| Salmonella enterica serovar Enteritidis(SE)is one of the most common pathogens associated with poultry health and foodborne salmonellosis worldwide.It can cause a major public health problem and economic losses in the world.The protective efficacy of vaccines is serotype specific.Antibiotics can cause intestinal flora imbalance and bacterial resistance in livestock and poultry.Therefore,improving genetic resistance in livestock is an effective complement to traditional prevention and control of SE.While regulatory mechanisms at the m RNA and mi RNA levels in chickens infected with SE have been investigated in our laboratory,the regulatory mechanisms at the protein level remain unclear.This study aimed to explore the regulatory mechanisms of SE infection in chickens at the protein level.The 2-day old SE-negative F1 generation of Jining Bairi chickens and Guangxi Yao chickens were randomly divided into two groups.The treated group was orally fed with 0.3 m L of SE solution at a concentration of 6.34×10~7cfu/m L,while the control group was orally fed with an equal amount of sterile phosphate buffered saline(PBS).The protein regulatory mechanism of SE infection was analyzed based on TMT(Tandem mass tags)labeled proteomics and phosphorylation modification proteomics of cecum tissues 3 days after inoculation in chickens.The may results are as follows:1 Regulation of protein expression in chickens postinfected with SEA total of 6,117 proteins were identified in the quantitative proteomic,with 4,995 being quantifiable.Among them,315 differentially expressed proteins were screened(fold change>1.2,P adj<0.05),which 166 proteins were up-regulated,149 proteins were down-regulated.To validate the proteomics results,eight differentially expressed proteins were randomly selected for parallel reaction monitoring(PRM),and the PRM results for FABP4,FABP1,ACACA,AGPAT2,CPA5,HBB,HBAD,and OASL were fully consistent with the sequencing results.Further analysis was performed of up-and down-regulated proteins based on protein subcellular localization,COG/KOG classification,Gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG),and protein structural domains.The up-regulated proteins were significantly enriched in 14 GO-biological process(BP)terms,which mainly related to protein activation cascade,defence response to bacterium,activation of immune response,and lipid transport.The significantly enriched in GO-BP terms of down-regulated proteins were related to transport and regulation of stimulus response,such as oxygen transport,organic anion transport,regulation of extracellular stimulus response,and negative regulation of biotic stimulus response.Specifically,the up-regulated proteins were significantly enriched in KEGG pathways such as PPAR signaling pathway and ABC transporters,while the down-regulated proteins were significantly enriched in metabolic-related KEGG pathways such as metabolic pathways,retinol metabolism,fatty acid metabolism,fatty acid biosynthesis,and steroid biosynthesis.The results of functional enrichment showed that the chicken infected with SE could regulate the metabolism process by regulating the expression of related proteins in the early stage,and after changing the immune-metabolism environment to resist SE colonized in the cecum.Furthermore,protein-protein interaction networks were mapped for differentially expressed proteins,including three major subnetworks:metabolic pathway,PPAR signaling pathway,and cardiac muscle contaction.In summary,the study provides new insights into the regulatory mechanisms of SE infection in chickens,shedding light on the alterations in immune regulatory processes.2 Regulation of protein phosphorylation modification in chickens postinfected with SEIn this study,a total of 10,722 phosphorylation modification sites corresponding to 3,868proteins were identified,with 7,329 sites on 3,155 proteins quantitatively informative,and338 sites identified as having significantly changed phosphorylation levels after differential modification significance analysis.Among them,213 up-regulated sites corresponding to 146proteins and 125 down-regulated sites corresponding to 97 proteins(fold change>1.5,P adj<0.05).18 differentially phosphorylation modification sites were randomly selected for PRM validation,out of which 4 phosphorylated sites were identical to the sequencing results,while the fold change of remaining 14 sites was less than 1.5,but the expression trends were consistent with the sequencing results.Motif analysis of protein phosphorylation modifications sites revealed 64 conserved motifs out of 7,181 serine phosphorylation(p S)sites and 7 conserved motifs out of 527 threonine phosphorylation(p T)sites.Functional enrichment analysis showed that the up-regulated phosphorylation modifications sites corresponding proteins were significantly enriched in 14 GO-BP terms(P adj<0.05),which could be classified into four main categories:defence responses,activation of immune cells,antioxidant-related responses and others.Three GO-BP terms(P adj<0.05)were enriched for down-regulated phosphorylation modification sites corresponding proteins,which were adherens junction organization,retrograde vesicle-mediated transport,Golgi to ER,and positive regulation of GTPase activity.The enriched KEGG(P<0.05)pathways for up-regulated phosphorylated modification sites corresponding proteins could be classified into three categories:metabolism-related pathways,immune,metabolic,and inflammatory response-related signaling pathways,and others.The down-regulated phosphorylated modification sites corresponding proteins was significantly enriched to ABC transporters(P adj<0.05).It was also observed that SE inoculation resulted in altered phosphorylation levels of metabolism-related proteins such as CARHSP1,CASK,KHSRP,RAD23A,RBM5,TMF1,FANCM,HECTD4,PPP2R5A,and transporter proteins ABCA3,ABCB1,and ABCC3.The enriched Wnt signaling pathway,NOD-like receptor signaling pathway,PPAR signaling pathway,MAPK signaling pathway and ABC transportor indicate that SE infection can affect the immune response and metabolism of chicken by changing the protein phosphorylation in the relevant signaling pathway.The constructed protein-protein interaction network consisted of 74 up-regulated and 43 down-regulated differentially phosphorylation modification sites corresponding proteins,which involved three subnetworks,protein processing,endocytosis and tight junction.3 Combining analysis of proteomics and phosphorylation modification proteomicsThis study counted proteins with insignificantly expressed in proteomics but significantly expressed in phosphorylation modification proteomics.The results showed that there were 139 proteins corresponding to up-regulated phosphorylation modification sites and94 proteins corresponding to down-regulated phosphorylation modification sites had no significant differences in proteomics.An interaction network was mapped between differentially expressed proteins in proteomic and phosphorylated modification sites corresponding proteins in phosphorylation modification proteomics,which have five subnetworks:PPAR signaling pathway,metabolic pathways,VEGF signaling pathway,Apelin signaling pathway and metabolism of xenobiotics by cytochrome P450.There were 30overlapped proteins between the 315 differentially expressed proteins identified in the proteomics and the 243 phosphorylated modification sites corresponding proteins in the phosphorproteomics.Only 4 proteins were regulated in the same type,while the remaining 24showed opposite regulation,and the phosphorylation modification sites of F1NLZ2 and E1C172 had two types of regelation.The BP terms enriched for the overlapped proteins were actin filament organization,actin filament polymerization,negative regulation of the platelet-derived growth factor receptor signaling pathway,and actin polymerization or depolymerization.Additionally,the KEGG pathway significantly enriched for these proteins was tight junctions,indicating that chickens infected with SE may maintain cellular homeostasis through the actions of actin and tight junctions.4 FABP1 interact with ATP1B1 and NPC2 and maintain homeostasis in chickens postinfected with SEThe study investigated the mechanism of action of FABP1,a protein associated with lipid metabolism,using the yeast two-hybrid technique to screen for interacting proteins.Non-toxic and non-self-activating FABP1 bait vectors were constructed,and the interaction protein of FABP1 was screened by co-transformation method.Two positive clones were screened.After sequencing,the sequence alignment was ATP1B1(sodium/potassium transport ATPase subunitβ-1)And NPC2(NPC intracellular cholesterol transporter 2),and then conduct one-to-one and dot plate verification to confirm that ATP1B1 and NPC2 are the interacting proteins of FABP1.And they jointly regulating the response to SE infection in chickens.However,further research is needed to determine how these interactions contribute to the regulation of homeostasis between the organism and SE.Overall,the differentially expressed proteins and phosphorylated modification sites in chickens infected with SE were analyzed.Protein-protein interaction regulatory network was constructed and interacting proteins of differentially expressed protein FABP1 using a yeast two hybrid system were screened to clarify the protein regulatory mechanism in chickens postinfected with SE.These findings provide theoretical support and scientific basis for SE infection resistance breeding in poultry. |
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