Identification and functional exploration of phosphodiesterase encoding gene LA_RS06960 in Leptospira interrogans
10.3760/cma.j.cn112309-20230808-00033
- VernacularTitle:钩端螺旋体磷酸二酯酶基因LA_RS06960的鉴定及其相关生物学功能初探
- Author:
Wenwu YAO
1
;
Zhangnyu YANG
;
Lingbo WANG
;
Zhuoying WU
;
Weilin HU
Author Information
1. 浙江省疾病预防控制中心微生物检验所，杭州 310051
- Keywords:
Leptospira interrogans;
Phosphodiesterase;
Innate immunity
- From:
Chinese Journal of Microbiology and Immunology
2024;44(8):672-679
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To identify the phosphodiesterase (PDE) activity of the gene product encoded by LA_RS06960 of Leptospira interrogans ( L. interrogans), and analyze whether dinucleotides that can be degraded by PDE can activate macrophages to express innate immune factors. Methods:The LA_RS06960 gene in L. interrogans strain 56601 was amplified by PCR, and the prokaryotic expression system was constructed for the protein expression. The expressed rPDE was purified by Ni-NTA affinity chromatography. High performance liquid chromatography (HPLC) was used to measure the degration of c-di-AMP or 5′-pApA to AMP by rPDE. Real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to detect the changes in the expression of target genes in Leptospira or THP-1 cells associated with innate immune factors during infection. qRT-PCR and ELISA were used to detect the changes in the expression and secretion level of the innate immune factors in macrophages treated with bacterial dinucleotide. Results:The prokaryotic expression system for LA_RS06960 gene of L. interrogans was constracted successfully, and the purified rPDE could degrate 5′-pApA and c-di-AMP into AMP in vitro. The mRNA level of leptospiral LA_RS06960 gene was significantly down-regulated, while IFN-β, TNF-α, IL-6 and IL-1β encoding genes of macrophages were significantly up-regulated during infection. The mRNA level or the secretion level of IFN-β and IL-6 of macrophages were increased after treated with the bacterial dinucleotide substrate of PDE. Conclusions:PDE encoded by LA_RS06960 gene has phosphodiesterase activity, and the bacterial dinucleotide substrate of the PDE could activate the innate immune response of macrophages.
