The Preliminary Study Of The B.abortus Major Outer Membrane Protein On Intracellular Survival Mechanism

Brucellosis is a zoonotic disease caused by Brucella which is gram-negative and intracellular bacteria. Brucellosis is widely popular in China as well as in most countries of the world. Main reservoirs of Brucella are human and animals. Human brucellosis is characterized by undulating fever,which if untreated, may result in localization of bacteria in various tissues leading to chronic disease with serious clinical manifestations, such as orchitis, osteoarthritis, spondylitis, endocarditis and several neurological disorders. Animal brucellasis mainly cause arthritis, abortion, and orchitis. In recent years, the diseases have an upward trend, which is seriously harm to human security and economic development. However; the mechanism behind the pathogenesis of Brucella is unclear. OMP10and OMP19are the two major outer membrane proteins of Brucella and they are also two key virulence factors closely related to the Brucella intracellular survival. Our study focused on the impacts of the two proteins on macrophage apoptosis and the fusion between Brucella-containing vacuole (BCV) and lysosomal. This study might reveal further pathogenic mechanisms of Brucella.1. Construction and identification of Brucella abortus2308(2308) omp10and omp19gene deletion strains (Aomp10and Aompl9). Using Bacillus subtilis as a template, we amplified SacB gene high-fidelity by PCR. The upstream and downstream sequence of the omp10gene was high-fidelity amplified and fused together by fusion PCR technology. The fused sequence and SacB gene were linked into the suicide vector pGEM-7xf+to construct the recombinant plasmid pGEM-7zf-Δomp10-SacB (p10S). The pGEM-7zf-Δomp19/9-SacB (p19S) was constructed by the same way. The recombinant plasmid p10S and p19S were electroporated into Brucella abortus2308. We screened the positive colonies by ampicillin and sucrose, tested their genetic stability and detected their intracellular and in vivo life. The results showed that the Aomp10and Aompl9at15generations did not back to parent strain. After4h infection, the number of2308in murine macrophages RAW264.7(RAW264.7) had an upward trend, while AomplO and Aompl9had a downward trend. Those results indicate that AomplO and Aompl9had genetic stability, and are weaker than parent strain2308.2. The effect of AomplO and Aompl9on the fusion rate between BCR and lysosomal. After the infection of RAW264.7by AomplO, Aompl9and2308, intracellular Brucella were labeled by sheep anti-B.abrotus IgG antibodies and Rhodamine (TRITC) conjugated AffiniPure donkey anti-sheep IgG antibody, and lysosomes was labeled by Cell NavigatorTM Lysosome Staining Kit Green Fluorescence. We used the confocal laser scanning microscope to observe the fusion rate between BCV and lysosomes. The fusion rate of AomplO and Aompl9infected groups were more than90%, while2308infected group about10%at early stage and about70%at later stage. Those results indicate that the Aomp10and Aompl9could not inhibit the fusion between BCV and lysosomes.3. The effect of Aomp10and Aompl9on the apoptosis of RAW264.7. After the infection of RAW264.7by Aomp10, Aompl9and2308, about1-5×105cells of each sample were collected, suspended with Binding Buffer and treated by Annexin V-FITC and PI at room temperature. After5-15min reaction in the dark we detected the apoptosis of RAW264.7using flow cytometer. The apoptosis rate of RAW264.7was about20%after the infection of2308and Aompl9, The apoptosis rate of RAW264.7was about30-40%after the infection of Aomp10. Those results indicate that the2308and Aompl9was able to suppress the apoptosis of RAW264.7, but the Δomp10could not inhibit the apoptosis of RAW264.7.4. The results of this study showed that Δomp10and Δomp19had genetic stability and the intracellular life of the Δomp10and Δomp19were significantly weaker than the parent strain2308. Both Δomp10and Δomp19could not obviously inhibit the fusion between BCV and lysosomes. With2308both Δomp10and Δomp19did not obviously inhibit the apoptosis of murine macrophages. Δomp19was able to suppress the apoptosis of RAW264.7, but Δomp10could not inhibit the apoptosis of RAW264.7.