Modulation of Gene Expression via Disruption Of NF- kB Signaling by a Bacterial Small Molecule

1. Modulation of Gene Expression via Disruption Of NF-kBSignaling by a Bacterial Small Molecule Kravchenko et al. Science 2008 Paulina Blazejewska

2. Innate Immunity & Pathogen Two phases of the innate response Recognition: nonspecific & specific effectors Innate Immunity 0-4 hours Infection Removal Early induced Innate response 4-96 hours Recognition: microbial-associated molecular patterns Recognition: microbial-associated molecular patterns Infection Removal Inflammation/ effector cells

3. LPS C12 ? TLR4-independent mechanism Conserved receptor e.g. TLR4 IkB NFκB IkB kinase (IKK) activity IκBα & RelA P proinflammatory cytokines e.g. TNFα p38 protein kinase pathway Pathogen elimination Bacteremia & persistant infection

4. Objectives • What is the strategy used by pathogens to attenuate the innate immune system ? • How to maintain local persistent infection ?

5. Experimental approach • Comparison the macrophages response and the phosphorylation of p38 after infection with Salmonella typhimurium , Staphylococcus aureus or Pseudomonas aeruginosa • Biochemical effect of LPS and C12 and their combination • C12 in the late stage of LPS stimulation • Role of C12 in mice

6. Comparison the macrophages response and the phosphorylation of p38 after infection with S. typhimurium , S. aureus or P. aeruginosa • similar amount of p-p38 • upon activation with S. typhimuriumand S. aureusthepatterns of IκBα degradation and resynthesis were comparable • substantial delay in IκBαresynthesis after infection with P. aeruginosa - P. aeruginosadeficient in lasL(C12 synthesis) showed the normal profile in IκBα C12 affects the NFκB pathway in macrophages

7. Experimental approach • Comparison the macrophages response and the phosphorylation of p38 after infection with Salmonella typhimurium , Staphylococcus aureus or Pseudomonas aeruginosa • Biochemical effect of LPS and C12 and their combination • C12 in the late stage of LPS stimulation • Role of C12 in mice

8. Biochemical effect of LPS and C12 and their combination • C12 impairs the expression and phosphorylation of IκBα • C12 modulates the phosphorylation of RelA but not expression - IκBβ degradation was disrupted in the presence of C12 • p-IκBβ remains bound to RelA in the absence of IκBα C12 reduces the early phase of IKK activation by LPS

9. Experimental approach • Comparison the macrophages response and the phosphorylation of p38 after infection with Salmonella typhimurium , Staphylococcus aureus or Pseudomonas aeruginosa • Biochemical effect of LPS and C12 and their combination • C12 in the late stage of LPS stimulation • Role of C12 in mice

10. C12 in the late stage of LPS stimulation • the addition of C12 to the macrophages (pretreated with LPS) resulted in rapid reduction of p-IκBαand p-RelA • C12 also impairs other NFkB-regulated genes - C12 acts asa general modulator of the NFkB pathway

11. Experimental approach • Comparison the macrophages response and the phosphorylation of p38 after infection with Salmonella typhimurium , Staphylococcus aureus or Pseudomonas aeruginosa • Biochemical effect of LPS and C12 and their combination • C12 in the late stage of LPS stimulation • Role of C12 in mice

12. Role of C12 in mice • LPS-induced responses were suppressed by C12 • inhibition of TNF by C12 in LPS-stimulated leukocytes may be beneficial for the survival of both pathogen and host • C12 mediated disruption of NFkB attenuates TLR-4-dependent innate responses – promoting persistent infection with P. aeruginosa What is a mammalian C12 receptor?

13. Thank you for your attention & Merry Christmas and a Happy New Year !