Chromatin Accessibility And Transcriptional Regulation Of IPEC-J2 Cells During Inhibition Of Salmonella Choleraesuis By Lactobacillus Reuteri

Salmonella choleraesuis(S.choleraesuis)is a ubiquitous foodborne pathogen and one of the most common pathogens causing pig diarrhea,which is prevalent in the pork production chain worldwide and seriously endangers the sustainable development of the pig industry.The infection rate of S.choleraesuis in weaned piglets is high,which can lead to weight loss and even death of pigs.Therefore,measures to reduce S.choleraesuis transmission and infection are essential for the pig industry.In recent decades,with the widespread use of antibiotics,many bacteria,including Salmonella,have become resistant to antibiotics,so alternative strategies to find antibiotics are urgently needed to control pathogen infections.Lactobacillus reuteri(L.reuteri)naturally colonizes many animals and is a probiotic with antibacterial effect,which can effectively inhibit the activity of pathogens.L.reuteri inhibits the growth of Salmonella and other pathogens by producing reuterin and other organic substances,and has physiological effects such as regulating intestinal microbial balance and enhancing body immunity.L.reuteri also has a similar antibacterial effect in the gut of pigs and is able to inhibit S.choleraesuis,which causes diarrhea in piglets,however,the specific mechanism by which L.reuteri inhibits S.choleraesuis infection remains unclear.Therefore,in this study,we used the porcine small intestinal cell line IPEC-J2 as a model to first verify the inhibitory effect of L.reuteri on S.choleraesuis in vitro,and then combined ATAC-seq and RNA-seq analysis was used to reveal changes in host cell chromatin accessibility and transcriptional processes during L.reuteri inhibition of S.choleraesuis,and candidate transcription factors and key signaling pathways during antibacterial processes were screened and identified.The main findings were as follows:1,In vitro detection of the inhibitory effect of L.reuteri on S.choleraesuisIn order to investigate the inhibitory effect of L.reuteri on S.choleraesuis in vitro,the Oxford cup test was used to detect the antibacterial effect of L.reuteri on S.choleraesuis in this study.The results showed that the inhibition zone diameter reached 15.31±0.50 mm,indicating that L.reuteri could inhibit the growth of S.choleraesuis in vitro.In order to investigate the effect of L.reuteri on IEPC-J2 cells infected with S.choleraesuis,108 CFU/mL was first determined as the subsequent treatment concentration using the CCK8 cell viability assay,and then co-culture of L.reuteri and S.choleraesuis with IPEC-J2 cells revealed that the adhesion of S.choleraesuis to IEPC-J2 cells was significantly reduced(P<0.05);while the number of adhesion of L.reuteri was significantly increased compared with the control group(P<0.05),and the expression detection of S.choleraesuis virulence factors hila and inva showed that the addition of L.reuteri significantly inhibited the expression of hila and inva genes(P<0.05),indicating that L.reuteri could effectively inhibit the infection of IPECJ2 cells by S.choleraesuis.2,Transcriptional changes of IPEC-J2 cells during L.reuteri inhibition of S.choleraesuisIn order to investigate the potential molecular mechanism during L.reuteri inhibition of S.choleraesuis,in this study,L.reuteri and S.choleraesuis treatments were divided into three groups:control group(CTL),S.choleraesuis group(SC),and S.choleraesuis plus L.reuteri group(LR+SC),with three replicates in each group and a total of nine samples for RNASeq.517 differentially expressed genes(DEGs)were screened between SC and CTL groups,and 598 differentially expressed genes were screened between LR+SC and SC groups.KEGG enrichment analysis showed that differentially expressed genes in S.choleraesuis and controls were mainly enriched in signaling pathways such as Ras,MAPK and Rapl;some diseaserelated pathways were enriched in the LR+SC group,and then further GSEA analysis revealed that the expression levels of Clusterl and Cluster3 genes were down-regulated by S.choleraesuis and then recovered by L.reuteri,while the expression levels of Cluster4 gene were up-regulated by S.choleraesuis and then recovered by L.reuteri.The results of enrichment analysis showed that Clusterl,3,and 4 genes were mainly enriched in cell cycle,MAPK and other pathways.Notably,the MAPK pathway was enriched not only in Cluster3 but also in Cluster2,indicating that genes of this pathway were both up-and down-regulated following S.choleraesuis treatment,with down-regulated genes partially restored by L.reuteri.The above results suggest that the inhibition of S.choleraesuis by L.reuteri may be associated with the regulation of host inflammatory factors,cell cycle and apoptosis and other signaling pathways.3,Chromatin accessibility changes in IPEC-J2 cells during L.reuteri inhibition of S.choleraesuisIn order to further understand the changes in chromatin accessibility of IPEC-J2 cells during L.reuteri inhibition of S.choleraesuis,ATAC-seq was simultaneously performed on a total of nine samples from CTL,SC,and LR+SC groups in this study.Differential chromatin accessible regions(DARs)were annotated to genes based on ATAC-seq,9340 DARs were screened between SC and CTL groups and significantly enriched in signaling pathways such as PI3K-AKT,MAPK,and Rap1;3714 DARs were screened between LR+SC and SC groups and significantly enriched in signaling pathways such as MAPK and Rap1,which were similar to enriched pathways in RNA-seq.UBTD2,LRRC39,GRIK4 and ENSSSCG00000014143 genes were randomly selected for Chip-qPCR to detect the enrichment of H3K4me3 and H3K27ac in their promoter regions,and it was found that the chromatin accessible region of UBTD2 was differentially enriched for H3K4me3 and H3K27ac between SC and CTL groups,but LRRC39,GRIK4 and ENSSSCG00000014143 genes did not change significantly in their chromatin accessible regions after S.choleraesuis and L.reuteri treatment,indicating that the transcriptional activation of host cells during the antibacterial process of L.reuteri was not completely consistent with chromatin opening.In addition,the combined analysis of ATACseq and RNA-seq screened 26 genes showing similar trends in transcript expression and chromatin accessibility changes,which may be potential regulatory molecules during L.reuteri antibacterial processes.4,Regulation of PI3K-AKT signaling pathway in antibacterial pathway of L.reuteriIn this study,we focused on PI3K-AKT,a potential signaling pathway,by MEME analysis,and qRT-PCR,IGV visualization,and Western blot results showed that PIK3R1 expression was up-regulated,while AKT phosphorylation was inhibited in IPEC-J2 cells infected with S.choleraesuis,and the addition of L.reuteri could inhibit PI3KR1 expression,thereby restoring AKT phosphorylation levels.Detection of downstream signaling pathways revealed that S.choleraesuis could activate the phosphorylation of JNK1 and ERK1,while L.reuteri could inhibit their phosphorylation levels.Detection of cell cycle and apoptosis in IPEC-J2 cells showed that S.choleraesuis could promote apoptosis and arrest the cell cycle to G2/G1 phase,while the addition of L.reuteri could restore S.choleraesuis-induced apoptosis,but had no significant effect on the cell cycle.Further treatment of IPEC-J2 cells with AKT activator SC79 revealed that the adhesion number of S.choleraesuis was significantly decreased(P<0.05),and hilA and invA expression showed a significant decrease(P<0.01).The above results suggest that PI3K-AKT signaling pathway may be a potential regulatory pathway for the antibacterial pathway in L.reuteri.In summary,in this study,we analyzed the chromatin accessibility and transcriptional changes induced by L.reuteri inhibiting Salmonella cholerae-infected porcine intestinal epithelial cells by ATAC-seq and RNA-seq,which not only provided new insights into the regulation of host gene expression patterns and epigenetic mechanisms when L.reuteri exerted bacteriostatic effects,but also excavated PIK3R1,a candidate molecular target,providing some basis and basis for further understanding the epigenetic and transcriptional regulation mechanisms triggered by L.reuteri inhibiting Salmonella cholerae-induced porcine intestinal epithelial cells,and also provided some reference for the genetic improvement of porcine bacterial diarrhea.