Study On The Relationship Between SLY And Streptococcus Suis Serotype 2 Infection

Background:Streptococcus suis is an important zoonotic pathogen,endemic in nearly all countries with an extensive pig industry.It is widely recognized in the veterinary world as a cause of rapidly progressive and fatal sepsis in infant pigs, associated with meningitis,polyarthritis,and sepsis.S.suis is capable of spreading to humans from pigs.Infection in humans is considered an occupational disease that affects persons who work in close contact with pigs or pork byproducts.The first case of human SS2 infection was reported in Denmark in 1968.Reported clinical presentations include acute bacterial meningitis,arthritis,sepsis,endocarditis,and endophthalmitis.Permanent hearing loss and vestibular dysfunction are commonly noted Sequelae.In 1998 and 2005,two S.suis outbreaks in humans occurred in China's Jiangsu and Sichuan,respectively.The latter one exhibited a much higher human morbidity and mortality,resulting in more than 200 cases with 38 deaths.A majority of those patients,and almost all of the fatal cases,had typical symptoms of Streptococcal toxic shock syndrome(STSS).Virulence factors of S.suis type 2 are not well characterized,several putative virulence factors have been described, including the capsular polysaccharide(CPS),muramidase-released cell-wall protein (MRP),extra-cellular protein factor(EF) and suilysin(SLY).The current understanding of the pathogenesis of S.suis remains limited,particularly concerning the mechanisms by which the bacteria survive and disseminate into the bloodstream and reach a variety of organs and deep tissues.These features are especially important for the strains causing outbreaks and human infections in China.Methods:In this work,the sly gene was cloned into pET28a vector,expressed as a His-tag fusion protein in Escherichia coli BL21,and the expressed fusion protein was found almost entirely in the insoluble form(inclusion bodies) in cell lysate.The inclusion bodies were solubilized with 8M urea,and the recombinant protein was purified by Ni-NTA column.The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea.A new purification strategy that produced SLY from S.suis 05ZY was developed by a combination of ammonium sulfate fractionation anion exchange column chromatography and hydrophobic interaction chromatography.Purified and refolded fusion proteins were characterized by hemolytic activity test.The suilysin(SLY) production of different SS2 strains were determined by ELISA and Western blot,the role of enhanced suilysin production on bacteremia was studied by using a murine peritonitis in vivo model and in vitro models.Results:In this study,a method for the purification of the natural SLY from S. suis05ZY culture supernatant is established and optimized.Through this method,we got the purified natural SLY and characterized its hemolytic activity.We also cloned the sly gene into pET28a vector,expressed rSLY as a His-tag fusion protein in Escherichia coli BL21,and purified by Ni-NTA affinity chromatography. Polyclonal anti-antibodies were obtained by immunized the rabbits and rats using purified fusion protein.We found,compared to the nonepidemic strains,the epidemic S.suis sequence type 7(ST7) strains exhibited an enhanced production of SLY.In accordance to the functions of SLY,animals infected with S.suis ST7 strain resulted in lower viability of PMN.SLY can enhance the ability of S.suis to invade into and survive in the bloodstream,and cause direct injury to peritoneal epithelial cells.Impaired function of neutrophil(PMN)-mediated phagocytosis by recombinant SLY protein was observed in vitro.Conclusions:A simple purification procedure is developed for purifying natural SLY protein from streptococcus suis cultured supernatant and it was also shown to have good biological activity.Compared to the nonepidemic strains,the epidemic S.suis sequence type 7(ST7) strains exhibited an enhanced production of SLY.Our findings suggest that the enhanced SLY production in S.suis ST7 strains facilitate to penetrate the epithelium and survive in the bloodstream,then contribute to the invasive infections,which is likely associated with the outbreaks occurred in China.