The Molecular Mechanism Of Atlantic Salmon Resistance Against Aeromonas Salmonicida By Multi-omics Analysis

Aeromonas salmonicida?A.salmonicida?is a major etiologic agent which induces furunculosis and is globally harmful in salmonid cultures,especially in Atlantic salmon?Salmo salar?farming.In order to improve knowledge of its poorly understood pathogenesis and select potential biomarkers from Atlantic salmon against this bacteria,methods including metabolomic,proteomic,phosphoproteomic and molecular biology were utilized in our work.As symptoms mainly existed in kidney,which was also the important organ provoking immune responsiveness in fish.Therefore,we set kidney of fish as the main target organ in our research.Otherwise,we purified recombinant protein ALDH7A1 which was obtained from omics data.And it could act in host immune defense against bacterial infection and decrease the mortality rate of Atlantic salmon at early stages of infection with A.salmonicida.All main results are as follows:1.Metabolic profiling in kidneys of Atlantic salmon infected with A.salmonicidaHere,we used ¹H nuclear magnetic resonance?NMR?to comprehensively analyze the metabolic changes in the kidney of Atlantic salmon infected with A.salmonicida.Through the NOESYPR1D spectrum combined with multivariate pattern recognition analysis,including principal component analysis?PCA?and orthogonal partial least-squares discriminant analysis?OPLS-DA?models,significant metabolic changes were observed in post-infection groups compared to control groups at seven and 14 days?Liu et al.2016?.Hence,the main objective of this study was to estimate the significant metabolites with resistance to furunculosis and further understand the mechanism of A.salmonicida in Atlantic salmon.Notably,substantial alterations of kidney metabolites were observed,such as with fumarate,alanine,valine,glycine,aspartate,choline,glycerophosphocholine and betaine,and summarized by metabolic pathways including the citrate cycle,glycolysis/gluconeogenesis,tryptophan metabolism,and urea cycle,respectively.After analyzing the alteration trend of these metabolites,we inferred that A.salmonicida caused absorption inhibition of amino acids and disturbed protein metabolism as well as cell metabolism in favor of its replication.2.Proteomic analyses in kidneys of Atlantic salmon infected with A.salmonicidaWe utilized high-throughput proteomics to display differentially expressed proteins in the kidney of Atlantic salmon challenged with high and low infection dose of A.salmonicida at 7 and 14 days.In this study,4009 distinct proteins were identified in three two-dimensional LC/MS/MS analyses.Then we chose 140 proteins?fold change ratio?1.5 and P<0.01?combined with protein–protein interaction analysis to ultimately obtain 39 proteins in network which could be considered as potential biomarkers for Atlantic salmon immune responses.Nine significant differentially expressed proteins were consistent with those at the proteomic level used to validate genes at the transcriptomic level by Real-Time quantative PCR?RT-qPCR?.Collectively,these data was first reported using an iTRAQ approach and expression of ten proteins including glyceraldehyde-3-phosphate dehydrogenase,ALDH7A1,ALDH9A1,L-lactate dehydrogenase B chain,HADH,hemoglobin subunit beta,hemoglobin subunit alpha-4,CCT7,triosephosphate isomerase A and complement factor B-like increased significantly in infected groups.3.ALDH7A1 is a protein that protects Atlantic salmon against A.salmonicidaAldehyde dehydrogenases?ALDHs?belong to a super-family of detoxifying proteins and perform a significant role in developing epithelial homeostasis,protecting cells from toxic aldehydes and drug resistance.However,the activity and function of these detoxifying proteins remain unknown.In our research,we had expressed and purified recombinant protein ALDH7A1 to study its function in Atlantic salmon infected by Aeromonas salmonicida.The rALDH7A1 protein was verified by SDS-PAGE and western blot.The molecular mass of the deduced amino acid sequence of rALDH7A1 is 58.9 kDa with an estimated pI of 7.09.And only a low complexity region(¹4141 yvegvgevqeyvdv ¹⁵³)without signal peptide was existed in rALDH7A1.Results of ELISA indicated that rALDH7A1 exhibited apparent binding activities with A.salmonicida.And ALDH7A1 expression was high in fish kidney,liver intestine and blood?decrease successively?,and low in gill and spleen.The quantification of bacteria copy numbers were obtained by a Real-Time quantative PCR?RT-qPCR?assay in kidneys to confirm that fish in this experiment were authentically infected and bacterial loads in rALDH7A1-adminsitered fish were significantly reduced at an early stage of infection.Meanwhile,the mRNA expression of NF-k B,P-38 MAPK,caspase-3 and TNF-?in tissues of kidneys,livers and spleens were quantified.Of the genes that were stimulated by rALDH7A1 that were mainly up-regulated in rALDH7A1+high-dose infection group at 72 h of fish kidneys and livers at 72 h,and the same variation trend was also found in fish spleens at 12 h.Consistent with these observations,neutralization experiments in vivo indicated that rALDH7A1 could obviously reduce the death rate compared to the BSA and control group.4.Phosphoproteomic analyses in kidneys of Atlantic salmon infected with A.salmonicidaPhosphoproteomic analyses were conducted to further clarify the dynamics changes of protein phosphorylation pattern triggered by bacterial infection.To our knowledge,this is the first study to characterize phosphorylation events in proteins from A.salmonicida-infected Atlantic salmon.Overall,we identified over 5635phosphorylation sites in 3112 proteins,and 1502 up-regulated and 77 down-regulated proteins,quantified as a 1.5-fold or greater change,respectively.Based on the combined data from proteomic and motif analyses,we hypothesize that five prospective novel kinases?VRK3,GAK,HCK,PKC?and RSK6?with common functions in inflammatory processes and cellular pathways to regulate apoptosis and the cytoskeleton could serve as potential biomarkers against bacterial propagation in fish.Therefore,further research could be processed to prove defensive mechanism of these proteins on their sites.Data from STRING-based functional network analyses indicate that fga is the most central protein which could act as an important regulatory factor in kidneys of Atlantic salmon infected with A.salmonicida.