Immunity of turbot Induced by inactivated vaccine of Aeromonas salmonicida from the perspective of DNA methylation

Abstract

Introduction: DNA methylation was one of the most important modification in epigenetics and played an important role in immune response. Since the introduction of Scophthalmus maximus, the scale of breeding has continued to expand, during which diseases caused by various bacteria, viruses and parasites have become increasingly serious. Therefore, the inactivated vaccines have been widely researched and used in the field of aquatic products with its unique advantages. However, the immune mechanism that occurred in turbot after immunization with inactivated vaccine of Aeromonas salmonicida was not clear.

Methods: In this study, differentially methylated regions (DMRs) were screened by Whole Genome Bisulfite Sequencing (WGBS) and significantly differentially expressed genes (DEGs) were screened by Transcriptome sequencing. Double luciferase report assay and DNA pull-down assay were further verified the DNA methylation state of the gene promoter region affected genes transcriptional activity after immunization with inactivated vaccine of Aeromonas salmonicida.

Results: A total of 8149 differentially methylated regions (DMRs) were screened, in which there were many immune-related genes with altered DNA methylation status. Meanwhile, 386 significantly differentially expressed genes (DEGs) were identified, many of which were significantly enriched in Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and C-type lectin receptor signaling pathway. Combined analysis of WGBS results and RNA-seq results, a total of 9 DMRs of negatively regulated genes are located in the promoter region, including 2 hypermethylated genes with lower expression and 7 hypomethylated genes with higher expression. Then, two immune-related genes C5a anaphylatoxin chemotactic receptor 1-like (C5ar1-Like) and Eosinophil peroxidase-like (EPX-Like), were screened to explore the regulation mechanism of DNA methylation modification on their expression level. Moreover, the DNA methylation state of the gene promoter region affected genes transcriptional activity by inhibiting the binding of transcription factors, which lead to changes in the expression level of the gene.

Discussion: We jointly analyzed WGBS and RNA-seq results and revealed the immune mechanism that occurred in turbot after immunized with inactivated vaccine of A. salmonicida from the perspective of DNA methylation.

Keywords: Aeromonas salmonicida; DNA methylation; Scophthalmus maximus; Whole Genome Bisulfite Sequencing; inactivated vaccine; transcriptome sequencing.

Figure 1

Screening of differential genes by transcriptome sequencing. (A) Venn diagram showed differential gene in the comparison of AsVSm and NVSm. (B) The bar chart showed statistics of the number of significantly DEGs. (C) Clustering heat map of significantly DEGs. The abscissa represents the sample name, and the ordinate represents the value of fpkm after homogenization. Red means high expression and green means low expression. (D) GO enrichment analysis of significantly DEGs. (E) Scatter plot of KEGG enrichment. The most significantly enriched top 20 pathways of significantly DEGs in AsVSm vs NVSm.

Figure 2

Screening of DMR and related genes by whole genome bisulfite sequencing. (A) The distribution of methylation level of samples at 2K upstream and downstream of gene region. (B) The results of thermographic analysis of methylation level of gene functional region in mCG sequence environment. (C) The number of intersections or unique gene sets of DMGs anchoring promoter regions in different sequence contexts (CG, CHG, CHH). (D) Clustering heatmap of DMR methylation levels in different sequence contexts (CG, CHG, CHH). (E) Go enrichment analysis of DMGs in the context of CG sequence. (F) The top 20 pathways significantly enriched for DMGs in the context of CG sequences.

Figure 3

Regulation of methylation modification on gene expression level. (A) Distribution of gene expression level and methylation level in CG sequence environments. (B) Statistics of overlapping genes between DMGs and DEGs. (C) GO enrichment analysis of overlapping gene in promoter region CG sequence environment. (D) Significantly enriched pathway statistics for methylation negatively regulated genes.

Figure 4

DNA methylation negatively regulated gene expression. (A) The relative expression levels of C5ar1-Like gene in AsVSm group and NVSm group. (B) The average methylation level of the promoter regions of the C5ar1-Like gene in AsVSm group and NVSm group. (C) The relative expression levels of EPX-Like gene in AsVSm group and NVSm group. (D) The average methylation level of the promoter regions of the EPX-Like gene in AsVSm group and NVSm group. *p<0.05.

Figure 5

The mechanism of DNA methylation inhibited immune-related gene expression. (A) Amplified fragments from C5ar1-Like and EPX-Like promoter regions. The different colors represent different gene fragments. (B) Effects of DNA methylation modifications on fluorescent activity of C5ar1-Like promoter regions. (C) Effects of DNA methylation modifications on fluorescent activity of EPX-Like promoter regions.*Representative significant difference (P < 0.05). (D) Coomassie blue staining of HEK293T nuclear proteins. (E) Silver staining of DNA pull down enriched proteins. The red box shows the different bands between the met-SmC5ar1-Like and the SmC5ar1-Like. (F) Total number of the met-SmC5ar1-Like and the SmC5ar1-Like interacting proteins identified by MS.