Prokaryotic Expression And Apoptosis-inducing Activity To Cells Of Leptospira Interrogans Sphingomyelinase Hemolysin Sph2

Objective To explore the change of expression level of Leptospira interrogans sph2 gene, cell apoptosis-inducing activities and mechanism of sphingomyelinase hemolysin Sph2.Methods A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. interrogans strain Lai infecting J774A.1,IAR20,L-02 and THP-1 cells. Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2 (rSph2) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatography. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to human and murine mononuclear-macrophage- like cell line (THP-1 and J774A.1) and hepatic cell line (L-02 and IAR20) was determined by flow cytometry. rSph2 labeled with FITC can identify the ability of invasion into the cells by laser confocal technique.Results The mRNA levels of sph2 gene was significantly elevated within 0.75～2 h of L.interrogans strain Lai infecting J774A.1,IAR20,THP-1 and L-02 cells, and the peak value is up to 4-8 than before infecting,and then the mRNA levels was quickly descended. The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern.10μg/ml rSph2 could induce the apoptosis of J774A.l,IAR20,THP-1 and L-02 cells, and the peak apoptotic rates were 23.96%, 32.92%,17.52% and 24.13% respectively. FITC-rSph2 have the ability of invasion into J774A.1,IAR20,THP-1 and L-02 cells within 15-30min.Conclusion The sph2 gene of L.interrogans strain Lai has a transient expression when the microbe contacts host cells, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira. rSph2 possesses activities of sheep erythrocyte lysis. rSph2 can caused the macrophage and hepatocyte cell membrane lesions and induce apoptosis, so Sph2 is a important virulence factor closely related with jaundice and pneumorrhagia caused by leptospirosis. The mechnasim of cell apoptosis induced by leptospirs interrogans is related with rSph2 which can hydrolyze sphingomyelin of the cell membrane into ceramide, and damage the mitochondrial membrane which give rise to the realease apoptotic factor or proapoptotic, at last activates mitochondrial apoptotic pathway and ceramide apoptotic pathway.