铁离子和储铁蛋白在钩端螺旋体生长繁殖和能量代谢中的作用与机制

Roles and mechanisms of ferri ion and ferritin in the growth,reproduction and energy metabolism of Leptospira interrogans

目的了解铁对问号钩端螺旋体生长繁殖和能量代谢的影响,确定LA_2690与LA_3598基因产物为问号钩端螺旋体储铁蛋白及其亚铁氧化酶功能。方法采用Petroff-Hausser计数法了解缺铁对问号钩端螺旋体黄疸出血群赖型56601株在EMJH培养基中生长繁殖的影响。分光光度法和化学发光法检测缺铁抑制问号钩端螺旋体DNA和ATP合成的作用。采用生物信息学软件分析问号钩端螺旋体LA_2690和LA_3598基因结构与功能。构建LA_2690和LA_3598基因原核表达系统,Ni-NTA亲和层析法提纯目的重组蛋白rLep2690和rLep3598。分光光度法检测rLep2690和rLep3598亚铁氧化酶活性。实时荧光定量RT-PCR检测问号钩端螺旋体56601株感染人脐静脉内皮细胞(HUVEC)和单核细胞(THP-1)时LA_2690和LA_3598基因转录水平变化。结果在缺铁EMJH培养基中,问号钩端螺旋体生长繁殖能力、DNA和ATP合成水平均显著下降(P<0.05)。LA_2690和LA_3598基因产物分别为细菌铁蛋白(Bfr)和细菌DNA结合铁蛋白(Dps),均含有双亚铁氧化酶中心,但后者缺乏亚铁血红素结合位点和亚铁氧化酶核心。LA_2690和LA_3598基因原核表达系统能高效表达目的重组蛋白rLep2690和rLep3598,提纯后经SDS-PAGE检测均显示为单一的蛋白质条带。rLep2690和rLep3598亚铁氧化酶活性分别为1238.619和60.052 U/L。感染细胞时,LA_2690和LA_3598基因mRNA水平均显著升高(P<0.05)。结论铁离子参与问号钩端螺旋体生长繁殖、DNA和ATP合成。LA_2690和LA_3598基因(Bfr和Dps)均为问号钩端螺旋体感染细胞时所需,其中LA_2690基因产物具有较强亚铁氧化酶活性。

Objective To investigate the influence of ferri ion on the growth,reproduction and energy metabolism of Leptospira interrogans(L.interrogans),and to identify whether the LA_2690 and LA_3598 gene products functioned as ferritin and ferroxidase.Methods Petroff-Hausser counting method was used to analyze the influence of ferri ion deficiency on the growth and reproduction of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain 56601 in EMJH medium.Spectrophotometry and Chemiluminescence method was used to detect whether ferri ion deficiency inhibited the synthesis of DNA and ATP in L.interrogans.The structures and functions of L.interrogans LA_2690 and LA_3598 genes were analyzed using bioinformatic softwares.Prokaryotic expression systems for LA_2690 and LA_3598 genes were established and the target proteins,rLep2690 and rLep3598,were extracted by Ni-NTA affinity chromatography.The ferroxidase activity of rLep2690 and rLep3598 was detected by spectrophotometry.After L.interrogans strain 56601 was used to infect human umbilical vein endothelial cells(HUVEC)and monocytes(THP-1),changes in the expression of LA_2690 and LA_3598 genes at transcription level were detected using real-time fluorescence quantitative RT-PCR(qRT-PCR).Results In the ferri ion-absent EMJH medium,the growth and reproduction of L.interrogans as well as the DNA and ATP synthesis levels were significantly decreased(P<0.05).The products of LA_2690 and LA_3598 genes were predicted as bacterioferritin(Bfr)and DNA-binding ferritin containing ferroxidase diiron centers,but the latter lacked the heme-binding site and ferroxidase core.The prokaryotic expression systems for LA_2690 and LA_3598 genes could efficiently express the target recombinant proteins.Both the purified rLep2690 and rLep3598 showed a single band on SDS-PAGE.The ferroxidase activity of rLep2690 and rLep3598 was 1238.619 U/L and 60.052 U/L,respectively.The expression of LA_2690 and LA_3598 genes of L.interrogans at mRNA level was significantly elevated during infection of the two types of cells(P<0.05).Conclusions Ferri ion participates in the growth and reproduction of L.interrogans as well as the synthesis of DNA and ATP.LA_2690 and LA_3598 genes were essential for L.interrogans to infect cells,and the product of LA_2690 gene possessed a stronger ferroxidase activity.