Brucella Type ? Secretory System Independent Effector BspI Induces ER Stress In Macrophages

Brucellosis is a zoonotic bacterial disease which can cause infection-allergenic reaction with the invasion of Brucella.Brucella spp.is a classical intracellular pathogenic bacterium.The replicative Brucella-containing vacuole(rBCV)formed by the interaction between Brucella and endoplasmic reticulum(ER)is the key factor for its proliferation in the host cells.In the process of Brucella replication within r BCV,Brucella infection can lead to the restructuring of the ER,causing the ER stress and unfold protein reaction(UPR).However,the exact mechanism by which Brucella infection induces ER stress remains unknown.Current studies have shown that the Brucella type IV secretory system(T4SS)dependent effector proteins play important roles in Brucella-induced ER stress.It is well known that Brucella effects contain T4SS dependent and independent effectors.However,we still do not know the role of T4SS independent effector proteins in mediating ER stress in host cells.Therefore,this study will investigate the effect of T4SS independent effector in regulating ER stress and explore its possible molecular mechanism.Brucella BspI protein is encoded by the BAB1-1865 gene of chromosome 1 of Brucella,and contains three unique structural domains: signal peptide domain(1-17Aa),transmembrane domain(17-33 Aa)and GTPase activation domain(98-220 Aa).Among them,GTPase activation domain can theoretically regulate the activity of ER small G protein and affect the assembly of ER,suggesting that it may be involved in ER stress caused by Brucella infection.Because BspI was identified as a T4SS independent effector,in this experiment,we selected BspI protein as the research object to explore role of T4SS independent effectors in mediating ER stress and the UPR caused by Brucella infection.Firstly,a prokaryotic expression vector pGEX4T-1-BspI was constructed to express GST-BspI fusion protein in E.coli.After the purification of GST-BspI protein,the Pull-down experiment and mass spectrometry were performed to identify specific proteins in RAW264.7 cell,which can bind to BspI protein.Our data showed that the BspI protein of Brucella can interact with the UPR molecular chaperone Bip.In order to further study whether the BspI protein can affect the UPR,anotherprokaryotic expression vector p ET-28a-BspI was constructed in this study,and the optimal condition for the highest expression of soluble recombinant BspI protein was determined according to the character of BspI.Akta protein purification system was used to purify the recombinant protein BspI.After dialysis,we obtain the unique soluble protein BspI.To determine the suitable stimulation concentration of BspI,the proliferative toxicity of BspI on RAW264.7 cells was detected by CCK-8 assay.After stimulating RAW264.7 cells with BspI protein,the expression of Bip was significantly increased by Western blot compared with the control group,suggesting that BspI protein may induce ER stress in RAW264.7 cells.To further verify this effect,ELISA kit was used to measure related cytokines production,such as IL-6 and TNF-?.The results showed that the expression levels of both cytokines were increased significantly compared with control group.Finally,q PCR,Western blot and immunofluorescence experiments were used to detect the biomarkers of the pathways related to the UPR,and the results showed that the expression of XBP-1 was increased at both the m RNA level and the protein level,suggesting that the BspI protein of Brucella can mediates UPR via the IRE-1pathway.In summary,this study found that the BspI,an independent effector protein of Brucella T4SS,can induce the UPR and ER stress in the host cells.Our data suggest that Brucella T4SS independent effectors also play an important role in mediating ER stress in Brucella-infected cells,which will increase our understanding about ER stress caused by Brucella infection.