Functional Study On The Regulation Of Innate Immunity By F-box Proteins In Drosophila Melanogaster

BackgroundInnate immunity is well-known to be the first line of defense against invading pathogens in multicellular organisms.The innate immune response of the host against invading pathogens is a complex process involving multiple genes,and is controlled at multi-levels,including transcription,post-transcription,translation and post-translation.Thus,in-depth study of these processes will help us to fully understand the way on how immune system works,and in the effort of developing the cure for treat diseases.Of particular interest to this study is innate immunity and the use of Drosophila melanogaster as a model organism.The Drosophila innate immune system involves cellular immunity includes coating,phagocytosis and melanization by blood cells,as well as antimicrobial peptides induced by humoral immunity.The major mechanisms of innate immunity in Drosophila has been systematically elucidated,but there are still a large number of key genes in these mechanisms remain unexplored as well as their functions which need to be further investigated their roles.As the largest family of E3 ligases,the SCF E3 ligase complex is comprised of Culins,Skp A and F-box proteins and is responsible for recognition of large number of target proteins in ubiquitin proteasome system.The ubiquitin-mediated degradation plays an important role in the regulation of immune response,signal transduction,cell cycle,cell growth,apoptosis,DNA repair and other important biological processes.Among the component of the SCF complex,the F-box protein family is considered to be an important variable component.To date,approximately fourty-five F-box proteins have been discovered in Drosophila,which are responsible for mediating the association between Skp A and the substrate proteins in SCF complex to recognize a large number of target proteins.ObjectiveTo systematically explore the important role of SCF E3 ubiquitin ligases in Drosophila innate immunity,and to further explore the molecular mechanism of F-box in innate immunity.MethodWe used the Gal4/UAS system to systematically knockdown the expression level of F-box gene in whole-body,and then infected with E.faecalis,S.marcescens or Ecc15 in order to select the key genes involved in the regulation of innate immunity according to the survival rates.ResultsAt present,our research findings are as follows:1)Thirteen F-box genes have been identified to potentially regulate innate immunity in Drosophila,and ten of them have not been revealed for their mechanism of action in immunity.2)ebi-knockdown or overexpression flies were more sensitive to E.faecalis infections than wild-type flies.Unexpectedly,knockdown or overexpression of ebi increased the bacterial load of E.faecalis by about five times higher compared to wild type.However,knockdown or overexpression of ebi did not affect the expression of antimicrobial peptides Drs and IM2,nor the phagocytic activity of blood cells.Our further experiments found that knockdown of ebi blocked the cleavage of PPO1 and thus affected melanization.3)Fsn-knockdown flies were more sensitive to E.faecalis or S.marcescens infections than wild-type flies.Furthermore,knockdown of Fsn increased the bacterial load of E.faecalisby about five times higher compared to wild type.However,knockdown of Fsn did not affect the expression of antimicrobial peptides Drs and IM2,nor the phagocytic activity of blood cells.However,the delay of melanin synthesis was observed in Fsn knockdown flies,but the shear of PPO1 was not affected.ConclusionThirteen F-box genes have been identified to potentially regulate innate immunity in Drosophila.ebi may play an important role in anti-gram-positive E.faecalis infection by regulating PPO1-dependent melanin,while Fsn may play an important role in anti-Gram-positive E.faecalis infection by PPO1-independent melanin.These results provide important evidences for further research on how F-box protein regulates innate immunity.