Comparative Genomic Analysis Of Brucella And Studies On Cytotoxicity For Macrophage

Brucellosis is a zoonosis worldwide, leading great economic loss in our country every year. Brucella is the agent of Brucellosis, and it could infect different animals, including humans, resulting in testitis in male animals and abortion in femal ones. Human Brucellosis is manifested by brucella fever, loss of labor force. According to host preferences, Brucella was divided into 6 species and 19 subtypes. Till present, whole genomes of 10 strains of Brucella were sequenced, providing new options for Brucella related researches. Different species of Brucella showed different host preferences. Four of these species could infect humans, including B. melitensis, B. abortus, B. suis, and B. canis. The differences in host preferences and virulence of Brucella are closely related with the differences in gene contents. Smooth and rough strains showed different virulence to human and animals: smooth strains showed higher virulence than rough strains. The intracellular survival capability is a key virulence characteristic of Brucella. Smooth strains have great survival capability in macrophage, and on the other hand, they inhibit apoptosis and death of macrophage for their replication. However, when compared with smooth strain, rough strain have greatly reduced survival capability, and showed cytotoxicity for macrophage. Recent studies showed that this cytotoxicity of rough strains is mediated by type IV secretion system (T4SS).To probe into Brucella speciation and genome diversity, in the present study, comparative genomic hybridization was used to analyze gene content differences among Brucella species. By using MLVA, the genetic diversity of these species was defined. The differences between virulent strains and vaccine ones will provide some clues for the attenuation and immunological protection mechanism of vaccine strains. To further analyze the molecular mechanism of virulence differences between smooth and rough strains, and the possible roles of T4SS in cytotoxicity, we compared the cytotoxicity of wild type strain, virB over-expression strain and virB mutant strains to macrophage, putatively testing whether the cytotoxicity is rough strain unique. With the above aims, we carried out the following experiments:Experiment 1. Development of whole genome microarray and comparative genomic hybridizationSequence of 16M and 9-941 was downloaded from genbank and sequences of their genes were extracted. A total of 3218 genes, including all genes from 16M and 941-unique ones, were chosen for primer design and synthesis. After several rounds of condition optimization and redesign of primers, PCR products of 3212 genes were obtained. The PCR products were purified and used for microarray printing. The microarray includes a total of 6912 probes, with each gene 2 spots, and positive and negative control. By using Cy3/Cy5 double staining and 16M and 941 as reference and test samples respectively, the comparative genomic hybridization was developed.Experiment 2. Comparative genomic hybridization of Brucella type strains19 type strains were analyzed by comparative genomic hybridization. Our results showed that genetic uniformity is very high in the Brucellae. Moreover,25 different fragment regions (DFRs), DNA fragment including at least sequential genes, was identified. 19 type strains showed different distribution profiles of DFRs. In addition to gene deletion, several genes were found to be multi-copied. With the gene content differences, the speciation and micro-evolution of Brucella was putatively explained.Analysis of Brucella species by comparative genomic hybridization is likely to identify factors responsible for diffences in host preference and virulence restriction between pathogenic and nopathogenic species.Experiment 3. Comparative genomic hybridization of Brucella vaccine strainsThe comparative genomic hybridization was also applied to 4 vaccine strains,104M, M5, S19 and S2, which are extensively applied in our country. As observed in type strains, a number of DFRs and duplications were found in vaccine strains. The differences in gene content of vaccine strains implied their possible mechanism of attenuation, immunogenecity and protection. This also provide target genes for differentiate virulent strains and vaccine strains.Experiment 4. Functional and cluster analysis of differential genesGene acquisition or deletion is an important process for Brucella evolution. By compare 19 type strains and 4 vaccine strains, a total of 25 DFRs was identified. These DFRs are located on I or II chromosome of different Brucella strains,12 DFRs were found located on chromosome I and 13 DFRs were found located on chromosomeâ…¡. The genes from different DFRs showed different function categories. And different strains showed different DFRs profiles, possibly explained their differences in biological characteristics, host preferences and virulence. The results of cluster anlysis can be used to identification different species and serotypes of Brucella.Experiment 5. Genotyping of Brucella strains by 15 MLVA locusThe method of 15 MLVA locus based genotyping was firstly developed, and then applied to 23 strains of Brucella laboratory strains (19 type strains and 4 vaccine strains) and 10 clinically isolated strains. With the 15 MLVA loci, these strains could be easily differentiated.8 out of the 10 clinical isolates are clustered with B. melitensis type 2, implying that the isolates epidemic in Songyuan City are mainly this types. Another two strains clustered with M5, implying their high similarity to M5 strain. The results of cluster analysis and MLVA assay were found conformity besides little difference.Experiment 6. Cytotoxicity effects of smooth strain to macrophageBy using plasmid rescue methods, the virB operon, which encodes T4SS, was cloned into multi-copy plasmid pBBR1MCS-5, which was transformed into smooth strain to generate over-expression strain BM-VIR. The cytotoxicity of wild type (BM), virB over-expression (BM-VIR) and virB mutant (BM-â–³virB) strains to macrophage was analyzed and compared. The results showed that at high MOI, smooth strain showed cytotoxicity, which is T4SS dependent. The BM-â–³virB strain showed greatly reduced cytotoxicity, and BM-VIR showed greatly increased cytotoxicity. BM-VIR had similar survival capability under stress conditions, implying that the cytotoxicity is mediated by effector proteins of T4SS, which is tightly regulated.