Mechanisms And Antibacterial Effect Of Autophagy Response In Grass Carp (Ctenopharyngodon Idella) Under Infection Condition

Edwardsiella piscicida(E.piscicida)and Aeromonas hydrophila(A.hydrophila)are common intracellular and extracellular pathogens respectively,which cause enumerous economic losses in aquaculture.However,the molecular and cellular mechanisms responsible for these two bacterial infection remains to be elucidated.At present,it is well known that autophagy represents evolutionarily conserved eukaryotic mechanisms in innate immunity for engulfment and disposal of harmful bacteria.Whether autophagy plays a role in bacterial clearance in fish remains puzzling.To address this issue,this dissertation tried to reveal the interaction between autophagy and these two pathogens as well as the related mechanisms.The detailed results are as follows:1.E.piscicida induced autophagy in grass carp primary monocytes/macrophages(monocytes/macrophages)and carp cell line Epithelioma papulosum cyprini(EPC)cells.(1)E.piscicida increased the quantity of microtubule-associated proteins light chain 3(LC3)puncta in monocytes/macrophage and EPC cells.(2)LC3 colocated with red fluorescent protein-lablled E.piscicida(RFP-E.piscicida)in monocytes/macrophages.(3)Lysosome colocated with RFP-E.piscicida in monocytes/macrophages.These results suggested the involvement of E.piscicida in the autophagic process including autophagosome formation,bacterical sequestering and degradation.2.The mechanism of E.piscicida triggering autophagy was elucidated.(1)NOD1,an intracellular pattern recognition receptor,colocated with RFP-E.piscicida in monocytes/macrophages.(2)In EPC cells,the colocalization of NOD1 and ATG16L1 was observed by immunofluorescence,and the interaction of NOD1 and ATG16L1 was proved by co-immunoprecipitation(Co-IP)assay.(3)In EPC cells,the colocalizaion of NOD1,ATG16L1,and E.piscicida was detected.These results uneviled the interplay of E.piscicida and autophagy as well as autophagic machinery targeting E.piscicida.The followed studies investigated whether E.piscicida regulated the expression of NOD1 that mediated autophagy as described above.3.E.piscicida decreased the expression of NOD1.(1)E.piscicida reduced the protein levels of NOD1 in the multiplicity of infection(MOI)-and time-dependent mannar in monocytes/macrophages.(2)E.piscicida down-regulated the gene-expression of NOD1 in EPC cells.4.The mechanism responsible for the regulatory effect of E.piscicida on NOD1 expression.(1)E.piscicida inhibited the gene expression of Estrogen-related receptor-α(ESRRA)in monocytes/macrophages.(2)Kaempferol,an ESRRA inverse agonist,attenuated the protein and gene expression of NOD1 in monocytes/macrophages and EPC cells.(3)Overexpression of ESRRA in EPC cells partially reversed the decrement of NOD1 expression modulated by E.piscicida.These results suggested that E.piscicida decreased the expression of NOD1 in an ESRRA-dependent mannar.(4)E.piscicida and its virulent factors,ESCB and EVPC,facilitated the ubiquitinoylation of NOD1 in EPC cells and monocytes/macrophages.(5)In EPC cells and monocytes/macrophages,the inhibitory effects of E.piscicida and its virulent factors on NOD1 protein expression were reversed by using a proteasome inhibitor,MG-132.These results uncovered E.piscicida and its virulent factors reduced NOD1 protein levels by ubiquitin-dependent proteasomal degradation pathway,thereby evading from host autophagy.These results demonstrated the interplay between autophagy and E.piscicida in fish cells,which prompted us to decode the intrinsic factor regulating the interplay of autophagy and E.piscicida and assess the antibacterial effect governed by autophagy.5.IFN-γ manipulated the interaction between autophagy and E.piscicida.(1)IFN-γamplified E.piscicida-induced LC3-Ⅱ protein expression in an additive manner in monocytes/macrophages.(2)IFN-γ enhanced the colocalization of LC3-labelled autophagosomes or NOD1 with E.piscicida in monocytes/macrophages.These results suggested the ability of IFN-γ to facilitate the interaction of autophagy with E.piscicida.(3)IFN-γ upregulated basal LC3-Ⅱ protein levels and LC3 puncta formation in monocytes/macrophages.(4)In monocytes/macrophages,IFN-γ promoted the eradication of intracellular E.piscicida,which was blocked by autophagy inhibitor 3-MA but not influenced by L-NMMA disturbing NO synthetize.These results indicated the enhancement of E.piscicida clearance in a short-term(within 8 h)treatment with IFN-γwas dependent on autophagy but not nitric oxide production in grass carp monocytes/macrophages.6.The effect of rapamycin-induced autophagy against E.piscicida in vitro and in vivo.(1)Rapamycin induced the expression of LC3-Ⅱ in monocytes/macrophages.(2)Rapamycin induced the colocalizion of LC3 and E.piscicida in monocytes/macrophages.(3)Rapamycin promoted the eradication of intracellular E.piscicida in monocytes/macrophages.(4)In vivo,grass carp pre-treated with rapamycin showed higher survival rate upon E.piscicida infection.These results revealed the protective role of rapamycin-induced autophagy against E.piscicida.These results implied the potential of IFN-γ and rapamycin in controlling E.piscicida infection.7.The preliminary exploration on the interplay between A.hydrophila and autophagy.(1)A.hydrophila induced LC3-Ⅱ expression and LC3 puncta formation in monocytes/macrophages.(2)A.hydrophila colocalized with autophagosomes in monocytes/macrophages.(3)Rapamycin promoted but 3-MA restrained the eradiction of A.hydrophila in monocytes/macrophages.In summary,this dissertation for the first time elucidated the interplay mechanisms between autophagy and aquatic pathogens,and particularly uncovered the roles of host PRR and bacterial toxic proteins during bacterial infection,which paved the new way for developing vaccines and drugs in aquaculture.Autophagy played essential role in the eradiction of both intracellular and extracellular aquatic pathogens,suggesting that enhanced autophagic response in host may be an effect way in the prevention of aquatic pathogen infection.