Comparative Proteomic And Transcriptomics Analysis Of Brucella Biofilm And Planktonic Cells

Brucellosis,which is caused by infection with Brucella,is one of the most common bacterial zoonotic diseases with potential lethal property in cattle and human worldwide.Animals,such as cattle,sheep and pigs,were the first host for brucella,and huamans were only acquired via touching or eating infected animals or their dairy products.Biofilm is a special condition for bacterium.With biofilm,the bacterium can adsorbed on the surface of objects,secrete large amounts of polysaccharide matrix protein,fiber,protein,fats and protein polysaccharide complex to adapt the bad living environment.With biofilm,bacteria can withstand heat,cold,disinfectants and antibiotics and to further cause a persistent infection and repeated infections in man and animals.Thus,the study of Brucella biofilm is important for brucellosis’ clinical investigation.In the present study,we constructed the Brucella biofilm model,and compared the drug resistance profile between biofilm and planktonic cells.Furthermore,we conducted 2-D electrophoresis,high-throughput sequencing and real time RT-PCR analyses in order to find differently expressed proteins and genes that are responsible for high pathogenicity of biofilm cells.1.Establishment of real time PCR methods to detect Brucella and the isolation and identification of BrucellaTwo kinds of genus real-time PCR and three kinds of Species differentiation real-time PCR were established based on the nucleotide sequences of Brucella.After optimization of the detection methods,the high specificity of the five methods was demonstrated by detecting 10 Brucella strains and 12 other bacteria strains,such as Listeria monocytogenes,Shigelladysenteriae,and so on.The maximal detection limits of those methods were all less than 43 copies of bacteria and DNAs.Therefore,the five real-time PCR methods developed here performed high efficiency,speediness,specificity and sensitivity in the detectin of Brucella.A large number of serum,aborted fetus and fresh milk samples from cattle and sheep were collected and were detected by real time PCR.Eight strains were isolated from aborted fetus and seven of them were Brucella which was identified by agglutination test and biochemical identification.Virulence test showed that A3313 strains exhibited the highest virulent.2.Prokaryocytic expression of outer membrane protein OMP25 and establishment of immunomagnetic beads method for brucella initiallyThe epitopes of OMP25 from Brucella was forecasted,it gene was synthesized and cloned into prokaryotic espression vector pET-3 2(+)and identified by enzyme digestion and DNA sequencing.The recombinant plasmid was transformed into E.coli BL21(DE3)and expression.The recombinant protein was purified and detected by Western blot.It was showed that the recombinant proteins possessed the good reactogenicity and reactogenicity.Its antiserum can react to thalli of Brucella.The antibody of the recombinant protein could be used to detect Brucella by immunomagnetic beads.3.Potential differentially expressed mRNA and associated molecular mechanism investigation on virulence of Brucella melitensis strain RM57The DEMs between samples in Brucella melitensis M1981 strains and RM57 strains were revealed based on a gene expression profile in GEO database.Then the enrichment analysis and PPI network analysis were preformed based on these DEMs.Finally,the protein subcellular localization(SCL)analysis was performed to reveal the location of potential hub proteins,followed by the signal peptides(SPs)prediction.A total of 403 DEMs were revealed between samples in two groups.These DEMs were mainly enriched in pathways like metabolic and functions like ribosome.A PPI network and unique module were constructed based on these DEMs.These DEMs including as rpsP and rpoA were mainly enriched in pathways like RNA polymerase and functions like ribosome.The SCL investigation showed that proteins encoding by these DEMs such as rpsP were located in cytoplasm.SPs prediction showed that the likelihood for the protein type of SP,TAT,LIPO and Other in rpsP was 0.1288,0.2109,0.0513 and 0.609,respectively.Cytoplasmic rpsP might play an important role in the virulence reduction of RM57 strain via participating in ribosome pathway and structural constituent of ribosome function.4.The culture and characteristic observation of Brucella biofilmBrucella abortus strain A3313,which was isolated from the abortus of cattle in Hohhot district,Inner Mongolia,China,was used in this study to culitivate biofilm.Phase constrast microscope and scanning electron microscope were used to reveal the formation and structure of biofim.After cultured with 9 difference antibiotics,a significant anti-drug ability was identified in Brucella biofilm while compared with planktonic cells.5.Comparative proteomics analysis of Brucella biofilm and planktonic cellsThe differentially expressed proteins between biofilm and planktonic cells of A3313 were investigated by 2-D electrophoresis and MS analysis.And the mRNA transcriptional levels of the identified proteins were revealed by real time RT-PCR.As a result,total of 47 differentially expressed protein points were identified more than 1.5,including 7 upregulated points and 40 downregulated points.After analyzed by MS,a total of 27 differentially expressed proteins were confirmed,and the real time RT-PCT results showed that the mRNA transcriptional levels of 19 proteins were downregulated in biofilm cells.It indicated that Brocella biofilm had a significant difference in the levels of protiens and mRNA while compared with planktonic cells.6.Comparative transcriptomics analysis of Brucella biofilm and planktonic cellsThe differentially transcribed mRNA of Brucella biofilm and planktonic cells were investigated by high-throughput sequencing analysis,and the bioinformatics was used for molecule level analysis,such as molecule pathway.Moreover,the mRNA quantitative analyses of selected genes and cirulence factor genes were conducted by real time RT-PCR.As a result,157 differentially transcribed genes in 3 kinds of biofunction were identified and mainly enriched in 6 biological pathways.Real time RT-PCR results showed that 15 upregulated genes,7 downregulated virulence factors genes and 2 upregulated virulence factor genes were identified in Brocella biofilm while compared with plaktonic cells.7.Evaluation of the differences between biofilm and planktonic Brucella abortus via metabolomics and proteomicsA number of bacteria evolve various responsive mechanisms such as biofilm stages and planktonic free-living lifestyles,which enables their adaptation to a wide range of adverse environments.Here,we aimed to analyze the difference between biofilm and planktonic Brucella abortus from metabolomics and proteomics dimensions.Biofilm formation showed different expression of metabolites and proteins.The differentially expressed proteins were primarily located in cytoplasma.Association analysis of proteomics and metabolomics revealed that fatty acid biosynthesis was the potential pathway implicated in biofilm formation.In conclusions,there are differences between biofilm and planktomic Brucella abortus at metabolic and proteomic dimensions,and our results shed novel and pioneering insights into the biofilm-forming process of Brucella abortus.