Moleculer Mechanism Research Of Suilysin By Inducing Inflammatory Factor From Host

Streptococcus suis is an important etiological agent of swine meningitis, and it isalso a zoonotic agent worldwide. Although sporadic cases of S. suis infections inhumans (mainly meningitis) have been reported during the last44years, in2005,China resulted in more than200human cases which were characterized by an unusualclinical presentation of STSLS with an increased death rate as high as20%[1]. Serumsamples reveals that IL-1β, IL-6, IL-8, IL-12p70, IFN-γ, and TNF-α weresignificantly higher in the patients with streptococcal toxic-shock-like syndrome(STSLS) than in the patients with meningitis only [2]. Over-expression of thesecytokines may lead to death.It was reported that heat-killed bacterial (European strain31533) can activateTHP-1, mouse macrophage cell lines J774A1and P388D1, by the release of cytokines[3,4,29]. Cell wall and suilysin can activate endotheliocyte release cytokines [33]. Afurther study speculated that rSLY can trigger the production of TNF-α by humanmonocytes, and IL-6from pig PAMs and monocytes [5].Sequence type7(ST-7) strains caused the human outbreak in China, which hadbeen reported to be more toxic for human PBMCs than a well-characterized Europeanvirulent strain, ST-1[1]. SLY is a major virulence factor that is expressed by clinicalisolates of the bacteria in China [1].This497amino acid residue protein belongs to thecholesterol-dependent cytolysin (CDC) family which over20members, such asperfringolysin O and streptolysin O, expressed by Clostridium perfringens andStreptococcus pyogenes, respectively [10]. Like other members of the CDC familyproduced by Gram-positive bacteria, a classical feature of these toxins is their abilityto create transmembrane pores in cholesterol-containing membranes and therebycause cell lysis [10,11]. Reserchers found that SLY can induces osmotic lysis oferythrocytes by forming transmembrane pores of approximately7nm diameters [11].To find the inflammatory component in this bacterium, we have constructed manygene knock-out mutants from05ZY.△SLY, isogenic non-suilysin mutant indicateobvious loss of inflammatory ability. Therefore, we purified the native SLY from thesupernatant of bacterial culture as previous experiments [16], and determined the hemolytic activity to make sure the usage on cell.In this research we choose several cell types to study the inflammatory ability ofsuilysin, such as PBMCs, THP-1MΦ, RAW264.7and peritoneal macrophage frommice. The results indicate that PBMCs were more sensitive to suilysin than other celltypes. Small amount of suilysin can activate PBMCs synthesis large amount of TNF-α.Cellular necrosis or clearage can induce inflammatory reaction, therefore theinflammatory activity of suilysin was determined whether depende on its hemolyticactivity. Therfore, recombinant suilysin (rSLY) and point mutation (P353V) wasconstructed and the proerin was purified as our previous study. Our studies indicatethat the inflammatory activity of suilysin is independent on its hemolytic activity; itmay depend on the recognition from host receptor.Suilysin had been studied for the inflammatory ability both in vitro [5] and in vivo.There is no deep study on this protein in inflammation. Pneumolysin（PLY）, amember of CDCs, which from Streptococcus pneumoniae induces proinflammatoryresponses in macrophages in a TLR4-dependent manner [12]. We asked whethersuilysin utilize the similar manner. In our study, we indicate that the inflammatoryactivity of suilysin is in a TLR4-dependent manner with TLR4specific inhibitorCLI-095, which can totally inhibit the inflammatory of suilysin on several cell types.We compared the response of WT and TLR4point mutant peritoneal-derivedmacrophages to nSLY. The results confirm that the inflammatory activity of suilysin isdependent on TLR4.We used both MyD88and TRIF specific inhibitors, Pepinh-MYD and Pepinh-TRIFrespectively, also their homotype control called Pepinh-Control to analyze the signalpathway. We use MAPK-pathway inhibitors and western blot technology to determinethe activation of MAPKs. Then we use luciferase as a reporter gene to study the roleof suilysin by activating the transcription factors NF-κB in RAW264.7. MyD88is themain adapter protein participates in SLY-TLR4signal pathway. Suilysin can directlyactivate MAPK p38in PBMCs. native Suilysin can activate NF-κB in RAW264.7.It had been reported heat-killed bacterial (European strain31533) can activateimmune cells by the release of cytokines. But a high bacterial concentration(109cfu/ml) was needed for maximal cytokine production [3,4,29], as TNFproduction quickly dropped when bacterial titre was decreased to108cfu/ml [4]. TNFis the first cytokine detected, but its levels drop much faster within5hours. But in ourstudy, small amount SLY (Mol/L) can induce large amount of TNF-α from human PBMCs, and persist for36hours. Our results indicate human PBMCs are moresensitive than THP-1macrophages, RAW264.7and primary peritoneal macrophagesfrom mouse. Macrophages, which presence in different tissues, are the first immunecell type meeting S.suis, the immune response as inflammatory reaction can help hostclearing the bacteria. It is very dangerous when the bacteria getting into blood,because mol/L of suilysin can activating human PBMCs by releasing large amountTNF-α all over the body. The bacteria can be easily separated from patient’s blood inChina. So we should take more attention of suilysin interact with PBMCs.We use several methods to eliminate the influence of LPS, Firstly, heat treatment ofnSLY, because LPS would have been expected to resist heat treatment. Secondly, theproteins were incubated with polymyxin B at4℃for1hour just before experiment,because polymyxin B can disable LPS[28]. Thirdly, we use Ttritoon X-114toremoval endotoxin from recombinant proteins. Finally, we obtain irrelevant proteinwith totally the same condition from prokaryotic expression to removal LPS. All thesetreatments support our final conclusion.The present study demonstrate for the first time that SLY activate immune cells viaTLR4. And the inflammatory activity of suilysin is independent on its hemolyticactivity. We think that S.suis acts as a double-edged sword during the host-pathogeninteraction. As the pore-forming activity of SLY, S.suis can damage host cells, and asthe inflammatory ability, it can activate immune reaction which can help the host toclear the bacteria. But when SLY in the S.suis over activated immune cells in the host,TSLS may be happened easily.This is first time to find the receptor of suilysin, and the signal pathway was studied.Suilysin can directly activate MAPK p38in PBMCs, which didn’t found inmacrophages. P38MAPK specific inhibitor showed different role in different celltypes, so p38can be used as an important target for prevention and treatment in China.Our studies indicate that the inflammatory activity of suilysin is independent on itshemolytic activity, so P353V can be used as protective antigen.