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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. Wich Gbps are involved in the control of virulent LM or Mb BCG ?.

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SCIENCE VOL 332 6 MAY 2011

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  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. WichGbps are involved in the control of virulent LM or Mb BCG? Infection with Listeria monocytogenes or Mycobacterium bovis + siRNA duplexes IFNγ treatement for 16-20h 4h RAW264.7 macrophages Gbp1 + Gbp7 Gbp 7 + Gbp10 Gbp10 + Gbp1

  3. WichGbps are involved in the control of virulent LM or Mb BCG? L. monocytogenes or M. bovis ± ± IFN-γ Gbp1 S52N Gbp7 S52N Gbp1 +/+ Gbp1 -/-

  4. Are thereconsequences of Gbps absence in vivo? Quantified CFU in liver and spleen and Weight loss L. monocytogenes Oral infected Gbp1+/+ or Gbp1-/-

  5. Are thereconsequences of Gbps absence in vivo? Quantified CFU in lung, liver and spleen Mycobacterium bovis i.V Gbp1+/+ or Gbp1-/- The 65-Gbps are a class of bacterial host defense proteins that operate in vitro and in vivo

  6. Where are localizatedGbpsduringbacterial infection? Infection with L. monocytogenes or M. bovis IFNγ treatement microscopy 30 min 2h Protective Gbps target bacteria-containing vacuoles

  7. Where are localizatedGbpsduringbacterial infection?

  8. Whatkind of cargo Gbps transport ? GST-Gbp7 (Cterminal domain) + IFNγ-induced macrophage lysate copreciptation

  9. Whatkind of cargo Gbps transport ? GFP-Gbp7 (full-length) + IFNγ-induced macrophage lysate copreciptation

  10. Could Gbp7 act as a bridge between p67phox-p40phox to gp91phox-p22phox ? HA-p67phox + IFNγ-induced macrophage lysate copreciptation (P67phox allows robust copreciptation of endogenous gp91phox)

  11. Does the absence of Gbp7 disturbe p67phox-p40phoxtargetingMCVs? ± siRNA or ± doxycycline IFNγ treatement Infection with M. bovis microscopy

  12. Whathappens to the O2- production in the absence of Gbp7? Infection with L. monocytogenes or M. bovis O2- production IFNγ treatement 16 hours Gbp7 is important for INF-γ-induced oxidant protection aganist intracellular bacteria

  13. Is thereanotherantimicrobialpathwayinvolvingGbps?

  14. After a Gbp-interaction screen… eYFP-GBp1/ eYFP-GBp7/ eYFP-GBp10 + IFNγ-induced macrophage lysate copreciptation P62 delivers ubiquitinated cargo to autolysosomes Gbp1 binds to p62 and delivery the autophagossome to the lysosome.

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

  16. Is Gbp7 important to autophagosome conformation? scRNAs, Gbp7 siRNAs or stably expressing eGFPGbp7 IFNγ treatement microscopy

  17. Conclusion The IFN-γ–inducible Gbps promote oxidativekilling and deliverantimicrobial peptides to autophagolysosomes

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