The Endoplasmic Reticulum Stress And Gonadal Hormonesecretory Influence Of Brucella VceC On Goat Trophoblast Cells

Brucella spp are Gram-negative facultative intracellular bacteria that cause brucellosis,a wide spread zoonosis that affects mainly domestic animals and humans,causing important economical and sanitary problems in many areas of the world.Brucellosis is a chronic disease and may last several weeks or months.If it is not treated effectively,the disease can lead to pathologies in the liver,spleen,lymph nodes,bone marrow,reproductive tract,and skeletal system.Trophoblast cells are the main target cells of Brucella except monocyte-macrophages.Previous study confirmed that Brucella survival in association with the endoplasmic reticulum(ER)of the target cells.Bacterial Type IV Secretion system plays crucial roles in the inhibition of the host innate immune response and in intracellular survival during infection,and effectors secreted by Type IV Secretion system mainly implement the regulation.VceC is one of the important effectors and major virulence factors secreted by Type IV Secretion system.It is very significant to clarify the mechanism of intracellular survival and the ability to evade the host immune of Brucella by revealing its role and mechanism in the infected host cells.In this experiment we constructed the eukaryotic expression vector of VceC and transfected it into goat trophoblast cells(GTC),then we detected(1)the main factors of ERS,GRP78 and CHOP,the activating of the unfolded protein response and IRE1 in order to explore the effect of ERS by VceC in the host cells in the process of Brucella infections;(2)the Progesterone and Estrogen secreted by goat trophoblast cells to research the hormone secretion influence by VceC when Brucella infects goat trophoblast cells.The main results are as follows:1.We cloned the Brucella VceC gene and constructed the VceC eukaryotic expression vector successfully using pEGFP-C1 vector,and transfected it into cells successfully by detecting VceC protein using immunofluorescence and Western Blot.2.Using Western Blot to detect the main factors of ERS,we found that GRP78 has a highly significant up-regulation at 12 h and 24 h(P<0.01),while CHOP down-regulating(P<0.05)when VceC eukaryotic expression vector expressed 24 h in goat trophoblast cells;the results suggest that VceC can activat ERS.Also it activats mainly the IRE1 pathway ofunfolded protein response to causing ERS by detecting the related molecules using RT-PCR and Western Blot..3.Using ELISA to detect Progesterone and Estrogen repectively,the content of Progesterone decreased significantly(P<0.01)at 12 h and 24 h than GTC group and pEGFP-C1 group,while the change of Estrogen content had little change.The results show VceC protein affectes the secretion of Progesterone of the GTC.