DNA Methylome And Transcriptome-Based Integration Analysis For Glaesserella Parasuis-Induced Meningitis In Pig

Glaesserella parasuis（GPS）is the causative agent of pig Gl?sser disease,which is one of the pathogenic microorganisms that are widespread and causing serious harm in the pig industry.And it is a gram-negative bacterium that colonizes the upper respiratory tract of pigs.This strain can induce systemic inflammatory responses in pigs under specific circumstances,causing pig Gl?sser disease,exhibiting clinical symptoms such as polyserositis,arthritis and meningitis.But the specific pathogenesis of meningitis in pigs caused by Glaesserella parasuis is still unclear.In this study,whole genome bisulfite sequencing and transcriptome sequencing for Glaesserella parasuis-infected porcine brain were performed to reveal the genome-wide DNA methylation pattern and transcriptome expression pattern in Glaesserella parasuis-induced meningitis,as well as the association between the methylome and transcriptome profiles.And the key functional genes regulated by DNA methylation in the pathogenic process of Glaesserella parasuis were idenfitied by functional enrichment of differential genes.By the combination of an integrated bioinformatic analysis,we explored the possible role of DNA methylation in the pathogenic process of Glaesserella parasuis-induced pig meningitis and lay the foundation for subsequent functional studies.In this study,six 28-day-old naturally farrowed early weaned piglets（Duroc×Landrace×Large white,weighing 8-10 kg）were randomly divided into two groups including the negative control group and Glaesserella parasuis-infected group（GPS group）according to similar weight and sex ratio,with three replicates in each group.1 m L of Glaesserella parasuis strain SH0165（serotype 5）was injected intraperitoneally with 2×10⁹CFU/m L in the GPS group.The control group were injected intraperitoneally with an equal amount of 0.9%Na Cl solution.Clinical autopsy was performed for 7 days and cortical tissue samples were collected and stored in liquid nitrogen for whole genome bisulfite sequencing and transcriptome sequencing.The expression levels of candidate genes and DNA methylation levels in the promoter regions were detected by quantitative real-time fluorescence PCR（q RT-PCR）and bisulfite sequencing seperately.Whole genome bisulfite sequencing showed that there were 12145 differentially methylation regions（DMR）and 316621 differentially methylation loci（DML）in the cerebral cortex tissue of Glaesserella parasuis-infected piglets（|diff Methy|>0.2,P<0.05）,in which 4649 DMRs and 132,557 DMLs were significantly hypermethylated（P<0.05）,and 7,496 DMRs and 184,064 DMLs were significantly hypomethylated（P<0.05）;DMLs were most common in intergenic regions（36.6%）and in introns（34.3%）,followed by promoters（16.3%）and exons（7.3%）;the highest proportion of DMRs were found in intergenic regions（64.7%）and in introns（26.1%）,with only a small proportion of DMRs occurring in promoter regions（3.9%）and exons（2.8%）;KEGG pathway enrichment analysis for the genes with DMRs showed that these genes were mainly involved in axon guidance,focal adhesion and phospholipase D signaling.The transcriptome sequencing showed that 1126 genes were significantly differentially expressed（|log₂FC|≥1,P<0.05）in the cerebral cortex of the piglets infected with GPS,compareing to the control group,in which 606 genes were significantly up-regulated and 520 genes were significantly down-regulated（P<0.05）.KEGG enrichment of differentially expressed gene（DEG）showed that these genes are mainly involved in cytokine-cytokine receptor interaction,phagosome,cell adhesion molecules,hematopoietic cell lineage and antigen processing and presentation signaling pathways.The integration analysis of whole genome bisulfite sequencing data and transcriptome sequencing data showed that 40 DEGs in the cerebral cortex tissue of Glaesserella parasuis-infected piglets were significantly negatively correlated with their promoter region DMRs（|log₂FC|>0.5,|diff Methy|>0.1,P<0.05）,and 33 DEGs were significantly positively correlated with their promoter region DMRs（|log₂FC|>0.5,|diff Methy|>0.1,P<0.05）.The KEGG pathway enrichment for DNA methylation-m RNA expression inversely correlated genes showed that these inversely correlated genes were mainly involved in signaling pathways such as cell adhesion molecules,hematopoietic cell lineage,RIG-I-like receptor signaling pathway and bacterial invasion of epithelial cells.Meanwhile,the protein-protein interaction network was constructed for the inversely correlated genes by String database analysis,and the result showed that CXCL10,CCL5,IRF7 and MX1were the core proteins of the interaction network.Based on the difference fold change and significance,11 genes（C1QA,FRMD1,GBP1,SEMA4D,FSCN2,KCNA1,STXBP2,VWA1,CD8B,SLC25A34 and VEGFD）were selected from the inversely correlated genes as the candidates,and validated by quantitative real-time fluorescence PCR.The result showed that the expression levels of SEMA4D and VWA1 genes were significantly up-regulated（P<0.01）,STXBP2 gene expression level was significantly up-regulated（P<0.05）,C1QA and GBP1 genes were not significantly different but showed an up-regulated expression trend in the cerebral cortex tissue of Glaesserella parasuis-infected piglets,FRMD1 gene was significantly down-regulated（P<0.01）,KCNA1 gene was significantly down-regulated（P<0.05）,while the difference of FSCN2,CD8B,SLC25A34 and VEGFD genes were not significant after GPS infection,but with a down-regulated expression trend.Furthermore,based on the quantitative results and gene functional correlations,5 of the 11 candidates（FRMD1,STXBP2,GBP1,SEMA4D and VWA1）were selected for the subsequent bisulfite sequencing.The result showed that the DNA methylation levels in the promoter regions of SEMA4D,VWA1,and FRMD1 genes were significantly down-regulated（P<0.01）,the DNA methylation level in the promoter region of STXBP2 was significantly down-regulated（P<0.05）,and the DNA methylation level in the promoter region of GBP1were significantly up-regulated（P<0.05）in the cerebral cortex tissue of GPS-infected piglets,comparing to the control group.The result indicated that the m RNA expression levels of SEMA4D,VWA1 and STXBP2 genes were negatively correlated with the DNA methylation levels in the promoter regions,and the m RNA expression levels of FRMD1and GBP1 genes were positively correlated with the DNA methylation levels in the promoter regions in the cortical tissue of GPS-infected piglets.In conclusion,by the integrated analysis of genome-wide DNA methylation and transcriptome analysis for the cerebral cortex tissues from Glaesserella parasuis-infected piglets,we found that the detailed regulation mechanisms of DNA methylation modifications to the gene expression during Glaesserella parasuis-infecte might be different from case to case.And we identified three DNA methylation-m RNA expression inversely correlated genes（SEMA4D,VWA1 and STXBP2）,which may be involved in the process of Glaesserella parasuis-infection by modulating the DNA methylation level in the promoter region and play a role in the host immune regulation and inflammatory responses.This work lays a foundation for further study of the molecular mechanisms of meningitis induced by Glaesserella parasuis in pigs from the perspective of epigenetic modifications,and also provides potential new therapeutic targets for the treatment of bacterial meningitis.