Transcriptome Analysis And Establishment Of The Models Of Cells And Mice Infected With B.suis Virulent Strain And Vaccine Strain

Brucella is an important zoonotic pathogen,and there are ten recognized Brucella species based on their host preference and different biochemical characteristics.B.suis is one of important Pathogenic strains that seriously threaten human health and livestock breed.Approximately 500,000 humans are infected worldwide every year according to the World Health Organization.The pathogenic mechanism of Brucella is still not very clear at present.To analyze pathogenic mechanism of Brucella infection in molecular level,the study investigated the transcriptome of host cells infected with B.suis virulent strain and vaccine strain by RNA-seq and comparative genomic technology and further proved important different expression genes related to virulence in infected mice.According to RNA-seq,the genes related to Brucella infection or virulence were systematically researched in order to lay the foundation for deeply analyzing Brucella pathogenic mechanism.1 RNA-Seq and establishment of the model of mouse macrophages RAW264.7 infected with B.suis virulent strain and vaccine strainFirstly,the study establish the model of mouse macrophages RAW264.7 infected with virulent B.suis 1330 strain and vaccine B.suis 2 strain on the condition that the multiplicity of infection is 200.The result showed that B.suis 1330 strain and B.suis 2 strain could constantly multiply.Their growth reached the peak in cells after 48h,and then gradually decreased.In order to construct cDNA libraries,we extracted separately total RNA from infected cells and uninfected cells after 48h.The different expression genes(DEGs)were found between different virulent Brucella infected cells and uninfected cells by RNA-seq.As a result,there were 8606 DEGs through S2 infected cells and control cells.There were 8889 DEGs between S1330 infected cells and control cells.Compared with S2 infected cells and S1330 infected cells,the number of DEGs was 440.GO analysis and Pathway analysis showed the DEGs between different virulent Brucella infected cells and control cells were basically the same.These DEGs were involved in DNA-templated transcription,DNA repair,cell division and apoptotic process,while taking part in lysosome,proteasome and other signal pathway related with disease.However,GO analysis and Pathway analysis of the DEGs between different virulent Brucella infected cells were different from the former.The DEGs were involved in innate immune response,phagocytosis,recognition,inflammatory response,cellular response to lipopolysaccharide and positive regulation of tumor necrosis factor biosynthetic process,while taking part in transcriptional misregulation in cancer,NF-kappa B,protein processing in endoplasmic reticulum,phagosome and Toll-like receptor signal pathway.Meanwhile gene co-expression networks of S2 and S1330 infected cells were built,then selected 10 and 26 key genes from the DEGs.The key genes played a role in innate immune response,inflammatory response,protein transport and RNA splicing.2 Establishment of mice models infected B.suis virulent strain and vaccine strain and transcription level analysis of some innate immune related genesThe different expression genes enriched in innate immune response were found between S2 strain infected cells and S1330 strain infected cells by RNA-seq.In order to research the differential expression level of some innate immune related genes in infected animals,we set up the Brucella infected BALB/c mice model and analyze the differential expression level of 13 innate immune related genes after infection of 7,14,21 and 28 days,while monitoring the amount of bacteria in the spleen,the antibody titer.Compared to the control group,there were 6 differentially expressed genes from 13 innate immune related genes in S2 infected mice and S1330 infected mice.These genes may play essential roles during Brucella infection,which are I123a,Clec4a2,Tmem173,Clec5a,Fcgr1,Cfp.Compared to S1330 group,there were 10 differentially expressed genes except genes such as Clrb,Nlrp10 and Irf7 in S2 infected mice,which could be associated with anti-infection of different virulent Brucella.As a result of the monitoring,the amount of Brucella S2 in the spleen gradually reduced after 7 days and dropped to zero after 28 days.However,the amount of Brucella S1330 in the spleen was the lowest in the 21th day and significantly increased in the 28th day.It’s worth noting that the antibody titers increased with the growth of infected time,either mice infected with S2 or mice infected with S1330.3 Comparative genomic and transcription level analysis of different genes between B.suis S2 strain and B.suis S1330 strainBased on comparative genomic analysis between S2 strain and S1330 strain,we found 54 points of difference located in the coding region,30 of which were in chromosome Ⅰ and the rest were in chromosome Ⅱ.There were 7 genes with multiple bases insertion or deletion and 47 genes changed due to SNPs.We detected the expression of different genes caused change of conformation and chemical characteristics under different conditions that included the culture and susceptible cells.Relative to 51330 genome,the transcription levels of 4 of 7 genes with multiple bases insertion or deletion were significantly changed in culture medium or infected macrophages.The expression of 10 of 28 ORFs,whose coding sequences were changed due to SNPs,was also significantly affected in culture medium and infected microphages.These differentially expressed genes were related to versatile functions.Based on gene function annotation,differentially expressed genes Fis encodes a Fis family transcriptional regulator that is a nucleic acid binding protein.It is also a bacterial global regulator,which take part in both the process of the structure and regulation of genes and the influence of bacterial virulent genes.Therefore,we speculated the different expression of Fis family transcriptional regulator might be the major cause for S2 virulence attenuation.Other different expressed genes related to metabolism pathways made the vaccine strain S2 unable to adapt to harsh intracellular environment,should be the main reason of its virulence attenuation.