listeria monocytogenes or Mycobacterium bovis Gbp1 + Gbp7 Gbp 7 + Gbp10 Gbp10 + Gbp1

3. Gbp1 S52N Gbp7 S52N Gbp1 +/+ Gbp1 -/- ± L. monocytogenes or M. bovis ± IFN-γ

4. Gbp1 +/+ or Gbp1 -/- Oral infected L. monocytogenes Quantified CFU in liver and spleen and Weight loss

5. 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

6. 2h 30 min Protective Gbps target bacteria-containing vacuoles Infection with L. monocytogenes or M. bovis IFNγ treatementmicroscopy

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

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

10. HA-p67 phox + IFNγ-induced macrophage lysate copreciptation (P67 phox allows robust copreciptation of endogenous gp91 phox )

11. Infection with M. bovis IFN γ treatement microscopy ± siRNA or ± doxycycline

12. Gbp7 is important for INF-γ-induced oxidant protection aganist intracellular bacteria Infection with L. monocytogenes or M. bovis IFN γ treatement 16 hours O 2 - production

14. 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…

15. eYFP-GBp1/ eYFP-GBp7/ eYFP-GBp10 + IFNγ-induced macrophage lysate copreciptation

16. IFNγ treatement scRNAs, Gbp7 siRNAs or stably expressing eGFPGbp7 microscopy

17. Conclusion