Roles and mechanisms of ferri ion and ferritin in the growth, reproduction and energy metabolism of Leptospira interrogans
10.3760/cma.j.cn112309-20200611-00307
- VernacularTitle: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
- Author:
Weiqun YANG
1
;
Aihua SUN
;
Jie YAN
;
Weiling HU
;
Xu′ai LIN
Author Information
1. 浙江大学医学院病原生物学系，杭州 310058
- From:
Chinese Journal of Microbiology and Immunology
2020;40(8):593-599
- CountryChina
- Language:Chinese
-
Abstract:
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 1 238.619 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.
