A Preliminary Study On The Mechanism Of Brucella Escape Fusion With Lysosomal

Brucellosis is a serious zoonotic infectious diseases by Brucella infection of human and animals on social security and widely popular in the world, especially in developing countries, causing huge economic losses. Brucella is facultative intracellular bacterium, which can promote cell autophagy and escape with lysosome fusion obtainning the ability of intracellular survival and replication.Type IV secretion system(T4SS) is one of the key of Brucella virulence factor and when Brucella interact with host cells, T4 SS can secrete a variety of effector proteins. 11 potential effector protenins have been identified and only Tcp B, and Ric A Cst A for which functions have been more clearly ascribed, yet the phenotypes and regulation mechanism of other T4 SS effector protein is also unclear. Based on the Brucella T4 SS effector protein gene DK63-887 and BMEI1135 as the research objective, through exploring and discussing the influence of DK63-887 and BMEI1135 on Brucella intracellular survival and reproduction, as well as mediation autophagy and escape fusion with lysosome, the study unveiled the molecular mechanism of Brucella sustaining infection in host cells, while laying the foundation for the prevention and anti-disease livestock breeding.Objects: In our study, we used the reference Brucella melitensis 16M(16M) strain as the research object, to explore the function and action mechanism of DK63-887 and BMEI1135 in pathogenic process of Brucella.(1) We constructed and identified mutants of the DK63-887 and BMEI1135 gene while preliminary analysised of their viability.(2) We investigated the effect of T4 SS effector protein gene DK63-887 and BMEI1135 on 16 M mediation autophagy of RAW264.7 macrophages.(3) We preliminary studyied the effect of T4 SS effector protein gene DK63-887 and BMEI1135 on the 16 M escape fusion with lysosome of RAW264.7 macrophages.Methods:(1) DK63-887, BMEI1135 gene upstream and downstream homologous arm were amplified with 16 M as template and kanamycin resistant gene was amplified with plasmid p UC19 K as template. Using fusion PCR and resistance replacement methods,we combined the above fragments then connected it to vector pmd18-t simple to construct recombinant plasmid, subsequently which was transformed into 16 M competent cells within and screened by PCR. Under the same conditions of shaking culture, we observed growth of the parental strain 16 M, vaccine strain M5-90, mutant 16MΔDK63-887 and 16MΔBMEI1135. And then, each strains was placed in different environment, the survival rate of which was observed. Mouse macrophage were infected and BALB / c mice were intraperitoneal inoculated with 106 CFU of with parental and mutant strains, then we compared their ability of surviving in host cells and in vivo at different times.(2)When Brucella infected macrophages RAW264.7 for 24 h, the number of autophagic vesicles containing 16 M were observed by transmission electron microscopy and the expression of ULK1 and Beclin1 gene m RNA and protein were detected by quantitative real-time PCR and Western blot. With the parent and mutant strains infection stable expression p EGFP-LC3 macrophages for 12 h, autophagic dots formation were observed by confocal microscope.(3)Then, we constructed the expression of green fluorescence of parental strain and mutants which RAW264.7 cells were infected with. At 24 h lysosomes, Golgi apparatus, endoplasmic reticulum were stained by red probe and the colocalizations of Brucella and they were observed by confocal microscope.Results:(1) The 16MΔDK63-887 and 16MΔBMEI1135 mutant strains were successfully obtained. The mutants were genetic stability within 20 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. At 4,12,24 and 48 h post-infection, mutants in cells viability and after BALB/c mice immunized with mutant strains from 4 to 10 weeks, the numbers of bacteria in spleen of mice were significantly lower than the parent strain group(P<0.01);(2) At 12 h post-infection, the green fluorescent particles of 16MΔDK63-887 and 16MΔBMEI1135 group were significantly fewer(P<0.01); at 24 h post-infection,the m RNA expressions and protein levels of ULK1 and Beclin1 in cells with mutant strain group were significantly lower than those of control cells treated with 16M(P<0.01); compared with the parent strain 16 M, the number of autophagic vacuoles of mutant strain group were also significantly decreased(P<0.01);(3)Compared with the parental strain, at 24 h post-infection, the colocalization rate of Brucella with lysosomes of mutants infected groups were significantly increased(P<0.01); yet the colocalization rate of Brucella with Golgi, endoplasmic reticulum were significantly decreased(P<0.01).Conclusions:(1)After deletion the type IV secretion system effector protein gene DK63-887,BMEI1135, the survival of 16 M were significantly decresed in vivo and in vitro which enhanced the host immune cells resisting 16 M infection.(2) DK63-887, BMEI1135 mutant strains can significantly inhibit 16M-mediated RAW264.7 macrophage autophagy.(3) DK63-887, BMEI1135 mutant strains in favor of 16 M fusion with lysosomes, and reduced the amount of bacteria which reach the Golgi apparatus and endoplasmic reticulum place.