The Role Of Lysosome-associated Membrane Protein 1 In Virus Invasion And The Functional Study Of DNA Methylation In Trained Immunity Of Shrimp

1.The role of lysosome-associated membrane protein 1 in virus invasion of shrimpLysosome participates in various cellular functions,including eliminating pathogens through endocytosis in innate immune cells.Meanwhile,pathogens have evolved mechanisms to evade digestion and elimination by lysosome.Lysosome-associated membrane protein 1(LAMP1)is one of the components of lysosome and is involved in endocytosis.Pathogens can use LAMP 1 to proliferate,but the specific mechanism is unclear.Two LAMPs,LAMP 1 and LAMP5,were identified in Marsupenaeus japonicus.Both LAMPs had a signal peptide,a LAMP domain,a transmembrane region,and a short C-terminal peptide.The C-terminal peptide of LAMP 1 contained an AYXX? motif,which was more similar to that of mammalian LAMPs.The C-terminal peptide of LAMP5 contained a NYDTM motif,which was less similar to mammals.Lamp1 was constitutively expressed in various tissues of the shrimp,while Lamp5 was only expressed in hemocytes and intestine.Lamp1 responded to white spot syndrome virus(WSSV)stimulation in hemocytes and intestine,while Lamp5 was upregulated only in hemocytes upon WSSV stimulation.Therefore,LAMP1 was chosen for further research.It was found that WSSV replication levels significantly decreased and shrimp survival rates increased evidently in Lamp1-knockdown shrimp.LAMP 1 interacted with four WSSV envelope proteins,VP 19,VP24,VP26,and VP28,suggesting that LAMP1 might be the cell membrane receptor of WSSV.Additionally,LAMP1 was found to remain on the cell membrane after knocking down Ap2m and Clathrin,suggesting that AP2M and Clathrin were involved in LAMP1mediated endocytosis.LAMP1 also interacted with RAB5 and RAB7,which are markers for early and late endosomes,respectively.This study revealed the mechanism that LAMP1 facilitated WSSV infection in shrimp and provide a novel target for disease prevention and control in shrimp aquaculture.2.The functional study of DNA methylation in trained immunity of shrimpMetabolic reprogramming and epigenetic modifications are involved in trained immunity in innate immune cells.As an important type of epigenetic modification,DNA methylation plays a crucial role in trained immunity.But the functions of DNA methylation and enzymes that regulate DNA methylation are not clear yet.In this study,VP28,a WSSV envelope protein,was used to establish a trained immunity model in M.japonicus,and MethylRAD-seq was used to detect the level of gDNA 5-methylcytosine(5-mC).It was showed that gDNA methylation sites in shrimp were mainly located in introns and exons.Furthermore,it was found that VP28induced trained immunity reduced shrimp gDNA methylation levels,while WSSV infection increased gDNA methylation levels.KEGG database enrichment analysis showed that VP28induced trained immunity downregulated methylation levels at sites in adhesion junctions,the MAPK signaling pathway-fly,and the NF-κB signaling pathway,while upregulating methylation levels at sites in focal adhesions,ErbB signaling,MAPK signaling,and insulin signaling pathways.On the other hand,WSSV infection downregulates methylation levels in RNA transport pathways,while upregulating methylation levels in cytoskeleton regulation,endocytosis,MAPK signaling,and spliceosome pathways.Two DNMTs(DNMT1 and DNMT3A)and one ten-eleven translocation(TET)dioxygenase were identified in shrimp,all of which showed tissue-specific expression and responded to WSSV stimulation.By using RNA interference and inhibitor,it was found that in Dnmt1 or Dnmt3a-knockdown shrimp,gDNA methylation levels and WSSV replication were decreased,indicating DNMTs were beneficial for WSSV infection.Knockdown of Tet,however,led to increased WSSV replication,suggesting that TET inhibited WSSV replication.Finally,it was found that injection and feeding of α-ketoglutarate significantly reduced shrimp gDNA methylation level and exhibited antiviral activity.This study revealed the functions of DNA methylation and the enzymes responsible for the modifications in shrimp trained immunity and provided vaccine and drug candidates for immune prevention and control of diseases in shrimp aquaculture.