Construction And Biological Characterization Of Two Brucella Abortus Mutant Strains With Deletion Of BAB_RS22915 Or BAB_RS27735 Gene

Brucella spp is a facultative intracellular,Gram-negative bacteria,which causes Brucellosis in animal and human with symptoms of undulant fever,arthritis and abortion,then poses great threat to both agriculture and public health.Brucella have no classic virulence factors like exotoxin,endotoxin,capsules,cytosol,etc.,thus are described as “pathogen without classic virulence factors”.Its pathogenicity depends on its capability to survive and proliferate within host cells.This attribution probably makes it more adaptable for intracellular environment and enables Brucella establish replication in susceptible cells without inducing strong immune response or cell death.Research on Brucella's pathogenesis and interaction with host immune system is focused on the identification of virulence genes which are related to in vitro or in vivo survival of Brucella.Besides,with the identified virulence genes,live attenuated strain of Brucella spp could be constructed for vaccine development.In this study,we identified the single mini Tn5 insertion in 8 STM-mutants' s genome and mutated genes respectivly using Southern-blot analysis and Genome Walking-PCR,then,constructed 8 site-directed deletion mutant strains.Two mutants were further characterized for their intracellular survival and in vivo survival capacities.Their potential application as live attenuated vaccine candidates was also evaluated.The results provide foundation for future research on Brucella pathogenesis and control strategies.In this study,we first identified the mutated genes in attenuated STM mutant strains.With Southern-blot detection,it was confirmed that all the 8 STM mutant strains' genome are inserted with single copy of transposon,which indicated the mutant harbours only one mutated gene.Then with Genome Walking-PCR,the specific insertion positions and mutated genes were identified.After site-directed deletion with suicide plasmid,all 8 genes deleted mutant strains were constructed respectively.In vivo survival assay indicated that 4 of them showed attenuated survival than wild type Brucella abortus S2308.And the mutant strains which harbours deletion of BAB_RS22915 and BAB_RS27735,genes with specific coding sequence,were selected for further research.BAB_RS22915 encodes a homologous protein to membrane-bound lytic murein transglycosylase.After deletion of this gene,the smooth type mutant strain ?22915 showed altered intracellular traffic and attenuated in vitro survival in RAW 264.7 after 8 hours post infection.In addition,transriptome sequencing results showed that multiple genes which are involved with “Nucleotide/Amino acid metabolism” are significantly upregulated in transcription;BAB_RS27735 encodes a homologous protein to substrate-binding protein of L-amino acid ABC transporter.After deletion of this gene,the smooth type mutant strain ?27735 showed attenuated growth at expotential stage in TSB media.The intracellular survival in RAW264.7 was attenuated between 2 and 24 hours post infection,but partially recovered at 48 hours post infection.And no significantly dis-transcript genes were detected in ?27735 with transriptome sequence.This indicates the deletion of BAB_RS22915 and BAB_RS27735 altered B.abortus S2308's phenotype and resulted in attenuation of survival in vitro or in vivo.Evaluation of vaccine application showed that ?22915 is more suitable for application as live attenuated vaccine.In vivo survival on BALB/c mice showed that both site-deleted mutant strains were attenuated before 4 weeks post infection,after that,?27735's survival gradually recoverd and even more that wild type.While ?22915's survival did not recovered,and no pathological damage was observed with ?22915's infection.?22915 and ?27735 can both induced strong humoral immune response and protected BALB/c from the challenge of wild type B.abortus S2308.In conclusion,based on previously constructed STM mutant strains,we identified 4 genes which are related to Brucella's virulence.Two of them were further studied for the changes in phenotype and virulence of B.abortus S2308.Both deleted mutant strains ?22915 and ?27735 were evaluated for potential application as live attenuated vaccine candidate against Brucella infection,and the result indicated ?22915 is better for further development.This study provides foundation for future research on Brucella pathogenesis and control strategies.