Expression And Regulation Of Proteins In The Response To Salmonella Enterica Serovars Enteritidis Infection In Chicken Cecum

Salmonella enterica serovars Enteritidis(SE)is one of the most common zoonotic bacteria that can invade poultry digestive system and cause inflammation and even death,causing great damage to the poultry industry.It can be found in all types of poultry and poultry products,infects human and causes food poisoning,poses a health risk to human.The effectiveness of vaccine protection is serotype specific and weak.Vaccine immunity will not be able to decontaminate the flock by serological methods at a later stage.Antibiotics can cause dysbiosis of the intestinal flora of livestock and bacteria to develop resistance,so improving the genetic resistance of livestock is an effective alternative to the traditional prevention and control of SE.The mechanisms of regulation of SE resistance at the m RNA and mi RNA levels in chickens have been investigated previously,but the mechanisms of regulation of protein are still not yet clear.A total of 120 crossbred F1 generation chickens between Jining Bairi and Guangxi Yao chickens were tested for SE negative at 1 day of age.100 F1 SE negative chickens were divided into two groups:49 chickens in the control group and 51 chickens in the test group.The test group was inoculated orally with 0.3 m L of 8.95×10~8cfu/m L SE solution,while the control group was inoculated with an equal amount of sterile Phosphate buffered saline(PBS).At 3 d after inoculation,cecum and cecum contents from 13 chickens in the control group and13 chickens in the test group were collected.Protein extraction was performed using TMT(Tandem mass tags)labeled proteomics and sequencing was performed separately to investigate the protein regulation mechanism of SE infection.The results showed that:(1)Protein quantification yielded a total of 3,723 proteins,including 192 significantly differentially expressed proteins,156 up-regulated proteins and 36 down-regulated proteins(P<0.05,fold change>1.2).The up-regulated and down-regulated proteins were then analyzed for subcellular localization,COG/KOG classification and enrichment for Gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG)and protein structural domains.(2)The up-regulated proteins were mainly enriched in immune-related biological processes such as response to virus,defense against virus,and response to external stimuli(P<0.05).The down-regulated proteins were mainly enriched in pathways related to growth and development such as cell morphogenesis,enzyme-linked receptor protein signaling pathway,and extracellular structural tissue(P<0.05).(3)The up-regulated proteins were mainly enriched in immune-related pathways,such as influenza A,herpes simplex virus type 1 infection,NOD-like receptor signaling pathway,RIG-I-like receptor signaling pathway,Toll-like receptor signaling pathway,and others.Meanwhile,the down-regulated significantly different proteins were mainly concentrated in ECM receptor interaction pathway,adhesion point pathway and other pathways.(4)The verification results of the differentially expressed proteins showed that the PRM verification results were consistent with the sequencing results.In particular,LDHA,XDH,LBP,PCK2,ASS1 and STAT1 were significantly different and consistent with the sequencing results.(5)Results of m RNA expression revealed that ASS1,LDHA,LBP,CNP,and STAT1exhibited significant differences between test and control groups(P<0.05).(6)Targeting relationship verification showed that the co-transfection of the constructed ADK double luciferase reporter vector and mi R-12259-5p mimic/NC into DF1 cells resulted in a significant decrease in the fluorescence value of the wild-type vector and mi R-12259-5p mimic group compared to other groups(P<0.05).These results demonstrated that SE infection mainly induced protein expression in the F1generation of Guangxi Yao chicken and Jining Bairi chicken.These differentially expressed proteins were mainly enriched in immune-related biological processes,such as influenza A signaling pathway,herpes simplex virus type 1 infection,Toll-like receptor signal pathway,Fox O signal pathway,NOD-like receptor signal pathway and RIG-I-like receptor signal pathway.Proteins LBP,STAT1,LDHA,HSP90AB1 and ADK play major role in the response to SE infection.The findings provide both theoretical and scientific basis for understanding the regulatory mechanisms of SE infection and disease resistance breeding in poultry.