ABSTRACT
Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. 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 chromatogaphy. 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 murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. 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 and IAR20 cells. Results 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. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.
ABSTRACT
Objective To determine the hemolytic activity of products of sphingomyelinase hemolysin encoding genes of Leptospira interrogaas, and the transcriptional level alterations in the infected host cells. Methods By using genomic DNAs of pathogenic L. interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai and serogroup Pomona serovar Pomona strain Luo, and non-pathogenic L. biflexa serogroup Sama-ranga serovar Patoc strain Patoc Ⅰ as templates, PCRs were performed to amplify entire sph1-sph4 genes. The amplified products were sequenced after T-A cloning. Prokaryotic expression systems of sph1-sph4 genes were re-spectively constructed, and the expressions of target recombinant proteins rSph1-rSph4 were examined by SDS-PAGE. Ni-NTA affinity chromatographic column was used to extract the expressed rSph1-rSph4. Hemolytic ac-tivities of rSph1-rSph4 on sheep blood agar plate were identified. Transcription alterations of sphl-sph4 genes in L. interrogans strain Lai after infected J774A. 1 cells were measured by real-time fluorescence quantitative RT-PCR. Results From genomic DNAs of both L. interrogans strain Lai and Luo, but not from that of L. biflexa strain Patoc Ⅰ , the target fragments of sph1-sph4 genes could be amplified. All the cloned sph1-sph4 genes had 100% nucleotide sequence identities compared to the corresponding reported sequences. The constructed pro-karyotic expression systems were able to efficiently express the target recombinant proteins rSph1-rSph4, respec-tively. All the rSph1-rSph4 had hemolytic activities, and among the four products rSph2 displayed the strongest hemolytic activity. After L. interrogaas strain Lai infecting J774A. 1 cells, the transcriptional levels of sph1-sph4 genes were remarkably up-regulated, especially for mRNA levels of sph2 and sph4 genes. Conclusion sph1- sph4 genes exist only in pathogenic L. interrogans species, and their products have hemolytic activity. The up-regulation of sph1-sph4 gene transcriptional levels in L. interrogans strain Lai after infected cells implies that the sphingomyelinase hemolysins may play important roles in the process of L. interrogans infection in hosts.