| Brucellosis, which caused by Brucella, is one of the most important bacterial zoonoses endemic in the world, especially in developing countries. These pathogens can affect a broad range of mammals and cause serious economic losses. Three Brucella species, B. melitensis, B. abortus and B.suis, are virulent for humans, causing a chronic, debilitating disease with severe and sometimes fatal outcomes. Moreover, as Brucella spp. can be easily aerosolized, this pathogen could be used to develop biological weapons.Brucella is an intracellular bacterium. Research on Brucella and its interaction between host reveals that Brucella activate reduced immune response. The virulence of Brucella depends upon its ability to survive and replicate in host cells. They replicate in endoplasmic reticulum (ER) and escape from the immune response. Effective immune response is mainly mediated by cellular immunity. Outer membrane proteins (OMPs) play important roles in stabilizing the structure of the outer membrane, adapting to external and intracellular environments, and Brucella virulence. OMPs are located on surface of bacteria and can be easily recognized by host immune system, therefore, some of them represent important immunoantigen.Although many attepts have been tried for development of different vaccine types, live attenuated vaccine is the most efficient vaccine for Brucellosis for the present. In the present study, to analyze the roles of major OMPs in Brucella virulence and immunological protection, outer membrane proteome was separated by 2D eletrophoresis and the proteins were identified by MS. A total of 67 proteins were identified to be possibly membrane proteins. Nine of these protein spots are products of Omp25 and seven were those of Omp31. Omp25 is a member of the OmpA protein family, which have immunogenicity. Outer membranes proteins play important roles in stabilizing the structure of the outer membrane and are relative to the virulence. To predict its function, the sequence of omp25 was analyzed by bioinformatics methods. In the genome of Brucella, there are four omp25 genes(BMEI1249(omp25), BMEI1007(omp25b),BMEI1829(omp25c) and BMEI1830(omp25d). Among the four genes, only omp25 (BMEI1249) have been shown to be related with brucella virulence .To further investigate the function of omp25, the mutants of the four omp25 genes were constructed by homologous recombination, and then their virulence phenotypes were tested. Firstly, the kanamycin gene of pBBR1MCS-2 was PCR amplified and cloned at the multicloning site of pUC19 to generate a new suicide plasmid pUC19K, which was then used to construct the mutant strains. By using resistance replacement method, mutants of the four omp25 genes were successfully constructed, named BMΔ1007, BMΔ1249, BMΔ1829 and BMΔ1830.In vitro growth curve assays showed that the four mutants and BM have similar growth curves. However, the mutants showed higher growth rate than the wild type strain at logarithmic phase. At stationary phase, BMΔ1007 showed the highest cell density. This implyied that omp25 negatively regulate growth of Brucella under the assaying conditions. When grow into stationary phase, BMΔ1249 autoaggregated to form clamps in culture, which were not observed in the other three mutant strains. Survival under stress conditions showed that the four mutants showed different sensitivity to the stress simulating intracellular environments, including high salt, high osmosis, low pH, heat shock and oxidative stress. Sensitivity of these mutants to polymyxin B and sodium deoxycholate were also increased. These data indicated that omp25 plays important roles in Brucella adaptation to hostile environments and maintaining integrity of membrane structure. Intracellular survival showed that the omp25 mutants could invade macrophage, but were rapidly cleared by host cell, implying that omp25 is important for Brucella intracellular replication and chonic infection. Balb/c mice was infected with M5 and omp25 mutants, and then challenged with BM and 16M to test their protection capability. When challenged with M5, no great differences were observed in protection efficiency between three mutants and M5, but the BMΔ1829 showed better protection. Then, BMΔ1249, BMΔ1829 and M5 was used to immunize mice and challenged with 16M. The mutant showed reduced protection than M5. The mice immunized with omp25 mutants showed lower antibody titer and IFN-v those infected with M5, implying that omp25 plays important roles in protection efficency of M5. At last, the four omp25 proteins were expressed in E. coli. Western blot result showed that all the proteins could react with brucellosis sera. Only omp25 protein could differentiate sera of M5 from that of the mutant strain.The results above showed that the omp25 play important roles in brucella adaptation to hostile environments, intracellular survival and maintaining integrity of outer membrane. These data also showed that omp25 proteins play important roles in protection of live attenuated strain. These findings expand our knowledge of the function of Omp25 in Brucella survival both in vivo and in vitro, virulence phenotypes and immuno-protection, providing important clues for the understanding of the molecular mechanism of intracellular survival and protection mechanism of Brucella. |
PDF Full Text Request