Nuclease activity and cytotoxicity to host cells of toxic protein VapC produced by Leptospira species
10.3760/cma.j.issn.0254-5101.2012.02.015
- VernacularTitle:钩端螺旋体毒性蛋白VapC核酸酶活性及其对宿主细胞毒性作用的研究
- Author:
Xiaoyang XIN
;
Xüai LIN
;
Liwei LI
;
Jie YAN
- Publication Type:Journal Article
- Keywords:
Pathogenic Leptospira species;
Toxin-antitoxin system;
Toxic protein;
Nuclease activity;
Cytotoxicity
- From:
Chinese Journal of Microbiology and Immunology
2012;32(2):166-171
- CountryChina
- Language:Chinese
-
Abstract:
Objective To determine the function of toxic protein VapC in toxin/antitoxin system of Leptospira species and the cytotoxicity to host cells of the toxic protein.Methods Using genomic DNA of pathogenic L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai as the template,several PCRs were performed to amplify entire vapB,vapC and vapBC genes.Subsequently,the prokaryotic expression systems of vapB,vapC and vapBC genes were constructed.Expression of the target recombinant proteins rVapB and rVapC was detected by SDS-PAGE and the expressed rVapB and rVapC were extracted by NiNTA affinity chromatography.Activity of rVapB and rVapC to lyse the DNAs or RNAs from L.interrogans strain Lai and THP-1 cells were then determined.The changes of transcription and expression of vapB and vapC genes of L.interrogans strain Lai before and after infection of THP-1 cells were detected by real-time fluorescent quantitative RT-PCR and Western blot assay.The eukaryotic expression vectors of the vapB and vapC genes were generated for transfection of host cells and CCK-8 agent was used to detect the effect of leptospiral VapB and VapC proteins on activity of host cells.Results The nucleotide and putative amino acid sequences of the cloned vapB and vapC genes were completely identical with the reported corresponding genes.The constructed prokaryotic expression systems could express rVapB and rVapC,respectively.rVapC displayed RNase avtivity but did not lyse DNA.When L.interrogans strain Lai infected THP-1 cells,the transcription and expression of vapB and vapC genes were upregulated and partial VapC protein was secreted from the leptospiral cells.The mass mortality was observed in HEK293 human renal tubular epithelial ceils containing the vapC gene through transfection.Conclusion VapC protein of L.interrogans strain Lai is a RNase and is secreted during infection of host cells with obvious cytotoxicity.
