布鲁氏菌T4SS效应蛋白BspJ的生物学功能研究

Biological function analysis of the Brucella T4SS effector protein BspJ

为探究布鲁氏菌IV型分泌系统(T4SS)效应蛋白BspJ的生物学功能,本研究利用同源重组和载体回补方法,以粗糙型牛种布鲁氏菌(RB51)为亲本株,构建布鲁氏菌BspJ基因缺失菌株(ΔBspJ)与BspJ基因回补株(pBspJ),并经PCR和测序鉴定。将ΔBspJ与p BspJ连续传代至15代,经PCR鉴定ΔBspJ与pBspJ的遗传稳定性,结果显示,正确构建了ΔBspJ与pBspJ,且各代次ΔBspJ均未扩增出目的条带、p BspJ均扩增出目的条带,二者遗传稳定性好。将RB51、ΔBspJ、pBspJ分别于酸性(p H2.5/37℃)、碱性(pH11.5/37℃)、高盐(1.5 mol/L NaCl/37℃)及热休克(pH7.0/50℃)等不同体外条件下培养,统计各菌株生存率;将RB51、ΔBspJ、p BspJ分别以感染复数(MOI)100侵染小鼠单核巨噬细胞白血病细胞(RAW264.7)后,采用q RT-PCR检测凋亡相关因子基因BAX、BCL2、CASP3、CASP8及炎症相关因子基因CDK2、m TOR、TRAF3IP2、P62、IL-8的转录水平;另外,于侵染后24 h、48 h时分别裂解上述3种侵染细胞,计数各菌株的细菌菌落总数(CFU),分析各菌株的胞内生存能力。结果显示,相比于RB51,ΔBspJ在pH2.5/37℃的TSB液体培养基中的生存率极显著下降(P<0.01),在pH11.5/37℃、pH7.0/50℃、1.5 mol/L NaCl/37℃的TSB液体培养基中的生存率极显著下降(P<0.001);相比RB51侵染的RAW264.7细胞,ΔBsp J侵染的RAW264.7细胞中BAX、CASP3、CASP8基因的转录水平极显著升高(P<0.001),BCL2基因的转录水平极显著下降(P<0.001),TRAF3IP2基因的转录水平极显著升高(P<0.0001),P62基因转录水平极显著升高(P<0.01),IL-8、mTOR基因的转录水平显著升高(P<0.05),CDK2基因的转录水平无显著差异(P>0.05)。相比RB51,ΔBspJ的胞内生存能力在侵染24 h后显著下降(P<0.01),48 h后极显著下降(P<0.001)。上述试验中亲本株RB51组与回补株p BspJ组检测结果均无显著差异(P>0.05)。综上所述,本研究首次证实BspJ参与调控布鲁氏菌酸碱稳态和渗透压稳态,揭示了BspJ通过抑制宿主细胞的凋亡与炎症反应显著提高布鲁氏菌的胞内生存能力,为T4SS效应蛋白BspJ对布鲁氏菌致病机制的研究奠定了基础。

To investigate the biological function of the T4SS effector gene Bsp J in Brucella,a Bsp J gene deletion strain(ΔBsp J)and a gene Bsp J deletion complementing strain(p Bsp J)were constructed by homologous recombination and vector replenishment methods using RB51 as parent plant.TheΔBsp J and p Bsp J were then continuously passaged for 15 generations,and their stability was identified by PCR.The results showed thatΔBsp J and p Bsp J were correctly constructed.The survival rate of RB51,ΔBsp J and p Bsp J was determined under different in vitro conditions,including acidic(p H2.5/37℃),alkaline(p H11.5/37℃),high salt(1.5mol/L Na Cl/37℃)and heat shock(p H7.0/50℃).The RAW264.7 cells were infected with RB51,ΔBsp J and p Bsp J with a multiplicity of infetion(MOI)of 100,and apoptosis related factors(BAX,BCL2,CASP3 and CASP8)and inflammation related factors(CDK2,m TOR,TRAF3IP2,P62 and IL-8)were detected by q RT-PCR.The RAW264.7 cells were infected with RB51,ΔBsp J and p Bsp J with MOI 100for 24 hours and 48 hours,and the total bacterial colony count(CFU)of each strain was measured to assess their intracellular survival rate.The results showed that compared with RB51,the survival rate ofΔBsp J in TSB liquid medium at p H2.5/37℃was significantly decreased(P<0.01),and the survival rate ofΔBsp J in TSB liquid medium at p H11.5/37℃,p H7.0/50℃and 1.5mol/L Na Cl/37℃was significantly decreased(P<0.001).Compared with RB51,deletion of Bsp J significantly increased the gene transcription of BAX,CASP3and CASP8(P<0.001),and significantly decreased the gene transcription of BCL2 in infected RAW264.7 cells(P<0.001).In addition,deletion of Bsp J gene significantly increased TRAF3IP2(P<0.0001),P62(P<0.01),IL-8(P<0.05)and m TOR(P<0.05)gene transcription but showed no significant difference in CDK2 gene transcription(P>0.05).Compared with RB51,the intracellular viability of Brucella was significantly decreased after 24 hours and 48 hours infection withΔBsp J.There was no significant difference between RB51 and p Bsp J infection group(P>0.05).In summary,this study confirmed that gene Bsp J may be involved in regulating the acidbase homeostasis and osmotic pressure homeostasis of Brucella.It was revealed that gene Bsp J significantly improved the intracellular viability of Brucella by inhibiting apoptosis and inflammation of host cells,which laid a foundation for the study of the pathogenic mechanism of Brucella by T4SS effector protein Bsp J.