Construction Of Brucella BtpB Deletion Strain And Its Effect On NLRP3 Inflammasome In Goat Alveolar Macrophages

Brucellosis caused by the Brucella infection is a zoonosis that seriously endangers the dairy industry in China.Due to the lack of safe and reliable vaccines and differential diagnosis methods,it has been receiving much attention.It is the key point that reveals the pathogenic mechanism of Brucella for the development of new vaccines and effective prevention and control measures also a hot spot in the current researches.Alveolar macrophages are target cells of Brucella.Brucella can replicate in it and interfere with the function of macrophages.Brucella has a variety of immune evasion mechanisms,and the role of interfering with pattern recognition receptors(PRRs)is a critical way for entering into host cells and inhibition of the innate immune response.BtpB is an effector molecule secreted by the Brucella type IV secretion system and is one of the major virulence factors currently known.As an intracellular pattern recognition receptor,NLRP3(NOD-like receptor family member pyrin domain-containing protein 3)plays an important role in the innate response with the pathogen invasion.The NLRP3 inflammasome is a macromolecular protein complex presented in the cytoplasm,which can sense molecular signals released from pathogenic microbial infections,tissue damage,and metabolic imbalances.NLRP3 inflammasome activation promotes the maturation and secretion of inflammatory factors such as IL-1?,thereby promoting the expression of various immune-related genes,and recruiting lymphocytes to the infected site to inhibit pathogenic microbial.It is shown that during the Brucella infection,the host's innate immune response is interfered by BtpB in a number of ways,one of crucial ways is to interfere to TLRs signaling to inhibit the downstream immune cascade.The NLRP3 inflammasome activation is triggered by PRRs after sensing PAMPs(pathogen-associated molecular patterns).However,the relationship between BtpB,a TIR domain protein of Brucella,and NLRP3 inflammasome remain unclear,and its mechanism of exerting immunoregulatory functions needs further research.In order to study the effect of type IV secretion system effector protein BtpB on the virulence phenotype of Brucella and the relationship between BtpB and NLRP3 inflammasome,the BtpB mutant and complementary strains of Brucella S2 strain(B.suis S2)were to be first constructed.To explore the effect of BtpB on the virulence phenotype of Brucella by observing the bacterial morphology of B.suis S2,BtpB deletion strains and complementary strains by Scanning electron microscopic,and detecting the growth curves of three strains and their survival under stress conditions such as acid,heat,hypertonic and oxygen stress.At the same time,qRT-PCR,Western blot,and cellular immunofluorescence were used to detect the effect of BtpB on the expression of pattern recognitio n receptor,NLRP3 inflammasome,and cytokine after B.suis S2 infection of GAMs.The results are as follows:1.The revived B.suis S2 and pBBR1MCS5 as templates,the homologous arms of the upstream and downstream BtpB,and the gentamicin gene were amplified by PCR,and then the three fragments were ligated to pUC19 vector to construct the recombinant plasmid pUC19G-BtpB.Then,the pUC19G-BtpB was transferred into competent cells of B.suis S2 by electroporation after it was identify by restriction endonuc lease digestion and sequencing.The BtpB mutant strain was obtained by gentamicin resistance screening and PCR identification.The BtpB gene was amplified by PCR and ligated into the expression vector pBBori-PZT1 to construct a recombinant plasmid pBB-PZT1-BtpB.After sequencing,it was transferred into the competent state of BtpB mutant strain by electroporation,and positive clones were screened by Carbenicillin resistance.After identification by Western blot,BtpB complementary strain was obtained.2.The morphology of B.suis S2,BtpB mutant strain,and the complementary strain was observed by scanning electron microscopy.It was found that BtpB mutant had no effect on the morphology of Brucella.By detecting the growth curves of B.suis S2,BtpB mutant strain and the complementary strain and their proliferation in goat alveolar macrophages,it was found that there was no significant difference in extracellular and intracellular proliferation curves of the three strains.The survival rates of the three strains under stress conditions such as acid,heat,hypertonicity,and oxygen pressure were measured.Compared with the original strain and the BtpB complementary strain,the survival rate of the mutant strain under oxidative stress was found to increase.3.By qRT-PCR,immunofluorescence,Western blot and ELISA,it was found that the expression of TLR2 and TLR4 in GAMs cells was up-regulated in the BtpB mutant group,which was significantly different from the original strain treatment group and the complementary strain treatment group.The expression of NLRP3,ASC and caspase-1 were significantly increased in ?BtpB treatment group.At the same time,the expression of IL-1? was significantly increased compared with B.suis S2 and the complementary strain infection,and the difference was extremely significant.In conclusion,BtpB has no significant effect on the growth characteristics of B.suis S2,and may plays an important role through the NLRP3 inflammasome in the process of inhibiting innate immunity.