Apoptosis Of Macrophages Induced By39KD Cell-wall-related Protein Of Streptococcus Suis Serotype2

Streptococcus suis type2(SS2) is one of the most important zoonotic pathogensfor both humans and swine. Meningitis, septicemia,endocarditis, arthritis or suddendeath were caused in humans and swine when infected by SS2.Human health washarmed seriously by SS2,which caused serious economic losses to the pigindustry,posed a serious theat to the public health and food safety,and were paidhighly attention in the word.39KD cell-wall-related protein is one of the importantvirulence factor of SS2.In order to find out the pathopoiesia mechanism of39KDcell-wall-related protein, This study was carried out as follows:Firstly,Experimental animal models of BALB/c mice were infected by Strep-tococcus suis.Chosed SPF-BALB/c mice (20-25g),Streptococcus suis type2-HA9801strains infected mice through different pathways and different bacteria quantity.Theresults showed that intra-abdominal infection was more susceptible than subcutaneousinfection and nasal infection, the most insusceptible pathway was nasalcavity.infected mice showed typical symptoms and pathological changes, such asmeningitis, arthritis and other symptoms. The same bacteria as the inoculated oneswere separated from tissues of infected pigs the.The repeatability of model was verygood. The LD50was7.2×106CFU.Secondly,the pathogenic role of39KD cell-wall-related protein on macrophagesis poorly understood.In present study,(1)The39KD cell-wall-related protein waspurified until presenting only one band by SDS-PAGE.(2)Murine peritonealmacrophages (MPMs), isolated from SPF Balb/c mice（20-25g）, were co-incubatedwith39KD cell-wall-related protein（4μg/mL and40μg/mL）in vitro followed bymicroscopy. Fluorescence micrograph showed no sign of abnormal morphologicalchanges of MPM treated at4μg/ml, whereas characteristic cell changes includingblebbing, cell shrinkage, nuclear fragmentation and apoptotic bodies, representingapoptosis at40μg/ml.(3) After MPMs were induced for6h by injecting39KD protein（4-50μg/mL respectively）into peritoneal cavity of mice, DNA was extracted andsubsequent electrophoresis demonstrated a typical ladder pattern for all20-50μg/mLinductions, whereas the same and only one high-molecular band appeared for bothnegative control at4μg/ml.(4)After MPMs were inducted by40μg/mL39KD proteinfor2、4、6、8h respectively in vivo, MPMs were collected and following flow cytometry revealed that, apoptosis of as high as8.7%appeared as early as2h postinduction, significantly high than negative control (P<0.05), and increased obviously(P<0.05) with each increase of inducing time, finally up to27.1%at8h, highlysignificant compared with negative control (P<0.01). Meanwhile, Caspase-3wasdetected and showed same dynamics. Significant increase started at2h (P<0.05),reached highest point at8h(P<0.01). Based on above analysis, we concluded that the39KD cell-wall-related protein could induce high level apoptosis of MPMs, andthe39KD Protein alone was capable of a virulence factor of SS2.