Omics Characterization Of Brucella Strain019

Object: Brucellosis is a seriou zoonotic disease. Xinjiang is a important province ofanimal husbandry in China, while the main epidemic areas of brucellosis. In the early80’s of the last century, contagious ovine epididymitis outbreaked in a plurality ofbreeding sheep farm. Then Brucella strain019was isolated from Merino ram semensuffered byepididymitis and was identified as Brucella ovis by traditionalmicrobilogical test. This is the first report of Brucella ovis isolation in Xinjiang.However, strain019can infect Rhesus monkey and the genes related with outermembrane were different from them of classical Brucella ovis. In addition, deadbacteria adjuvant vaccine of strain019can trigger good cellular and humoral immuneresponse in sheep and provide good protection against Brucella ovis. To address theconfusion including Brucella ovis infecting Rhesus monkey, molocular differencewith cllasical Brucella ovis, dead bacteria adjuvant vaccine of strain019providinggood protection against Brucella ovis, we analyzed the charactorization of Brucella019by genome and proteome technology.Methods: The genome of strain019was sequenced by Illumina hiseq2000. Genomeassembled with SOAP1.05, ORFs were predicted with Glimmer3.02, annotation wasdone by Nr database. SNPs were analyzed between strain019and other15Brucellastrains. Synteny was analyzed between strain019and other Brucella strains. Thewhole genome phylogenetic tree was constructed based on homologous genes from51genomes from Brucella and strain019. High SNPs from intergenic space as sRNAoccured in strain S2were predicted in secondary structure and target genes. Then,iTRAQ quantitative proteomics were carried out to analyze the difference betweenstrain019, B. melitensis16M, B. abortus2308and B. ovis63/290. The differentproteins were clustered by STRING v9.0(protein protein interaction) and KEGGpathway. To screen the genes related to virulence, we analyzed the differenceproteome between strain019and B.suis vaccine strain S2by2-D and MS/MS. TetRfamily transcriptional regulator Br1381and sensor histidine kinase Br0613wereanalyzed in multiple stress such as starvation, acid, alkali, low and high temperature,high oxidation, intracellular macrophage RAW264.7cells.Results: Whole genome sequence of strain019yieled330M clean data. Assembleresults showed31scaffolds,722contigs. All of3,529ORF were predicted andannotated. There were7073SNPs between strain019and B. ovis63/290, while150SNPs between strain019and B. suis1330. The whole genome phylogenetic treeshowed that the most close strain was B. suis vaccine strain S2. Futhermore,20SNPswere shared by strain019and S2, while44SNPs position. ZK-2and ZK-3high SNPsin intergenetic space of strain S2were highly conserved in Brucellae andOchrobactrum anthropi. Secondary structure of ZK-2and ZK-3occurred hugechange after mutation. The target mRNA of ZK-2and ZK-3increased high aftermutation. And they expaned their regulation of metabolism largely. Total179differentproteins（99up，80down）were identified between strain019and B. ovis63/290,134 different proteins（60up，74down）between strain019and B. melitensis16M,118different proteins （49up，69down）between strain019and B. abortus2308. Theprotein protein interaction net showed that Brucella strain019over expressedribosomal related proteins. Total17different proteins were identified between strain019and B. suis vaccine strain S2. Their function occupied transport of sugar andamino acids, signal sensing and transduction, transcriptional regulation and responseto host environment. Among them, OMP31, ALPC, Tu and Ts were genes related tovirulence. Transporter periplasmic sugar-binding protein, amino acid ABC transportersubstrate-binding protein and Twin-arginine translocation pathway signal sequencedomain-containing protein may be virulent genes in Brucella. In addition, TetR familytranscriptional regulator and sensor histidine kinase were different. Furthermore, TetRfamily transcriptional regulator Br1381was sensitive to stavation, acid, starvation,acid, alkali, low and high temperature, high oxidation and high transcriptional level inRAW264.7. While sensor histidine kinase Br0613was sensitive to both stress acidand alkali in stavation.Conclusion: Results of strain019whole genome analysis showed Brucella019belonged to B. suis and was close to B. suis vaccine strain S2. And this cllassificationis easy to explain strain019infection Rhesus monkey et al. According to reportsavailable, Brucella strain019may come from abroad. ZK-2and ZK-3high SNPsintergenetic space in vaccine strain S2expanding their regulation may attenuate itsvirulence. In addition, according to the reports of dead bacteria adjuvant vaccine fromstrain019, together with the finding in the genome of strain019and S2, reformingstrain019into potential effect vaccine against brucellosis by molecular biologytechnology. Quantitative proteomics results showed the difference proteins occupiedamino acid metabolism, carbon metabolism, energy metabolism and nucleotidemetabolism. Among them, amino acid metabolism may trigger immunity of host andform different host adaption. Carbon metabolism, energy metabolism and nucleotidemetabolism were involved in virulence of Brucella. Total17different proteinsfunction as out membrane, metabolism, transduction and et al. Among them, ABCtransporter related genes, Twin-arginine translocation related gene, YaeC may benovel virulent factors. TetR family transriptional regulator Br1381sensitive tomultiple stress may be global transcriptional factors and plays imprtant role insurvival of macrophages. Sensor histidine kinase Br0613sensitive to acid and alkalinein starvation palys imprtant role in sensing pH changes of environment.