Construction Characterization Of M-like Gene Knock-out Mutant Of Streptococcus Equi Ssp. Zooepidemicus

Streptococcus equi ssp.zooepidemicus made substantial economic losses for pig industry and became a substantial threat to human health especially those involved in the pig industry.M-like protein(szp) of Streptococcus equi ssp.zooepidemicus is a major surface protein that conveys the resistance to phagocytosis and it is a virulent factor with opsonsin function.In the present study,a szp-gene knock out mutant of Streptococcus equi ssp.zooepidemicus was constructed,at the molecular levels we investigated the adhensive machanism of M-like protein,evaluated the pathogenicity and innunogenicity of the mutant strain in animal model of infection.To knock out the entire M-like protein gene of Streptococcus equi subsp zooepidemicus ATCC35246 strain via and construct a M-like protein-deleted mutant strain of Streptococcus equi ssp zooepidemicus.Two DNA segments,respectively derived from the upperstream 1178bp and 1172bp downstream of szp gene,were obtained by genome walking from gene DNA of of Streptococcus equi subsp zooepidemicus ATCC35246 strain, and these two segments were used to construct puc-18 deletion vector.The vector was then used to delete a 1090bp fragment of szp gene from a stain of SEZ(ATCC35246) through homologous recombination.The szp-knockout strain was evaluated for virulence and antigenicity in mice model of infection.Results showed,comparing with the wild type,the szp-knockout strain had a dramatically decreased LD50.Most mice innocuated with the szp-knockout strain can resis the challenge of the virulent strain.Semi-quantitative RT-PCR analysis showed a marked increased in the level of IL-2 and IFN-γmRNA in immunized mice with mutant strain.In the same time,a complementation assay was performed to detect the restoration of the wild-type phenotype from the szp gene knockout mutant.This mutant strain could be used as an attenuated vaccine candidate. HEp-2 cells,a kind of typical cell model,was used in the adhesion and adhesive inhibition experiments to evaluate the adhesion function of M-like protein from Streptococcus equi ssp.zooepidemicus strain ATCC35246 by ELISA.The adhesion results showed that M-like protein could adhere to HEp-2 cells.Treating recombinant M-like protein with the monoclonal antibody can inhibit the adhesion of M-like protein to HEp-2 cells.When concentration of M-like protein was up to3.2μg/200μL,the complete adhesive was obtained.Pretreatment of M-like protein with the monoclonal antibody against purified recombinant M-like protein at 1.8μg/500μL could inhibit its adhesion completely; Adhesion counting assay demonstrated high adhesive levels of both strains.Scanning electron microscope observation further showed a dense adhering of ATCC35246 at cellular membrane and microvilli,Lysis counting assay revealed a invasion of ATCC35246 and weak invasion of ATCC35246△szp to HEp-2 cells.These findings suggested,firstly,the epithelial cell served as the colonizing site and an entrance in establishment of infection;secondly,the direct invasion into epithelial cells following their adhesion was one of their mechanisms of breaking through the epithelial cell barrier.