| Brucella and O.anthropi are closely related and belong to the α-2 subclass of Proteobacteria.But the pathogenicity is very different,Brucella is a cause of zoonotic common pathogens,can be around the world of public health and cause serious harm;and O.anthropi is a kind of conditional pathogens,less harmful to humans.Two-component systems(TCS)are a kind of important virulence control system widely found in bacteria,which can play their regulatory role by sensing and processing environmental signals.The system consists of histidine protein kinase(HK)and response regulator(RR).Comparative genomics found that Brucella and O.anthropi were less diverse in the genome sequence,and TCS was one of their major differences.Differences in bacteria control system TceSR and TcfSR on brucella chromosome II in bacillus.Previous studies have shown that 12 pairs of TCS located on chromosome I can regulate the energy metabolism and transport of Brucella,biological macromolecule synthesis,bacterial division and bacterial survival and reproduction and brucella virulence are closely related to the gene,but located in the chromosome II on the binary control system TceSR and TcfSR virulence phenotype and pathogenesis is unclear.In this study,the mutant 16M△ TceSR and 16M△ TcfSR as the research object,through the analysis of the transcriptome sequencing and comparative proteomics,revealed by Two-component systems(TCS)TceSR and TcfSR regulation of Brucella virulence genes and pathogenicity protein,lay the foundation for the research and development of the pathogenic mechanism of Brucella and effective treatment drugs and vaccines.Objective:In this study,the parental strains of Brucella melitensis 16 M and mutant strain16M△ TceSR and 16M△ TcfSR as the research object,By simulating the environment of macrophages in vitro and to find out the conditions for efficient expression of TCS TceSR and TcfSR in vitro.In this condition,the parental strain 16 M and the mutant 16M△ TceSR and 16M△ TcfSR were cultured.The regulatory network of TCS TceSR and TcfSR in Brucella was studied by transcriptomics and comparative proteomics.And to explain how TCS TceSR and TcfSR affect the expression of target gene transcription and target protein after sensory and treatment of environmental signals,thereby promoting the survival of Brucella.The study of this experiment provides important information for the pathogenesis of Brucella.Methods:(1)By simulating the environment in macrophages in vitro,the parent strain 16 M was cultured in TSB to the logarithmic growth phase,and the bacteria were resuspended in high salt,low p H,high temperature,nutritional deficiencies and other environmental pressuresunder the pressure of 0.5h,extracting RNA,reverse transcription into c DNA.Tce S,Tce R,Tcf S,Tcf R four primers were designed to determine the optimal stimulation conditions using q RT-PCR technology.After the optimal stimulus conditions determined,will be different in the time of bacterial stimulation conditions(0.5h,lh,3h,4.5h,6h),under this condition the bacterial RNA was extracted and reverse transcribed into c DNA for real-timequantitative PCR to determine the best stimulation time.(2)Samples of whole samples were prepared by culturing the parent strain 16 M and the mutant strain 16M△TceSR and 16M△TcfSR in the best expression in vitro.Using Label-free technology and bioinformatics analysis,the differentially expressed proteins were screened out to reveal the relationship between regulatory network and pathogenicity of TCS TceSR and TcfSR in Brucella.(3)The parental strain 16 M and 16 M △ TceSR and 16 M △ TcfSR were cultured in the best environment in vitro.When the culture was carried out to the best time,the cells were collected and RNA was extracted.using RNA-seq technique was used and the biological information The differentially expressed genes between the parent strain 16 M and the mutant strain were screened to reveal the relationship between the regulatory network and pathogenicity of TCS TceSR and TcfSR in Brucella.Results:(1)By simulating the environment of macrophages,the expression of TceS,TceR,Tcf S,Tcf R four genes was highest when the cells were stimulated with 3h under the condition of nutrient deficiency and p H value of 4 by q RT-PCR method Therefore,the conditions of high efficiency in vitro were identified as 3h stimulation in GEM4.0.(2)The results of comparative proteomics showed that there were 465 differentially expressed proteins in the parental strain 16 M and mutant strain 16M△ TceSR,multiples of more than 2 of the 257.In the mutant strain 16M△ TceSR more than two times differentially expressde protein down-regulated a total of 147,a total of 110 up-regulated protein.These proteins were mainly related to the carbon and nitrogen metabolism,two-component regulatory system,cell division,heat shock protein,membrane protein,oxidative phosphorylation,ABC transporters and transcription related proteins.Differentially expressed proteins of parental strains 16 M and 16M△ TcfSR a total of 137,we select the protein fold difference in expression of more than two times as the research object of differentially expressed proteins,a total of 86,compared to the the parental strain 16 M there are 44 proteins are up-regulate of the expression of the differentially expressed proteins and there are 42 proteins are down-regulate.These differentially expressed proteins are invovled in metabolism,oxidative phosphorylation,membrane transport,type IV secretion system,transcription and protein transport and RNA degradation.(3)the transcriptome results showed that each strain was detected in 3121 genes in the 16 M and16M△ TceSR,compared to the 16 M,of 331 genes were changed expression in the 16M△ TceSR including137 genes decreased significantly,and 194 a gene(p<0.05)were increased significantly in the mutant strain 16M△ TceSR.Among these differentially expressed genes were mainly involved in two-component regulatory system system,carbon and nitrogen metabolism,oxidative phosphorylation,ABC transporter,ribosome synthesis related genes,lipopolysaccharide and secretion system etc..A total of 3117 genes were detected in each of the 16 M and 16M△ TcfSR strains.The expression of 342 genes was significantly changed in 16M△ TcfSR compared with 16 M.Of these,177 genes were significantly different in the mutant(p <0.05).The expression of 165 genes was up-regulated(p <0.05).Among these differentially expressed genes are mainly involved in ribosome size subunit composition related genes,carbon metabolism,oxidative phosphorylation,bacterial secretion systems,fatty acid synthesis,ABC transport systems,amino acid metabolism,purine,pyrimidine metabolism and DNA replication,repair and RNA degradation,TCS and so on.Conclusions:(1)The effective expression of TCS TceSR and TcfSR in vitro was stimulated by GEM4.0 for 3h;(2)the whole cell protein label-free and transcriptome sequencing results showed that two mutant and parental strains are compared,expression of many proteins and genes have been changed,indicating that there is a complex regulatory network in order to adapt to various complicated environment in brucella of TCS TcfSR and TceSR.Through this study,a preliminary understanding of the molecular mechanism of TCS TceSR and TcfSR and its regulation in survival of Brucella infection,provide important clues for further research on TCS TceSR and TcfSR and brucella cell survival and pathogenicity. |
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