Multiomics Analysis To Reveal The Transcriptional And Microbial Regulation Of Salmonella Enteritidis Infection In Chicken Cecum

Salmonellosis is a kind of zoonotic disease caused by Salmonella bacteria,which seriously threatens animal husbandry and public health safety.China is rich in genetic resources of livestock and poultry.The local chicken breeds have unique disease-resistant genes and regulatory mechanisms.To reveal the genetic resistance against Salmonella infection in local chicken and provide theoretical basis for genetics and breeding of disease resistance,the study was performed.The reciprocal crosses derived from two China local chicken breeds Guangxi Yao and Jining Bairi were orally inoculated with 0.3 m L（1×10⁸cfu/m L）Salmonella Enteritidis per chick at 2-day old.Based on the transcriptome of cecum tissues and the microbiome and metabolome of cecal contents at 3 days post-inoculation（dpi）,we drew out the main results as follows:1.Specific transcriptional regulation of S.Enteritidis infection was initiated in chicken cecum tissuesThe result showed that 3.06%m RNA species were altered significantly（|log₂FC|>1,Padj<0.05）,of which 394 were up-regulated and 131 were down-regulated at 3 dpi in cecum tissues.Meanwhile,0.62%of mi RNA species were altered significantly（|log₂FC|>0.678,Padj<0.05）,of which 8 were up-regulated and 5 were down-regulated.Gene Ontology（GO）enrichment analysis of 525 differentially expressed genes showed that three biological processes with the highest significance level were Innate immune response（P=1.10E-17）,Inflammatory response（P=3.65E-15）,and Immune response（P=1.60E-12）,including 36,30,and 24 genes,respectively.Except for the down-regulation of two genes in the Immune response,all the genes in the other two items were up-regulated.Notably,three anti-inflammatory genes ACOD1,TNIP3,and IL-10 were increased sharply in the challenge group.2.Chicken mi R-146b-5p and its isoforms were activated to modulate S.Enteritidis infection by targeting ubiquitin specific peptidase 3 gene（USP3）After analysis of mi RNA isoforms（isomi Rs）,some isomi Rs of mi R-146b-5p were significantly up-regulated in the challenge group（|log₂FC|>1,Padj<0.05）.The dual-luciferase reporter assay confirmed that both the annotated sequence of mi R-146b-5p and its3’+UU isoform can target USP3 in chicken fibroblast cell line（DF-1）and inhibit its expression.What’s more,the isoform showed a stronger inhibition effect than the annotated one（P=0.018）.3.Specific microbial regulation of S.Enteritidis infection was initiated in chicken cecal microbiotaUpon challenge,the Firmicutes phylum increased from 66.91%to 76.17%（P=0.041）at the expense that the Proteobacteria phylum decreased from 32.80%to 23.64%（P=0.044）.The decrease of Proteobacteria was almost entirely caused by Escherichia/Shigella genus（from 30.32%to 18.75%,P=0.005）,which was presumably restrained by the“sequestrating”effect of host EXFABP protein on bacterial siderophore.In respect of Firmicutes,multiple species belonging to Negativibacillus,Flavonifractor,Clostridium and two unclassified genera of Lachnospiraceae thrived in context of inflammation.4.Symbiotic bacteria stabilize the intestinal environment by promoting phenylpropanoid productionIn comparison of multi-omics,the responses of reciprocal crosses to S.Enteritidis invasion were different in metabolome.The variation of the Cross was obviously smaller than that of the Reverse-cross,and some phenylpropanoid metabolites were abnormally increased in cecal contents of the Cross.We identified the causal relation to microbiota,particularly to12 species of bacteria.In summary,2-day-old chicken can initiate inflammatory response and innate immune response to S.Enteritidis invasion at transcription level,and strengthen the control of the inflammatory response at 3 dpi for the tolerance switch.Some symbiotic bacteria proliferate in the process of Salmonella invasion,then form symbiosis with the host and exert resistance to pathogenic bacteria by promoting the production of some secondary metabolites such as phenylpropanoids,and contribute to the intestinal homeostasis.Our research can help to understand the interplay among host,microbiota,and S.Enteritidis,and provide a reference for further exploring the potential regulatory mechanisms.