Construction Of Brucella Vcec Gene Mutant And Its Effects On Endoplasmic Reticulum Stress In Goat Trophoblast Cells

Brucella are gram-negative facultative intracellular pathogen that can cause brucellosis in worldwide.Brucellosis has an important influence on the aquaculture and public health.Brucella mainly infects cattle,sheep and goats,causing abortion and infertility.In humans,the infection is characterized by undulant fever,fatigue,excessive sweating,and arthritis.Brucella as an intracellular bacteria,it has an unique intracellular survival strategy which plays a significant role in the pathogenicity.type ? secretion systems(T4SS)is an important virulence factor that plays crucial roles in survival of Brucella,and manipulating the host immune responses.VceC is secreted by the Brucella T4 SS,studying its survival and mechanism in host cells is of great importance for elucidating the mechanism of brucellosis and its effective prevention and control.In this experiment,a homologous recombination vector was constructed,we transformed it into B.suis S2 competent cells,and then the VceC gene mutant strain(?VceC)was constructed;The effects of ?VceC on goat trophoblast cells(GTC)apoptosis were investigated.We utilized FCM detected apoptosis of ?VceC infected GTC,and we detected the changes of apoptosis marker cleaved caspase3?pro-caspase9 and CHOP by Western Blot.In addition,we investigated effects of endoplasmic reticulum stress(ERS)and Unfolded protein response(UPR)pathway,using ?VceC infected GTC,detected the expression changes of the ERS marker molecule GRP78 and related proteins in the UPR pathway;Using Tm and 4-PBA to change the ERS states of GTC,detected the effect of ?VceC infected GTC on its intracellular proliferation.The results are as follows:1.We cloned the upper and lower homologous arms of the VceC and Gentamicin gene of Brucella,and constructed the Brucella homologous recombination vector PUC19G-VceC,and transformed it into Brucella competent cells,and constrcted Brucella VceC deletion strain ?VceC by PCR.2.Using FCM detected the apoptosis of ?VceC-infect GTC at 12 h?24 h?48 h,the results show that ?VceC promotes GTC apoptosis.Furthermore,apoptosis related protein cleaved caspase3?pro-caspase9 and CHOP were investigated by Western Blot.The results showed that cleaved caspase3 and CHOP were significantly increased.However,pro-caspase9 have no differences.The results showed that ?VceC could induced apoptosis of host cells through the ERS pathway.3.Using Western Blot detected GRP78,we found the expression of GRP78 has a significantly decreased(P<0.01).It was proved that ?VceC infected GTC can induce ERS.In further,we detected the related proteins that involved UPR signal pathway.The results showed that ?VceC infected GTC after 24 and 48 hours,the expression of phosphorylated IRE1 were down-expression,while the e IF2??ATF6-? and ATF6-? have no significant difference.It was found that ?VceC could activate IRE1 signal pathway.In addition,we detected ?VceC intracellular proliferation by using Tm and 4-PBA to change the ERS states of GTC.The results showed that pretreatment of GTC with 0.5 ?g/m L Tm,increased the expression of GRP78 in GTC compared with B.suis S2,meanwhile,significantly reduced the proliferation of ?VceC in GTC.On the contrary,pretreatment of GTC with 1 ?M 4-PBA reduced the expression of GRP78,and significantly increased the proliferation of ?VceC in GTCs.These results confirmed that different ERS states have effects on the proliferation of ?VceC in GTCs.