Nuclease Activity And Cytotoxicity To Host Cells Of Toxic Protein VapC Produced By Leptospira Species

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 Ni-NTA affinity chromatography. Activity of rVapB and rVapC to lyse the DNAs or RNAs from L. interrogans strain Lai and THP-1cells 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-1cells were detected by real-time fluorescent quantitative RT-PCR and Western Blot assay. The expression of VapB and VapC proteins in L. interrogans strain Lai-infected THP-1cells was examined by immunofluorescence confocal microscopy. The eukaryotic expression vectors of the vapB and vapC genes were generated for transfection of host cells and CCK-8agent 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-1cells, the transcription and expression of vapB and vapC genes were upregulated and partial VapC protein was secreted from the leptospiral cells. VapC protein could be detectable in the L. interrogans strain Lai-infected THP-1cells and the mass mortality was observed in HEK293human renal tubular epithelial cells 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.