Study of the platelet activating factor acetylhydrolase activity of a gene product encoded by LA2144 gene of Leptospira interrogans
10.3760/cma.j.issn.0254-5101.2015.08.001
- VernacularTitle:钩端螺旋体LA2144基因产物血小板活化因子乙酰水解酶活性的研究
- Author:
Meirong TANG
;
Jinfang ZHAO
;
Shilei DONG
;
Weilin HU
;
Xu′ai LIN
;
Jie YAN
;
- Publication Type:Journal Article
- Keywords:
Leptospira interrogans/LA2144 gene;
Platelet activating factor acetylhydrolase;
Gene expression/Protein secretion;
Hemorrhage
- From:Chinese Journal of Microbiology and Immunology
2015;(8):561-567
- CountryChina
- Language:Chinese
-
Abstract:
Objective To analyze the platelet activating factor acetylhydrolase ( PAF-AH) activity of a gene product encoded by LA2144 gene of Leptospira interrogans ( L. interrogans) , to investigate the ex-pression and secretion of LA2144 protein in various cell cultures and to further understand its function in in-ducing internal hemorrhage in an animal model. Methods The DNA sample containing LA2144 gene was extracted from L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai and used as the template for gene cloning by PCR. The LA2144 gene without the signal sequence coding region was amplified by PCR and inserted into a prokaryotic expression construct for the protein expression. The expressed recombinant protein, rLep-PAF-AH, was purified by Ni-NTA affinity chromatography. Spectrophotometry was used to measure the hydrolytic activity, hydrolytic efficiency, Km and Kcat values of the rLep-PAF-AH protein in hydrolyzing PAF substrate. Real-time fluorescent quantitative RT-PCR ( qRT-PCR) and Western blot assay were performed to measure the expression of LA2144 gene at mRNA and protein levels in human umbilical vein endothelial cells (HUVEC), human monocytes (THP-1) and murine macrophages (J774A. 1) with L. interrogans strain Lai infection, respectively. Each syrian hamster was intravenously injected with 100 μg of LPS-free rLep-PAF-AH for two times. Hemorrhage in the lungs, livers and kidneys were observed in three days after the injection. Results The constructed prokaryotic expression system for LA2144 gene of L. inter-rogans strain Lai could highly express the rLep-PAF-AH upon the induction of IPTG. The purified rLep-PAF-AH showed high purity with a single protein band in gel as indicated by SDS-PAGE. The efficiency of 5 μg of rLep-PAF-AH in hydrolyzing PAF substrate was 26. 6 U/L with a Km value of 82. 79 μmol/L and a Kcat value of 0. 24 S-1 . The expression of Lep-PAF-AH at mRNA level in HUVEC, THP-1 and J774A. 1 cells were significantly elevated after co-culture with L. interrogans strain Lai for 1 or 2 hours (P<0. 05). A large amount of Lep-PAF-AH were detected in the supernatants from co-cultures of L. interrogans strain Lai with the three cell lines, but not from the culture of the spirochete in EMJH medium. The signs of hemor-rhage were observed in the lung of hamsters injected with rLep-PAF-AH, but not in tissue samples from liver and kidney. Conclusion The LA2144 gene product was characterized by a stronger PAF-AH activity. The expression of LA2144 gene at mRNA and protein levels in various cell lines were enhanced during L. interro-gans infection. Moreover, the rLep-PAF-AH could induce the pulmonary hemorrhage in hamsters. This stud-y indicated that the protein encoded by LA2144 gene was an important virulence factor causing hemorrhage in hosts during L. interrogans infection.
