| Brucellosis is a serious hazard to safety of zoonotic infectious disease, which caused by Brucella. It is widely popular in the world; especially to bring great economic burdens for developing countries. Brucella is facultative intracellular bacterium. The virulence of Brucella relies primarily on its ability to survive and multiply within host cells. The two-component regulatory system(TCS) is a single transduction system which serves to induce, transmit and process environmental signals. More recently, 15 TCSs were identified in the genomes of Brucella. TCSs, which are located in chromosome I, are essential for virulence in Brucella; nevertheless the virulence phenotypes and regulatory mechanisms of TCSs that located in chromosome II are still unclear. Based on the Brucella TCS TceSR and TcfSR as the research objective, through exploring and discussing the influence of TCS TceSR and TcfSR on Brucella virulence phenotypes and target genes regulation and intracellular survival and reproduction, as well as the virulence of TCS TceSR and TcfSR in the mice, the study unveiled the molecular mechanism of Brucella sustaining infection in host cells, and laid the foundation for the new vaccine development, prevention and anti-disease livestock breeding.Objects: In our study, we used the reference B. melitensis 16 M strain as the research object, to explore the function and action mechanism of TCS TceSR and TcfSR in pathogenic process of Brucella.(1) We constructed and identified inactivation mutants of the TCS TceSR and TcfSR promoters.(2) We analyzed the intracellular and extracellular transcriptional levels of the virulence genes regulated by TCS TceSR and TcfSR, and then, we compared the differentially transcriptional genes of parental and mutant strains. By according to the function of the genes and differences in transcription level, the alternations of the virulence related phenotypes in the mutants were putatively explained.(3) We analyzed and compared the difference in intracellular survival related phenotypes of parental and mutant strains to define the ones affected by TCS TceSR and TcfSR.(4) We analyzed the virulence of mutants in mice, and investigated their roles in establishement of chronic infection of Brucella in vivo.Methods:(1) We used homologous recombination and resistance replacement methods to construct inactivation mutants of the TCS TceSR and TcfSR promoters. The mutants obtained were identified by PCR tests, and then, their genetic stability was detected by continuous bacteria culture.(2) RAW264.7 cells were infected with Brucella. At 24 h post-infection time, the intracellular transcription levels of virulence-related genes(these genes include lipopolysaccharide, outer membrane proteins, type IV secretion system, transporter system, flagella, regulatory system, stress proteins/chaperones and nitrogen metabolism) in Brucella were detected by using real-time quantitative PCR(qRT-PCR). Then, Parental and mutant strains were subjected to different in vitro stress treatments. At 30 min post-incubation, the intracellular transcription levels of virulence genes omp25, virB1, vjbR, gntR, dnaK, Hfq and htrA were analyzed by qRT-PCR.(3) Then, the related virulence phenotypes of parental and mutant strains were compared. RAW264.7 murine macrophages were infected with different Brucella. The invasion and intracellular survival capability in macrophage were assessed. RAW264.7 cells were infected with parental and mutant strains. The apoptosis and autophagy levels were detected by using qRT-PCR, flow cytometry and laser scanning confocal microscopy. Then, we constructed parental strain and mutants, which could express green fluorescence. RAW264.7 cells were infected with these strains, and observed the fusion situation of Brucella–containing vacuole(BCVs) and lysosomes.(4) Female 4-6 week-old SPF BALB/c mice were inoculated intraperitoneally(i.p.) with a total of 106 CFU of M5-90, 16MΔTceSR and 16MΔTcfSR. At different times post-inoculation, serum were collected and determined the cytokine levels of IFN-γ and the total IgG in mice by iELSA. Spleens of vaccined mice were removed aseptically, and the CFU of Brucella were calculated and pathological changes were observed using biopsy technique to determine the virulence difference between mutants and vaccine. Evaluation of protective immunity of mutants and vaccine was analyzed after challenge with 105 CFU B. melitensis 16 M. We cloned, expressed and purified TCES and TCFS proteins. Purified proteins could be used as diagnosis antigen. Western blot analyzed specific antibodies in mice immunized with 16MΔTceSR and 16MΔTcfSR for differential diagnosis.Results:(1) The 16MΔTceSR and 16MΔTcfSR mutant strains were successfully obtained. The mutants were genetic stability within 15 passages. The growing trend of mutant and parent strains are coincident in vitro. Compared with the parental strain, the mutants had decreased survival rate under the stress conditions. The mutants were sensitive to acidic, alkaline, high salt, high-temperature, nutrition defect and drying stresses.(2) Cells were infected with mutant and parental strains, at 24 h post-infection, a total of 66 differentially expressed virulence-related genes of Brucella were found: 41(62.12%) were over expressed and 25(37.88%) were less expressed in the 16MΔTceSR mutant and 47(71.21%) were over expressed and 19(28.79%) were less expressed in the 16MΔTcfSR mutant. Under the stress conditions, the transcriptional levels of intracellular survival-related virulence genes in mutants were lower than those of parental strain.(3) The adherence and invasion and intracellular survival of mutants were decreased in macrophages. Results from the apoptosis that mutants and parental strain induced apoptosis of host cells are similar. Results from the autophagy that ability of muntants was lower than that of parental strain. Compared with the parental strain, at 24 h post-infection, the fusion rate between BCVs and lysosomes of mutants infected groups were increased.(4) BALB/c mice were inoculated with mutant strains, and mutants induced the secretion of IFN-γ and elicited IgG response. The numbers of bacteria in spleen of mutants’ inoculated-mice were lower than parental strain inoculated-mice. The protection ability could be provided by mutants immunized-mice. Recombinant proteins TCES and TCFS could be used as diagnosis antigens for diagnosing of mutant strains and parental strain vaccined-mice. These results implied that the survival capability of mutants in vivo was lower than that of parental strain, and recombinant proteins TCES and TCFS have good reactionogenicity, which could be used to diagnosis brucellosis.Conclusions:(1) TCS TceSR and TcfSR play an important role in resistance to hostile environments.(2) Host cells were infected with TCS TceSR and TcfSR mutants, and the expressions of a large number of target genes were infected. TCS TceSR and TcfSR promote the adapation of Brucella to various harsh environments for intracellular survival.(3) TCS TceSR and TcfSR mutant strains could induced cellular immunity and humoral immunity in mice, their virulence was lower than that of parental strain. The mutants need to further transform. |
PDF Full Text Request