Construction Of Brucella GntR Gene Mutant Strain And Its Effect On Endoplasmic Reticulum Stress In Goat Alveolar Macrophages

Brucella,a Gram-negative bacterium,belongs to ?-Proteobacteria and it presents a small ball rods or short rods.Brucella can infect a variety of animals and people,such as cattle,goats,sheep and swine,and it causes significant zoonosis,brucellosis.Not only causing huge losses to the development of animal husbandry,but also seriously threat to human health and development.Brucella is a facultative intracellular bacterium,and mainly parasitizes in phagocytic cells,such as macrophages and trophoblast cells.In addition,Brucella could efficiently exclude lysosome and proliferation within phagocytic cells,causing a long-term infection.Originating from monocyte cells,goat alveolar macrophages(AMs)have the function of phagocytosis,immunization and secreting cytokines.In this study,prokaryotic expression vector of GntR transcription regulation factors BSS2_II0438(GntR)was constructed,and polyclonal antibody are produced,which could screen the gntR-deleted strain(?GntR).The GntR gene of B.suis.S2 was deleted by homologous recombination to observe ?GntR intracellular growth after ?GntR infected AMs and to investigate the effect of T4 SS after ?GntR infected AMs.For analyzing GntR function between Brucella and endoplasmic reticulum stress interaction,the protein of endoplasmic reticulum stress was detected;meanwhile,TNF-?,IFN-?,IL-1? and IL-10 of media was detected to investigate effect of GntR on changes of macrophage cytokines after ?GntR infected AMs.The results are as follows:1.For acquiring polyclonal antibody,gntR gene was cloned and prokaryotic expression vector of PET32a-GntR was constructed.Then,The GntR was produced and purified.The BALB/c Mice was immunized with purified GntR,after three primary immunization and one enhanced immunization,the titer of GntR polyclonal antibody determined by ELISA.Then,the serum was segregated and acquired GntR polyclonal antibody.2.For acquiring ?GntR,recombinant vectors PUC19-GntR was constructed.Then,PUC19-GntR was transferred into competent cells of B.suis.S2 by electro transformation after it was identification by restriction endonuclease digestion and sequencing.The potential ?GntR was selected by plating on TSA containing 50 ?g/m L gentamicin,which was then verified by PCR and Western blotting.To investigate the regulatory role of GntR on the T4 SS,6 genes related to T4 SS were chosen for the qRT-PCR assay.We find three genes(virB2,6,8)were decreased,and three genes(vir B1,9,11)showed no significant difference.These results deduces that T4SS-related genes are among the most GntR-regulated genes.3.The bacterial intracellular survival of ?GntR was also not significantly different compared with B.suis.S2 groups.AMs were infected with B.suis.S2 or ?GntR,and the ER stress marker molecules were analyzed by qRT-PCR(GRP78 and CHOP)and western blotting(CHOP).The mRNA and protein expression of GRP78 was increased after ?GntR mutant infection during the early phase of infection and was decreased during the lately phase of infection compared with the uninfected and B.suis.S2 groups.CHOP mRNA expression in the ?GntR group was elevated compared with that in the uninfected group during the lately phase of infection.These results showed that GntR is involved directly or indirectly in ER stress and triggered UPR.Meanwhile,we changed ER stress by Tm and 4-PBA to detect ?GntR intracellular proliferation,we find decreasing ER stress with 4-PBA significantly inhibited GRP78 protein expression and promoted ?GntR proliferation compared with B.suis.S2.In addition,we measured the secretion levels of inflammatory cytokines IFN-?,TNF-?,IL-1? and IL-10 by ELISA.we find the supernatant from AMs infected with ?GntR produced higher amounts of TNF-? than those from the B.suis.S2-infected and uninfected groups at 12 h,and remained higher than those from the B.suis.S2-infected group at 24 h.The result indicate that ?GntR could induce the secretion of TNF-? during the early phase of infection.