Identification of Sigma N signaling system target genes in Leptospira interrogans / 中华微生物学和免疫学杂志

Objective:To confirm the Sigma N transcription factor activity of a gene product encoded by LA2404 gene of Leptospira interrogans ( L. interrogans) and to identify the target genes of Sigma N signaling system. Methods:L. interrogans LA2404 gene and its regulated target genes were predicted using bioinformatic analysis according to the promoter sequence signature in Sigma N-regulated genes. A LA2404 gene-knockout (ΔLA2404) strain of L. interrogans was constructed based on homologous sequence recombinant of suicide plasmid. Real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to detect the changes in the expression of target genes at mRNA level in the ΔLA2404 mutant. A prokaryotic expression system for LA2404 gene was established and the target recombinant protein rSigma N was extracted by Ni-NTA affinity chromatography. Gel electrophoresis mobility shift assay (EMSA) was used to screen out the target genes regulated by rSigma N. Results:Pathogenic L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai carried one Sigma N gene and 22 Sigma N promoter sequence-containing target genes. Qualitative examination of the ΔLA2404 mutant by microscopy revealed no defect in motility and appearance. Expression of LA1188, LA2306, LA3426, LA1968, LA1313, LA3806 and LA0773 genes at mRNA level in the ΔLA2404 mutant was significantly down-regulated ( P<0.05), but no significant changes in the expression of other target genes at mRNA level were detected. EMSA results confirmed that rSigma N could bind to the promotor sequences of the target genes mentioned above. Conclusions:Sigma N transcription factor was encoded by LA2404 gene. LA1188, LA2306, LA3426, LA1968, LA1313, LA3806 and LA0773 genes contained Sigma N promoter sequence and the expression of them was regulated by Sigma N signaling system.

Expression of T and B cells combined epitopes protein of Leptospira interrogans and the immunity analysis / 中华微生物学和免疫学杂志

Objective To construct the T-cells and B-cells combined epitope peptide gene based on the LipL32,OmpL1 and LipL21 protein from Leptospira interrogans and E.coli expression system,and better understanding of the immunological activity of the recombinant protein. MethodsThe immunodomaint T- and B-cells combined epitopes of LipL32,OmpL1 and LipL21 were identified and used to synthetic a new gene and then construct its prokaryotic expression system.The expression of recombinant protein was determined by SDS-PAGE; MAT was used to determine the titer of the antiserum to L. interrogans standard strains of China ; Western blot and ELISA were used to identify the immunity activity of the recombinant protein.Results The synthetic gene was effectively expressed in E.coli BL21 ( DE3 ) strain and mainly presented in dissoluble protein.Western blot result showed that the expression protein react well with the antibodies from immunized rabbit by Leptospira or recombinant protein.ELISA and MAT results showed that the multiepitope protein could cross-react with different serogroup or serovar of Leptospira.Conclusion In this study,we successfully constructed the recombinant T- and B-cells combine epitope gene of leptospires and expressed it in E.coli.The recombinant protein had a good immune activity,and could cross-reacted with antibodies from different serogroups Leptospira infected patients.

Comparison of loop-mediated isothermal amplification and real-time PCR for the detection of Leptospira interrogans / 中华微生物学和免疫学杂志

Objective To compare the sensitivity and specificity of Leptospira interrogans using loop-mediated isothermal amplification (LAMP) and real-time PCR technology, then looking for a rapid,sensitive and specific methods for the detection of Leptospira interrogans. MethodsIn accordance with lipL41 gene from Leptospira interrogans, primers for LAMP and real-time PCR were designed and used to detect Leptospira interrogans in cultured 15 reference strains of 15 serogroups in China, then compared the sensitivity and specificity of the two methods in the detection of Leptospira interrogans. ResultsThe LAMP reaction could be completed within 30 min, and whole process of it less than 60 min. The whole real-time PCR reaction could be finished at about 60 min. Both of them had the same detection sensitivity and specificity,the lower detection limits in the reactions was approximately 100 copies and there was no false positive occurred. ConclusionBoth LAMP and real-time PCR were time-saving and had the same sensitivity and specificity. But LAMP reaction could be done under a constant temperature conditions, and need not a special expensive equipment. Therefore, as a sensitive and specific method for quantifying Leptospira interrogans,the LAMP assay was more rapidly and convenient than conventional methods.

Expression pattern and apoptosis-inducing activity to murine macrophages and hepatocytes of Leptospira interrogans Sph2 hemolysin / 中华微生物学和免疫学杂志

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.

Modality of Leptospira interrogans invading mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation / 中华微生物学和免疫学杂志

Objective To determine the modality of Leptospira interrogans invading human and murine mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation. Methods Transmission electron microscopy was applied to observe the invasion of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai into murine mononuclear-macrophage-like cell line J774A. 1 and PMA-activated human monocyte line THP-1 and the formation of leptospiral phagocytotic vesicles. By using immunofluorescence plus either laser confocal microscopy or fluorescence spectrophotometry, the changes of intracellular leptospiral numbers in J774A. 1 and PMA-activated THP-1 cells before and after block with endocytosis inhibitors monodansylcadaverin (MDC), phenylarsine oxide (PAO) and clathrin antibody were investigated. Results The leptospires in J774A. 1 cells were located in phagocytotic vesicles while the leptospires in THP-1 cells had no package with phagocytotic vesicle membrane. Both MDC and PAO presented the effect inhibiting endocytosis of L. interrogans into J774A. 1 and THP-1 cells in dose-dependent manner. The numbers of leptospires in J774A. 1 and THP-1 cells that pre-blocked with 10 μmol/L or above MDC and 1 μmol/L or above PAO were significantly less than that in the two cells untreated with MDC and PAO (P＜0.05＝. After J774A. 1 and THP-1 cells were blocked with clathrin antibody, the numbers of intracellular leptospires were also remarkbly decreased ( P<0.05 ).Conclusion Leptospira interrogans can invade into both human and murine mononuclear-macrophages through the way of clathrin-dependent endocytosis. There is an opposite diversity of leptospiral phagocytotic vesicle formations in human and murine mononuclear-macrophages, which may result in the difference of pathogenesis in human and mice after infected with L. interrogans.