Cenome-wide Screening Virulence-associated Genes And Essential Genes Of Brucella

Brcellosis is a zoonotic disease caused by the bacteria of the genus Brucella. In animals,brucellosis causes epididymitis in males and abortion in pregnant livestorck. In humans, brucellosis causes recurrent fever, flue-like infection, joint pain and so on. This disease causes a significant economic loss to animal husbandry production and is a big threat to human healthy. the pathogenesis is still unclear for brucellosis. So, in this paper, in order to identify the virulence associated genes, we identified the transposon insertion sites of the transposon mutant library of B. melitensis NI which was constructed by our laboratory. And also, the potential essential genes of Brucella were predicted via comparative genomics. Last, the virulence related genes were genome wide screenned of Brucella melitensis biovar 3 strain NI by using this mutant library.The 96-well plates of transposon mutant library constructed by our laboratory were inactivated,and digested the extracted genome, amplified the ligation product successively, sequenced the PCR product. Identify the transposon insertion sites of Brucella via Protein BLAST the PCR product sequence against the whole genome of Brucella NI. Finally, 10,832 sequence identified mutants were acquired from 32,640 mutants. 20 mutants randomly chosen in the mutant library were re-sequenced using the same method, the coincident rate was 100%, which indicated that the transposon insertion sites were accurate.The transposon insertion sites data were analyzed, after that we found the transposon insertion sites were evenly distributed in the whole genome of Brucella. Of the 10,832 sequenced defined mutants,8514 mutants were inserted in the intragenic region, among them 5,330 mutants are at Chromosome I(Chr I )and 3,184 mutants are at Chromosome Ⅱ ( Chr Ⅱ), disrupted 1,398 and 883 genes, respectively.There are 2,317 mutants with the insertions at the intergenic regions, amongst them 1,643 mutants had insertions at Chr I and 675 mutants had insertions at Chr II, which had transposon insertions at 724 and 325 intergenic regions, respectively. Averagely, every 304 bp in Chr I and every 305 bp in Chr II had one transposon insertion, which indicated that the coverage of the transposon insertion sites were acceptable.Genes in the mutant library without transposon insertion may be essential for Brucella survival.After analysis of the 10,832 sequenced defined mutants, we found 948 mutants had no transposon insertion, which were taken as candidate genes of Brucella for identification of essentiality. Due to essential genes are more evolutionarily conserved, in this study, 183 candidate essential genes of Brucella from 948 candidate genes were predicted by using comparative genomics analysis. These results will provide the basis for a target for drug research of brucellosis.In the mutant library, the same gene in different mutants may have same insertion sites and/or may have several insertion sites. In order to reduce workload and improve work efficiency, and provide convenience for the study on gene function and pathogenesis of Brucella, mutants with the same insertion sites and mutants with insertions in the same genes but different insertion sites were removed.6,782 selected mutants were rearranged successively in 96-well plates. The transposon of mutant array covered 71% ORF (Open reading frame) of the whole genome in Brucella NI. Macrophage killing assay were performed by using this mutant array, through immunofluorescence assay virulence-associated genes were screened, 275 genes relative to Brucella survival were identified after twice screened. After analyzed these genes, we found that 121 genes were identified as virulence-associated genes before, 154 genes were novel virulence related genes for the first time identified in this study.To sum up,10,832 B. melitensis NI sequence defined transoposon mutant library would provide a rich resource for the research on gene function and pathogenesis of Brucella. 183 putative essential genes for Brucella survival would provide a basis for developing Brucella drug target. 154 novel virulence associated genes would lay the foundation for molecular pathogenesis of Brucella.