Preliminary Study On The Response Mechanism Of Streptococcus Agalactiae Infection In Danio Rerio Mediated By L-amino Acid Oxidase

In recent years,it has been found that fish L-amino acid oxidase(LAAO)had significant inhibitory activity against a variety of pathogens in vitro,but the molecular mechanism of fish LAAO responding to pathogen infection in vivo has not been thoroughly studied.This study conducted a preliminary study on the response mechanism of Streptococcus agalactiae infection mediated by LAAO in model animal zebrafish(Danio rerio).Bioinformatics analysis of zebrafish LAAO(ZF-LAAO)showed that ZF-LAAO was a homologous dimer with molecular weight of about 56 k Da,was an exocrine protein with two Nglycosylation sites and two O-glycosylation sites,mainly located in the endoplasmic reticulum.A phylogenetic tree was established between ZF-LAAO and other reported bony fish LAAOs.It was found that ZF-LAAO had the highest similarity with Anabarilius grahami LAAO(79.45%),and LAAO showed obvious species difference at the Order level;q RT-PCR was used to analyze the expression profile of LAAO in zebrafish infected with S.agalactiae.The results showed that the expression of ZF-LAAO was significantly up-regulated within 6h after bacterial infection,and reached to peak at 12 h,and the expression level was significantly higher than lysozyme;Three prokaryotic heterologous expression strains of ZF-LAAO,AE-p ET28-LAAO,AE-p ET32-LAAO and AE-p Cold TF-LAAO were constructed,recombinant proteins of about 60,70 and 120 k Da were obtained,respectively.The results of mass spectrometry showed that the recombinant protein was the target protein ZF-LAAO.Solubility analysis of recombinant proteins showed that,AEp ET28-LAAO and AE-p ET32-LAAO were expressed as inclusion bodies,AE-p Cold TF-LAAO can express soluble ZF-LAAO recombinant protein.By immunizing mice with the recombinant protein,the polyclonal antibody was successfully prepared to recognize ZF-LAAO,which can recognize the natural protein LAAO in zebrafish liver;The ZF-LAAO-knockout zebrafish was obtained using CRISPR/Cas9 method;Transcriptome analysis was performed on liver,gill and spleen of wild-type zebrafish and LAAO-knockout zebrafish before and after S.agalactiae challenge.Compared with wild-type zebrafish,5 260 up-regulated and 3 081 down-regulated genes were detected in liver tissues,1 725 up-regulated and 2 612 down-regulated genes were detected in gill tissues.After challenged with S.agalactiae,899 up-regulated genes and 872 downregulated genes were detected in liver tissue,1 118 up-regulated genes and 794 down-regulated genes were detected in gill tissue,1 374 up-regulated genes and 511 down-regulated genes were detected in spleen tissue.GO analysis results showed that compared with wild-type zebrafish,the functions of differentially expressed genes in LAAO-knockout fish were mostly classified into muscle,ribosome and immune-related functions,and after challenged with S.agalactiae,differentially expressed genes were mostly classified into functions of lipid metabolism,immunity,mitochondria and respiratory chain.KEGG analysis showed that,both the differentially expressed genes of LAAO-knockout zebrafish compared with wild-type zebrafish,and them after challenged,were enriched in amino acid metabolism and synthesis related pathways,carbohydrate metabolism related pathways and lipid related pathways,while immune-related pathways were enriched in redox-related PPAR signaling pathway and glutathione metabolism pathway,revealing that ZFLAAO played an important role in the redox homeostasis of cells and participated in the regulation of cell metabolism and immune function.The results of this study provide a basis for further study of LAAO signaling regulatory molecular network after pathogen infection.