Proteomics Study Of Piglet Diarrhea Caused By Clostridium Perfringens Type C And Its Combined Analysis With Transcriptome Data

Diarrhea is a intestinal disease that causes poor performance of piglets,results in huge economic losses in the pig industry.Clostridium perfringens type C(C.perfringens type C)is one of the common pathogens causing bacterial diarrhea of piglets.C.perfringens type C infects piglets by oral route,then grows in the intestine of piglets quickly and destroies intestinal tissue by releasing a variety of toxins,eventually leads to piglet diarrhea.Although the use of drugs or vaccination to control the C.perfringens diarrhea in piglets has certain effects,however,long-term use of drugs may cause drug residue problems,and probably lead to increase resistance of pathogenic bacteria and the emergance of more virulent mutant strains,posing a huge risks to health farming and food safety.It is well known that differences in the genetic basis of individuals play an vital role in the resistance/susceptibility of pathogen infection.At the same time,individuals also initiate biological processes such as immune response and metabolic regulation in response to pathogenic bacteria infection,which involve in the expression of a large number of proteins and the activation or closure of key signaling pathways.Therefore,understanding the biological events of piglets respond to C.perfringens type C diarrhea in the overall level,and analyzing the genetic basis of C.perfringens type C diarrhea resistant piglets,is of great theoretical significance for breeding new strains of resistant pigs.In view of this,7-day-old binary piglets were used as research objects in this study,and 25 piglets were infected with C.perfringens type C culture solution.According to the total score of piglet diarrhea,3 piglets were selected as susceptible group(IS)and resistant group(IR),respectively,3 piglets were seclected as control group(IC)with uninoculated strain culture solution.The main research is as follows:(1)Identification and screening of protein expression profiles and differentially expressed proteins in ileum tissue of IR,IS and IC piglets using Label-free quantitative proteomics technology and bioinformatics technology,and differentially expressed proteins were performed GO and KEGG signal pathway enrichment analysis;(2)Combined proteomics data and previous RNA-Seq sequencing data to screen associated genes and co-expressed genes on mRNA and protein levels in ileum tissue of IR vs IS、IS vs IC and IR vs IS comparison group piglets.Bioinformatics technology was used to perform GO and KEGG signal pathways enriched analysis of the co-expressed genes to explore the molecular regulation mechanism of piglet tolerance/susceptibility to C.perfringens type diarrhea.The main results are as follows:1.A total of 38,899 peptides were identified,and the number of quantified proteins were 4327,4391,and 4262 in the IR,IS,and IC groups,respectively.There were 59 significantly up-regulated proteins and 52 significantly down-regulated proteins in the IR vs IC group,178 proteins were specific expression;There are 154 significantly up-regulated proteins and 85 significantly down-regulated proteins in the IS vs IC group,210 proteins were specific expression;There are 10 significantly up-regulated proteins and 7 significantly down-regulated proteins in the IR vs IS group,120 proteins were specific expression.2.The results of GO function and KEGG signaling pathway enrichment analysis of differentially expressed proteins in piglets among IR,IS and IC groups showed that:(1)The differentially expressed proteins in IR vs IC group were significantly enriched in 112 GO functions,mainly involved in processes of immune system,regulation of sodium ion export,and cell proliferation and death,involving some immune-related proteins and receptors.For example,S100 protein family S100A2,SLA-2 and transmembrane protein CD82;the result of KEGG signaling pathway enrichment analysis showed that the differentially expressed proteins significantly enriched in cell adhesion molecule,carbohydrate digestion and absorption,steroid hormone biosynthesis and so on,including ICAM1,MHC type ? antigen SLA8 and OCLN.These GO functions and KEGG signaling pathways may play a biological role in resistance of piglets to C.perfringens type C infection.(2)The differentially expressed proteins in the IS vs IC group were significantly enriched in 336 GO functions,mainly involved in antigen processing and expression,lipid transport and MHC protein complexes,including SLA-1 and SLA-DQA,myeloid differentiation protein MyD88 and actin;KEGG signaling pathway enrichment analysis showed that the differentially expressed proteins were mainly enriched in 16 KEGG signaling pathways,such as staphylococcus aureus infection,cells Adhesion molecules and cytochrome P450 metabolism of foreign organisms,etc.These GO functions and KEGG signaling pathways may be related to the susceptibility to C.perfringens type C in piglets.(3)The differentially expressed proteins in IR vs IS group were significantly enriched in 126 GO functions,mainly involved in MHC protein complex,bacterial defense response and Antigen processing and presentation,etc.The related proteins included SLA1,IL-6 and IFI30;KEGG signaling pathway enrichment analysis showed that the differentially expressed proteins were mainly enriched in protein digestion and absorption,pancreatic secretion,and were also enriched in NOD-like receptor signaling pathway,NF-κB signaling pathway and Toll-like receptor signaling pathway.These GO functions and the KEGG signaling pathway may have explained the molecular genetic basis of piglets resistant/susceptible to C.perfringens type C.3.In the combined analysis of transcriptome and proteome,we founded that there are 3945,3930 and 4013 associated genes in the IR vs IS,IS vs IC and IR vs IS groups,respectively,which contained 69,120 and 0 co-expressed genes.4.The results of GO function and KEGG signaling pathway enrichment analysis of co-expressed genes in IR vs IC and IS vs IC groups in the ileum of piglets showed that:(1)The co-up-regulated genes were significantly enriched in 108 GO functions in IR vs IC group,mainly involved in processes of ATP synthesis,catalytic activity and acute inflammatory response,including energy metabolism and immune associated proteins.The KEGG pathway enrichment analysis showed that the co-up-regulated genes were significantly enriched in 14 KEGG signaling pathways,such as metabolism pathway,peroxisome and endocytosis which including Saa2,Itih4 and Slc15a1 genes,etc.The co-down-regulated genes were mainly enriched in 65 GO functions in IR vs IC group,mainly involved in processes of transcriptional factor binding,regulation of biosynthetic process and response to bacterium,including Ocln,Mbnl and Elmo1 genes,etc.(2)The co-up-regulated genes were significantly enriched in 129 GO functions in IS vs IC group,mainly involved in processes of the integral component of cell membrane,ion transmembrance transporter activity,and organic subtance transport.KEGG signaling pathway enrichment analysis showed that the co-up-regulated genes were significantly enriched in 20 KEGG signaling pathways,such as metabolism pathway,fatty acid degradation and peroxisome.The co-down-regulated genes were significantly enriched in 143 GO functions in IS vs IC group,mainly involved in processes of integrin complex,T cell receptor,T cell activation via T cell receptor contact with antigen bound to MHC molecule on antigen presenting cell,and immune system,etc.KEGG signaling pathway enrichment analysis showed that the co-down-regulated genes were significantly enriched in 20 KEGG signaling pathways,such as human T-cell leukemia virus 1 infection,staphylococcus aureus infection,rap1 signaling pathway,and regulation of actin cytoskeleton,including Apbb1 ip,Stat5 and Alox15 genes,etc.In summary,through proteomic analysis of piglet ileum tissue and its combined analysis with transcriptome data,it is speculated that changes in energy metabolism,immune response,cell membrane structure,and intestinal inflammation may mediate piglets resistant/susceptible to C.perfringens type C,and genes such as Apbb1 ip,Stat5,and Wipf1 may play important roles in Clostridium perfringens type C diarrhea.This study can provide important foundation for molecular regulation mechanism of piglet diarrhea caused by C.perfringens type C and the breeding of diarrhea-resistant pig strain.