| As one of the most important foodborne microorganism,Campylobacter jejuni(C.jejuni)leads to the gastroenteritis of human and neurogenic disease like Guillain-Barre syndrome(GBS).Macrolides,such as erythromycin,are the first choice for the treatment of Campylobacter infections.However,resistance to macrolides is caused for the abuse of antibiotics,which reduces the therapeutic efficiency of antibiotics and threatens the clinical health of human.Substitution of bases,mutations of ribosomal protein and efflux pump are the main reasons of macrolides resistance to Campylobacter.Besides,methylase coded by erm genes is one of the main causes of macrolides resistance.In our research,transcriptomics,proteomics and metabolomics protocols were performed for the study of comparative transcripts,proteins and metabolites and associated biofunctions and pathways between ermB-positive and negative strains of C.jejuni,which explains the regulation mechanism of fitness cost of ermB gene expression in C.jejuni from RNA,protein and metabolite layers.Transcriptome analysis was carried out by the RNA-Seq high-throughput sequencing technology after extracted the total RNA of ermB-positive and negative strains of C.jejuni followed by generating cDNA library.According to the referent genome of the standard bacterial strain C.jejuni NCTC 11168,preprocessing was performed to filter the raw data,and the gene expression quantity FPKM was standardized for relatively quantification of transcripts.A total of 58 transcripts were identified,and 1 was up-regulated by 5.52 fold while 57 were down-regulated by 3.33-426.95 fold.After biofunction analysis,the up-regulated gene cj1477c participated in glyoxylate and dicarboxylate metabolism;the down-regulated genes flgE,flgK,flaA,flaB,fliD,flgS,flgD,flgI,flgE2,flgH,cj0887c,flgB,flgG,flgG2,fliS,flgC were associated with flagellar assembly,hisH,hisF were connected with histidine metabolism,cj0489 was relevant to glyoxylate and dicarboxylate metabolism,pseB,pseC,ptmB took part in amino sugar and nucleotide sugar metabolism.qRT-PCR was performed to validate the relative quantification of RNA-Seq,and the results were coincident to the consequence of RNA-Seq,which proved the accuracy and reliability of sequencing method.In proteomics studies,total proteins of ermB-positive and negative strains of C.jejuni were extracted by ultrasonic equipment and enzymolyzed to peptides by FASP workflows.Then the proteins were relatively quantified by MRM-MS mode after the selection of suitable peptides,optimization of precursor ions and product ions and so on.20 comparative proteins including 4 up-regulated(fold change was 1.52-4.71)and 16 down-regulated(fold change was 1.66-79.64)proteins were identified.GO and KEGG searching revealed that up-regulated protein Cj1450 was associated with ATP/GTP binding,FlgG2 was connected with flagellar basal-body rod and cilium or flagellum-dependent cell motility,Cj0850c participated in the transport process and Cj 1422c was relevant to the sugar transfer process;meanwhile,the down-regulated proteins FlgD,FlgE2,FlgI,FlaB,FlaA,FlgG were connected with structural molecular activity,FlgE2,FlaB,FlaA,FlgD were associated with cilium or flagellum-dependent cell motility,FlgE2,FlaB,FlaA,FlgD,FlgI,FlgG,PtmA participated in flagellar assembly,Cjl477c,Cj1476c,HisF,PtmA,HisH were connected with metabolic pathways,Cj1477c,HisF2,HisHl were related to biosynthesis of secondary metabolites,PseB,PseC joined in amino sugar and nucleotide sugar metabolism,HisF2,HisH1 were connected with histidine metabolism and biosynthesis of amino acids,FlaB,FlaA,FlgS belonged to two-component system.The flagella were dyed and observed by transmission electron microscope,and the images illustrated that the filament was lost in ermB-positive strain while the sensitive strain had complete flagella at double or single end.Besides,the capability of motility,autoagglutination,adhesion and invasion to HEp-2,VERO and IEC-6 cells reduced in ermB-positive strain.Results of ELISA demonstrated that the concentration of glucose and ATP increased,while the concentration of hypoxanthine decreased with the expression of ermB gene in C.jejuni.Metabolomic profiling was operated by quenching metabolism of bacteria with liquid nitrogen and extracting metabolites with cold methanol by freezing-thawing cycles.C18 and HILIC columns were utilized for separation of metabolites,and QTOF mass was used for the determination of features followed by data processing by MarkerLynx software with multivariate statistical analysis.After searching in databases,36 putative discrepant metabolites were identified.14 metabolites were up-regulated(fold change was 1.50-10.65)and 22 were down-regulated(fold change was 1.51-12.70).HMDB and KEGG were applied for the biofunctions and pathways analyzing of the putative compounds,and they were mostly related to cell signaling(3 metabolites were up-regulated while 5 were down-regulated),membrane integrity/stability(3 metabolites were up-regulated while 5 were down-regulated),fuel and energy source/storage(3 metabolites were up-regulated while 5 were down-regulated)and nutrient(4 metabolites were up-regulated while 3 were down-regulated).According to the biofunctions and cell locations of comparative metabolites,the determination of biofilm formation capability was performed and the results demonstrated that capability of biofilm formation in resistant strain was inferior than that in susceptive strain.From the multi-omics data,we can get a conclusion that ermB gene can induce the loss of filament and reduce the capability of motility,autoagglutination,adhesion and invasion after it expressed in C.jejuni and caused resistant phenotype.The concentration of glucose and ATP increased in resistant strains.Simultaneously,ermB gene also obviously decreased the concentration of hypoxanthine.These findings further elucidated the regulation mechanism of resistance caused by ermB gene in C.jejuni,meanwhile,our study provided scientific basis for the prevention and control of the resistance to Campylobacter. |
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