Innate Immunity. Insects rely solely on an innate immune system for defense against infection

Transcription

1 Innate Immunity Insects rely solely on an innate immune system for defense against infection The importance of mammalian innate immunity has only recently become appreciated The innate immune response is well conserved between insects and mammals

2 Part of the Drosophila immune response is the synthesis of a battery of antimicrobial peptides (AMPs) Different types of infection result in the synthesis of different AMPs At least two signaling pathways (Imd and Toll) are involved which distinguish between and combat Gram-negative bacterial pathogens (Imd) and fungal/ Gram-positive bacterial pathogens (Toll)

3 Drosophila Toll and Imd Pathways MyD88 Revision of Tzou,et al Current Opinion in Microbiology, Volume 5, Issue 1, Pages

4 My Research Aims To elucidate the signaling proteins required for immune peptide induction in the antifungal/ Gram-positive bacterial response To investigate the potential role of bifurcation and heterodimerization in the Toll signaling pathway

5 Experimental Setup 1) Cultured Drosophila S2* cells are transiently transfected with luciferase reporter constructs 2) Co-transfection with lac-z reporter plasmid controls for transfection efficiency 3) Cells are treated with dsrna 4) Infection is simulated and cells are harvested 4 hours later 5) Luciferase and lac-z activity are assayed with a luminometer

6 1 & 2) Transfection of Luciferase Reporters Quantitating pathway output a) Construct DNA plasmids consisting of upstream AMP regulatory elements and expressing a light-emitting luciferase protein b) Cells take up the plasmid constructs and the luciferase proteins are co-expressed with AMP c) Assay downstream gene expression of immune induced reporter constructs through measurement of luminescence Induction of AMP reporter constructs. S2* cells were transiently transfected with luciferase reporter plasmids containing the upstream region of either IM1 or Drs. After a 4 hour simulated infection, cells were harvested and reporter activity was assayed with a luminometer and adjusted by bgal activity. Fold induction was calculated by dividing the induced reading by the uninduced value.

7 AMP Induction: Drosomycin and IM1 Drs IM

8 3) RNA interference Powerful technique that permits targeted inhibition of protein expression a) Synthesize double stranded RNA (dsrna) that corresponds to pathway component a-dorsal No dsrna Dorsal dsrna b) Treat cells with dsrna c) dsrna is utilized by cells as a template for the destruction of identical RNAs d) Expression of targeted signaling component is blocked Expression of Dorsal protein with and without Dorsal dsrna treatment. Cultured Drosophila S2* cells were treated with Dorsal dsrna (lane 2) or no dsrna (lane 1). Cells were harvested after 4 hours of simulated infection and Dorsal protein expression was analyzed by anti- Dorsal antibodies on a Western Blot. Equitable cell numbers were verified through sample expression of lac-z plasmid.

9 Differential regulation of IM1 and Drosomycin (Drs) RNAi against MyD88 and Dif reduces Drs induction less substantially than IM1 induction (compare relative to the uninduced for both reporters) While IM1 activity is unaffected by IKK and Dorsal RNAi, Drs activity is reduced to 60% Relish (Rel) RNAi has a moderate affect on IM1 activity whereas Drs activity is reduced to nearly uninduced levels, suggesting a significant dependence on the transcription factor Rel for Drs induction

10 Drs Reporter Activity with RNAi Uninduced No RNAi Rel Dif Dl Dif+Dl Easter dsrna

11 Heterodimerization Rel family transcription factors, Dif, Dorsal, and Relish bind in different combinations to induce gene expression Rel dsrna treatment decreases Drs activity to nearly uninduced levels while RNAi with Dif or Dorsal (Dl) alone have only moderate effects. Treating cells with a combination of Dif and Dl dsrna reduces activity to a level similar to that of Rel RNAi alone This suggests a possible heterodimerization scheme requiring Rel/Dif or Rel/Dl dimers for Drs induction

12 Heterodimerization Model for Drs Gene Expression Relish Dorsal or Relish Dif Drosomycin

13 Conclusions While both IM1 and Drs are induced through Toll Pathway activation, they rely on different downstream components Drs transcription appears to be regulated through Rel/Dif and Rel/Dorsal heterodimers

14 Acknowledgments Chancellor s Scholarship Cal-(IT)2 Wasserman Lab