猪溶素诱导宿主产生炎症因子的分子机制研究
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摘要
猪链球菌可以引起猪脑膜炎，是世界公认可引起动物传染病的重要病原体。作为一种人畜共患病病原菌，猪链球菌感染人类的散发病例（主要是脑膜炎）在过去的四十五年已有报道。在2005年，中国报道超过200人感染猪链球菌，病死率高达20%。死亡病例表现为罕见的中毒性休克样综合征（TSLS），而且这类病人血清中细胞因子（IL-1β，IL-6， IL-8， IL-12p70， IFN-γ和TNF-α）水平明显高于临床表现为单纯脑膜炎的病人。这些细胞因子的过量释放可能是引起TSLS的重要原因。
众多国外学者发现，欧洲的猪链球菌致病株31533经过热灭活性后，可以激活人类单核细胞系THP-1以及小鼠巨噬细胞系J774A1和P388D1，释放大量的细胞因子。细菌的细胞壁成分及其毒力因子猪溶素（SLY）可以激活内皮细胞释放细胞因子。纯化的重组SLY蛋白可以刺激人单核细胞释放TNF-α，也可以刺激猪PAMs释放IL-6。
在中国引起暴发流行的猪链球菌为ST7型，与欧洲流行株ST1型相比，ST7型对人外周血单个核细胞（PBMC）的毒性较强；而且SLY蛋白在我国的ST7型菌株中表达量高于ST1型，是重要的毒力因子。本研究通过毒力基因筛选，发现SLY是中国流行株中引起宿主产生炎症因子的重要毒力蛋白。SLY由497个氨基酸残基组成，归属于依赖胆固醇的细胞溶素（CDC）家族；CDC家族目前具有23个成员，包括产气荚膜梭菌释放的外毒素（PFO）,以及化脓性链球菌释放的外毒素（SLO）。CDC家族所有成员都是由革兰氏阳性菌释放的一种外毒素，他们共同的特征是可以在富含胆固醇的细胞膜上打孔从而引起细胞通透性改变或者导致细胞裂解。据报道，SLY可以在人红细胞表面打孔，形成直径约为7nm的小孔，从而引起红细胞裂解，即我们通常所说的溶血。
以2005年分离的中国猪链球菌致病株05ZY为模板，本室构建了多株毒力基因缺失突变株。本研究首先通过构建细胞模型，对缺失突变株进行筛选，实验结果表明野生菌株05ZY可以诱导内皮细胞产生炎症因子，sly基因敲除后，不能诱导内皮细胞产生炎症因子。紧接着，对其编码的蛋白进行纯化和鉴定；深入研究了SLY蛋白诱导细胞产生炎症因子的机制。
随后，选用了PBMC、THP-1MΦ、RAW264.7和小鼠腹腔分离的原代巨噬细胞为细胞模型，对SLY的作用进行深入研究。实验结果表明PBMC对SLY蛋白敏感性强于巨噬细胞，具体表现为低浓度的SLY即可诱导PBMC产生高浓度TNF-α。由于细胞碎片也可以诱导细胞产生炎症因子，本研究还构建了重组SLY蛋白（rSLY）以及完全缺失溶血活性的点突变蛋白（P353V），进一步研究SLY的作用是否依赖其溶血活性。实验结果表明SLY的作用不依赖其溶血活性，很可能是被宿主重要模式识别受体识别，从而导致炎症因子的释放。
国内外学者通过体内和体外实验，均证明了SLY可以诱导宿主产生炎症因子，但是却没有进一步的深入研究其机制。作者通过文献调研，发现同家族的PLY(肺炎链球菌分泌的外毒素)诱导巨噬细胞产生炎症因子通过TLR4受体发挥作用。SLY是否与PLY有相似的作用值得进一步研究。本研究应用了多种受体抑制剂，结果发现TLR4特异性抑制剂可以完全抑制SLY对多种细胞的作用，推测SLY的作用很可能依赖于TLR4受体。紧接着，本研究引入了TLR4点突变小鼠，结果显示，SLY诱导细胞产生炎症因子依赖于TLR4受体。
随后，应用MyD88、TRIF以及MAPKs特异性抑制剂来分析SLY-TLR4的下游信号通路。结果显示，MyD88是SLY发挥作用的重要胞内接头蛋白分子。而p38-MAPK抑制剂可以完全抑制SLY对PBMCs的作用。应用western blot检测技术来分析SLY对细胞MAPKs的直接激活情况，结果表明SLY在短时间内即可直接激活PBMCs细胞内的p38-MAPK分子。最后利用荧光素报告基因检测技术来研究SLY对转录因子的激活作用，结果表明SLY可以激活转录因子NF-κB。NF-κB激活后从胞浆内进入细胞核，从而调控炎症因子的表达。
虽然有报道称热灭活的欧洲菌株31533可以激活单核巨噬细胞释放多种炎症因子。但是细菌的浓度必须达到109cfu/ml才可以诱导细胞产生高水平的炎症因子，当细菌浓度降至108cfu/ml时，TNF的水平很快降落下来。TNF是最早被检测的细胞因子，其动力学分析显示，其水平可于5小时内迅速降至基线水平。而国外学者从31533提取的细胞壁成分，浓度高达150ug/ml才可以诱导THP-1MΦ细胞产生TNF-α，本研究应用SLY浓度为100ng/ml时激活THP-1MΦ产生TNF-α水平与其相当。值得一提的是，12.5ng/ml的SLY蛋白即可诱导PBMCs产生大量TNF-α，并且此效应可以持续长达36小时。
本研究中所用的细胞模型包括单核巨噬细胞（THP-1MΦ，RAW264.7，小鼠来源的原代腹腔巨噬细胞）和PBMC。结果显示PBMC对SLY的敏感性明显高于巨噬细胞。巨噬细胞主要位于不同组织中，属于人体第一道防线的免疫细胞，此部位发生炎症反应通过炎症因子的释放，可以帮助机体有效清除病原菌，将感染控制在局部。但是，细菌一旦进入血液中将会是致命的，因为mol/L的SLY蛋白短时间内即可诱导PBMC产生大量TNF-α，这种全身性释放TNF-α可以引起休克。而在中国的猪链球菌病人血中可以轻易分离出该菌。所以SLY蛋白与PBMC的相互作用应当引起足够的重视。
由于LPS可通过TLR4受体激活免疫细胞，释放炎症因子。因此本研究运用多种手段排除内毒素LPS的影响。第一，基于LPS的热稳定性，作者对天然SLY蛋白进行加热处理，再与细胞共孵育；第二，基于多粘菌素B可以使LPS失去诱导细胞产生炎症因子的作用，作者将蛋白与多粘菌素B在4℃提前作用1小时，再与细胞共孵育；第三，本研究运用Ttritoon X-114抽提去除重组蛋白中的LPS，使内毒素的含量符合国际标准（＜5EU/mg）；最后，应用无关蛋白做对照，无关蛋白是猪链球菌的细胞壁蛋白，其诱导表达纯化与去除LPS过程与rSLY和P353V均一致。经过以上处理后，实验结果均证明，SLY可以诱导细胞产生炎症因子。
本研究首次揭示出SLY蛋白诱导细胞产生炎症因子不依赖其溶血活性，并且此作用通过TLR4受体介导。S.suis与宿主相互作用具有双刃剑的作用。S.suis分泌的毒力蛋白SLY具有溶血活性可以对真核细胞造成直接损伤，而SLY诱导细胞产生炎症因子的作用又可以激活机体的免疫反应，从而帮助宿主清除病原菌。但是若S.suis分泌的SLY蛋白激活真核细胞过度释放炎症因子，则宿主容易发生TSLS。
SLY蛋白诱导细胞产生炎症因子的细胞受体和胞内信号通路首次被揭示。p38-MAPK特异性抑制剂在不同细胞类型表现迥异，SLY可以直接激活PBMC中p38-MAPK。TLR4和p38-MAPK可以作为预防或治疗中国猪链球菌病的重要靶点。由于SLY诱导细胞产生炎症因子的作用不依赖其溶血活性，因此P353V可以作为保护性抗原进行深入研究。
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.
众多国外学者发现，欧洲的猪链球菌致病株31533经过热灭活性后，可以激活人类单核细胞系THP-1以及小鼠巨噬细胞系J774A1和P388D1，释放大量的细胞因子。细菌的细胞壁成分及其毒力因子猪溶素（SLY）可以激活内皮细胞释放细胞因子。纯化的重组SLY蛋白可以刺激人单核细胞释放TNF-α，也可以刺激猪PAMs释放IL-6。
在中国引起暴发流行的猪链球菌为ST7型，与欧洲流行株ST1型相比，ST7型对人外周血单个核细胞（PBMC）的毒性较强；而且SLY蛋白在我国的ST7型菌株中表达量高于ST1型，是重要的毒力因子。本研究通过毒力基因筛选，发现SLY是中国流行株中引起宿主产生炎症因子的重要毒力蛋白。SLY由497个氨基酸残基组成，归属于依赖胆固醇的细胞溶素（CDC）家族；CDC家族目前具有23个成员，包括产气荚膜梭菌释放的外毒素（PFO）,以及化脓性链球菌释放的外毒素（SLO）。CDC家族所有成员都是由革兰氏阳性菌释放的一种外毒素，他们共同的特征是可以在富含胆固醇的细胞膜上打孔从而引起细胞通透性改变或者导致细胞裂解。据报道，SLY可以在人红细胞表面打孔，形成直径约为7nm的小孔，从而引起红细胞裂解，即我们通常所说的溶血。
以2005年分离的中国猪链球菌致病株05ZY为模板，本室构建了多株毒力基因缺失突变株。本研究首先通过构建细胞模型，对缺失突变株进行筛选，实验结果表明野生菌株05ZY可以诱导内皮细胞产生炎症因子，sly基因敲除后，不能诱导内皮细胞产生炎症因子。紧接着，对其编码的蛋白进行纯化和鉴定；深入研究了SLY蛋白诱导细胞产生炎症因子的机制。
随后，选用了PBMC、THP-1MΦ、RAW264.7和小鼠腹腔分离的原代巨噬细胞为细胞模型，对SLY的作用进行深入研究。实验结果表明PBMC对SLY蛋白敏感性强于巨噬细胞，具体表现为低浓度的SLY即可诱导PBMC产生高浓度TNF-α。由于细胞碎片也可以诱导细胞产生炎症因子，本研究还构建了重组SLY蛋白（rSLY）以及完全缺失溶血活性的点突变蛋白（P353V），进一步研究SLY的作用是否依赖其溶血活性。实验结果表明SLY的作用不依赖其溶血活性，很可能是被宿主重要模式识别受体识别，从而导致炎症因子的释放。
国内外学者通过体内和体外实验，均证明了SLY可以诱导宿主产生炎症因子，但是却没有进一步的深入研究其机制。作者通过文献调研，发现同家族的PLY(肺炎链球菌分泌的外毒素)诱导巨噬细胞产生炎症因子通过TLR4受体发挥作用。SLY是否与PLY有相似的作用值得进一步研究。本研究应用了多种受体抑制剂，结果发现TLR4特异性抑制剂可以完全抑制SLY对多种细胞的作用，推测SLY的作用很可能依赖于TLR4受体。紧接着，本研究引入了TLR4点突变小鼠，结果显示，SLY诱导细胞产生炎症因子依赖于TLR4受体。
随后，应用MyD88、TRIF以及MAPKs特异性抑制剂来分析SLY-TLR4的下游信号通路。结果显示，MyD88是SLY发挥作用的重要胞内接头蛋白分子。而p38-MAPK抑制剂可以完全抑制SLY对PBMCs的作用。应用western blot检测技术来分析SLY对细胞MAPKs的直接激活情况，结果表明SLY在短时间内即可直接激活PBMCs细胞内的p38-MAPK分子。最后利用荧光素报告基因检测技术来研究SLY对转录因子的激活作用，结果表明SLY可以激活转录因子NF-κB。NF-κB激活后从胞浆内进入细胞核，从而调控炎症因子的表达。
虽然有报道称热灭活的欧洲菌株31533可以激活单核巨噬细胞释放多种炎症因子。但是细菌的浓度必须达到109cfu/ml才可以诱导细胞产生高水平的炎症因子，当细菌浓度降至108cfu/ml时，TNF的水平很快降落下来。TNF是最早被检测的细胞因子，其动力学分析显示，其水平可于5小时内迅速降至基线水平。而国外学者从31533提取的细胞壁成分，浓度高达150ug/ml才可以诱导THP-1MΦ细胞产生TNF-α，本研究应用SLY浓度为100ng/ml时激活THP-1MΦ产生TNF-α水平与其相当。值得一提的是，12.5ng/ml的SLY蛋白即可诱导PBMCs产生大量TNF-α，并且此效应可以持续长达36小时。
本研究中所用的细胞模型包括单核巨噬细胞（THP-1MΦ，RAW264.7，小鼠来源的原代腹腔巨噬细胞）和PBMC。结果显示PBMC对SLY的敏感性明显高于巨噬细胞。巨噬细胞主要位于不同组织中，属于人体第一道防线的免疫细胞，此部位发生炎症反应通过炎症因子的释放，可以帮助机体有效清除病原菌，将感染控制在局部。但是，细菌一旦进入血液中将会是致命的，因为mol/L的SLY蛋白短时间内即可诱导PBMC产生大量TNF-α，这种全身性释放TNF-α可以引起休克。而在中国的猪链球菌病人血中可以轻易分离出该菌。所以SLY蛋白与PBMC的相互作用应当引起足够的重视。
由于LPS可通过TLR4受体激活免疫细胞，释放炎症因子。因此本研究运用多种手段排除内毒素LPS的影响。第一，基于LPS的热稳定性，作者对天然SLY蛋白进行加热处理，再与细胞共孵育；第二，基于多粘菌素B可以使LPS失去诱导细胞产生炎症因子的作用，作者将蛋白与多粘菌素B在4℃提前作用1小时，再与细胞共孵育；第三，本研究运用Ttritoon X-114抽提去除重组蛋白中的LPS，使内毒素的含量符合国际标准（＜5EU/mg）；最后，应用无关蛋白做对照，无关蛋白是猪链球菌的细胞壁蛋白，其诱导表达纯化与去除LPS过程与rSLY和P353V均一致。经过以上处理后，实验结果均证明，SLY可以诱导细胞产生炎症因子。
本研究首次揭示出SLY蛋白诱导细胞产生炎症因子不依赖其溶血活性，并且此作用通过TLR4受体介导。S.suis与宿主相互作用具有双刃剑的作用。S.suis分泌的毒力蛋白SLY具有溶血活性可以对真核细胞造成直接损伤，而SLY诱导细胞产生炎症因子的作用又可以激活机体的免疫反应，从而帮助宿主清除病原菌。但是若S.suis分泌的SLY蛋白激活真核细胞过度释放炎症因子，则宿主容易发生TSLS。
SLY蛋白诱导细胞产生炎症因子的细胞受体和胞内信号通路首次被揭示。p38-MAPK特异性抑制剂在不同细胞类型表现迥异，SLY可以直接激活PBMC中p38-MAPK。TLR4和p38-MAPK可以作为预防或治疗中国猪链球菌病的重要靶点。由于SLY诱导细胞产生炎症因子的作用不依赖其溶血活性，因此P353V可以作为保护性抗原进行深入研究。
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.
引文
[1] Ye C, Jing H, Du H, Segura M, Zheng H, Kan B, Wang L, Bai X, Zhou Y, Cui Z,Zhang S, Jin D, Sun N, Luo X, Zhang J, Gong Z, Wang X, Wang L, Sun H, Li Z, SunQ, Liu H, Dong B, Ke C, Yuan H, Wang H, Tian K, Wang Y, Gottschalk M, Xu J.Streptococcus suis sequence type7outbreak, Sichuan, China. Emerg Infect Dis. Vol.(2006)12:1203-1208.
[2]Changyun Ye, Ji Zhang, Huaiqi Jing,Lei Wang,a Yanwen Xiong,Wei Wang,ZheminZhou, Qiangzheng Sun, Xia Luo, Huamao Du, Marcelo Gottschalk, and Jianguo Xu.Clinical, Experimental, and Genomic Differences between Intermediately Pathogenic,Highly Pathogenic, and Epidemic Streptococcus suis. The Journal of InfectiousDiseases(2009)199:97–107.
[3]Richard Graveline MS, Danuta Radzioch and Marcelo GottschalkTLR2-dependent recognition of Streptococcus suis is modulated by the presence ofcapsular polysaccharide which modifies macrophage responsiveness. Internationalimmunology (2007)Vol.19:375–389.
[4]M. SEGURA NVMG CD14-dependent and-independent cytokine andchemokine production by human THP-1monocytes stimulated by Streptococcus suiscapsular type2. Clinical and experimental immunology(2002)127:243–254.
[5]Shichun Lun JP-C, Wayne Connor, P.J. Willson Role of suilysin in pathogenesisof Streptococcus suis capsular serotype2. Microbial Pathogenesis (2003)34:27–37.
[6]Akira OTaS Pattern Recognition Receptors and Inflammation. Cell(2010)140,:805–820.
[7]Shizuo Akira SU, and Osamu Takeuchi Pathogen Recognition and InnateImmunity. Cell biology international (2006)124,:783–801.
[8]Akira S&Takeda K Toll-like receptor signalling. Nature reviews. Immunology(2004)4(7):499-511.
[9]Gottschalk M SM, Xu J Streptococcus suis infections in humans: the Chineseexperience and the situation in North America. Anim Health Res Rev (2007)8:29–45.
[10]Jacobs AA, Loeffen PL, van den Berg AJ,&Storm PK Identification,purification, and characterization of a thiol-activated hemolysin (suilysin) ofStreptococcus suis. Infection and immunity (1994)62(5):1742-1748.
[11]Gottschalk MG, Lacouture S,&Dubreuil JD Characterization of Streptococcussuis capsular type2haemolysin. Microbiology(1995)141(Pt1):189-195.
[12]Richard Malley PH, Sarah C. Morse, Michael J. Cieslewicz, MarcLipsitch,Claudette M. Thompson, Evelyn Kurt-Jones, James C. Paton, Michael R.Wessels, and Douglas T. Golenbock Recognition of pneumolysin by Toll-likereceptor4confers resistance to pneumococcal infection. Proc. Natl. Acad. Sci.USA(2003)100,:1966-1971.
[13]Chaitali Basak SKP, Asima Bhattacharyya, Shresh Pathak, Joyoti Basu, andManikuntala Kundu The Secreted Peptidyl Prolyl cis,trans-Isomerase HP0175ofHelicobacter pylori Induces Apoptosis of Gastric Epithelial Cells in a TLR4-andApoptosis Signal-Regulating Kinase1-Dependent Manner. The Journal ofImmunology(2005)174,:5672–5680.
[14]Akira TKaS TLR signaling. Cell death and differentiation (2006)13,:816–825.
[15]Yong-Chen Lu a,b, Wen-Chen Yeh a,b, Pamela S. Ohashi LPS/TLR4signaltransduction pathway. Cytokine(2008)42:145–151.
[16]LV Qing yu, HAO Huai jie, BI Li li, ZHENG Yu ling,JIANG Yong qiang, LVShu xia Purification and biological activities a nalysis of Streptococcus suis Serotype2suilysin. Chin J Cell Mol Immunol(2011)27(4):374-376.
[17]Huamao Du LfX, Xiaoling Wang, Xuemei Li, Changyun Ye, Jianguo XuBiological prof iles of recombinant Suilysin. Acta Microbiologica Sinica (2009)49(6):792-798.
[18]SHIGUI LIU RT, SHANNON McCLURE, GARTH STYBA, QINWEI SHI,andGEORGE JACKOWSKI Removal of Endotoxin from Recombinant ProteinPreparations. Clinical Biochemistry(1997)Vol.30, No.6:455–463,.
[19]Henne DG TK, Lowry SF, et al. Cytokine appearance in human endotoxaemiaand primate bacteremia. Surg Gynecol Obstet (1988)i66:147-153.
[20]Tracey KJ LS, Fahey TJ Ill, etal. Cachectin/tumour necrosis factor induces lethalshock and stress hormone responses in the dog.Surg Gynecol Obstet (1987)164:415-422.
[21]Evans Cytokines in Disease. CLIN.CHEM(1990)36/7;:1269-1281.
[22]Lingfeng Xu BH, Huamao Du, Xuejun C. Zhang, Jianguo Xu, Xuemei Li, ZiheRao. Crystal structure of cytotoxin protein suilysin from Streptococcus suis. Protein&Cell(2010)1(1):96–105.
[23]Giddings KS ZJ, Sims PJ, Tweten RK. Human CD59is a receptor for thecholesterol-dependent cytolysin intermedilysin. NAT Struct Mol Biol(2004)11,:1173~1178.
[24]Han J LJ-D, Bibbs L, Ulevitch RJ. A MAP kinase targeted by endotoxin andhyperosmolarity in mammalian cells. Science(1994)265,:808.
[25]Hambleton J WS, Lem L, DeFranco AL.Activation of c-Jun N-terminal kinase inbacterial lipopolysaccharide-stimulated macrophages. Proc Natl Acad Sci USA(1996)93,:2774.
[26]Wei Zhu JSD, Yingqiu Li, Jiahuai Han Co-ordinated regulation of TNFexpression by multiple MAP kinase pathways. Journal of Endotoxin Research,(1999)Vol.5, No.4:239-243.
[27]Masayuki Ii NM, Kaoru Hazeki, Kazuyo Nakamura, KatsunoriTakashima,Tsukasa Seya, Osamu Hazeki, Tomoyuki Kitazaki, and Yuji IizawaAnovelcyclohexenederivative,Ethyl(6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate(TAK-242), Selectively Inhibits Toll-Like Receptor4-Mediated Cytokine Production through Suppression of Intracellular Signaling. MolPharmacol(2006)69:1288–1295.
[28]COOPERSTOCK' MS Inactivation of Endotoxin by Polymyxin B.ANTMMICROUAL AGzmNs mAD CHMOnrPY (1974)Vol.6, No.4:422-425.
[29]MARIELA SEGURA JS, AND MARCELO GOTTSCHALK Heat-KilledStreptococcus suis Capsular Type2Strains Stimulate Tumor Necrosis Factor Alphaand Interleukin-6Production by Murine Macrophages. INFECTION ANDIMMUNITY,(1999)Vol.67, No.9:4646–4654.
[30]D. AL-NUMANI MS, M. DORé&M. GOTTSCHALK Up-regulation ofICAM-1, CD11a/CD18and CD11c/CD18on human THP-1monocytes stimulated byStreptococcus suis serotype2. Clinical and experimental immunology(2003)l133:67–77.
[31]Shin-Ichi Tanabe LB, Nahuel Fittipaldi, Louis Grignon, Marcelo Gottschalk,Daniel Grenier Pleiotropic effects of polysaccharide capsule loss on selectedbiological properties of Streptococcus suis. The Canadian Journal of VeterinaryResearch(2009)73:65–70
[32]Judith Branger SK, Sebastiaan Weijer,Jaklien C. Leemans,Jennie M. Pater,PeterSpeelman,Sandrine Florquin,and Tom van der Poll Role of Toll-Like Receptor4inGram-Positive and Gram-Negative Pneumonia in Mice. INFECTION ANDIMMUNITY,(2004) Vol.72, No.2:788–794.
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[2]Changyun Ye, Ji Zhang, Huaiqi Jing,Lei Wang,a Yanwen Xiong,Wei Wang,ZheminZhou, Qiangzheng Sun, Xia Luo, Huamao Du, Marcelo Gottschalk, and Jianguo Xu.Clinical, Experimental, and Genomic Differences between Intermediately Pathogenic,Highly Pathogenic, and Epidemic Streptococcus suis. The Journal of InfectiousDiseases(2009)199:97–107.
[3]Richard Graveline MS, Danuta Radzioch and Marcelo GottschalkTLR2-dependent recognition of Streptococcus suis is modulated by the presence ofcapsular polysaccharide which modifies macrophage responsiveness. Internationalimmunology (2007)Vol.19:375–389.
[4]M. SEGURA NVMG CD14-dependent and-independent cytokine andchemokine production by human THP-1monocytes stimulated by Streptococcus suiscapsular type2. Clinical and experimental immunology(2002)127:243–254.
[5]Shichun Lun JP-C, Wayne Connor, P.J. Willson Role of suilysin in pathogenesisof Streptococcus suis capsular serotype2. Microbial Pathogenesis (2003)34:27–37.
[6]Akira OTaS Pattern Recognition Receptors and Inflammation. Cell(2010)140,:805–820.
[7]Shizuo Akira SU, and Osamu Takeuchi Pathogen Recognition and InnateImmunity. Cell biology international (2006)124,:783–801.
[8]Akira S&Takeda K Toll-like receptor signalling. Nature reviews. Immunology(2004)4(7):499-511.
[9]Gottschalk M SM, Xu J Streptococcus suis infections in humans: the Chineseexperience and the situation in North America. Anim Health Res Rev (2007)8:29–45.
[10]Jacobs AA, Loeffen PL, van den Berg AJ,&Storm PK Identification,purification, and characterization of a thiol-activated hemolysin (suilysin) ofStreptococcus suis. Infection and immunity (1994)62(5):1742-1748.
[11]Gottschalk MG, Lacouture S,&Dubreuil JD Characterization of Streptococcussuis capsular type2haemolysin. Microbiology(1995)141(Pt1):189-195.
[12]Richard Malley PH, Sarah C. Morse, Michael J. Cieslewicz, MarcLipsitch,Claudette M. Thompson, Evelyn Kurt-Jones, James C. Paton, Michael R.Wessels, and Douglas T. Golenbock Recognition of pneumolysin by Toll-likereceptor4confers resistance to pneumococcal infection. Proc. Natl. Acad. Sci.USA(2003)100,:1966-1971.
[13]Chaitali Basak SKP, Asima Bhattacharyya, Shresh Pathak, Joyoti Basu, andManikuntala Kundu The Secreted Peptidyl Prolyl cis,trans-Isomerase HP0175ofHelicobacter pylori Induces Apoptosis of Gastric Epithelial Cells in a TLR4-andApoptosis Signal-Regulating Kinase1-Dependent Manner. The Journal ofImmunology(2005)174,:5672–5680.
[14]Akira TKaS TLR signaling. Cell death and differentiation (2006)13,:816–825.
[15]Yong-Chen Lu a,b, Wen-Chen Yeh a,b, Pamela S. Ohashi LPS/TLR4signaltransduction pathway. Cytokine(2008)42:145–151.
[16]LV Qing yu, HAO Huai jie, BI Li li, ZHENG Yu ling,JIANG Yong qiang, LVShu xia Purification and biological activities a nalysis of Streptococcus suis Serotype2suilysin. Chin J Cell Mol Immunol(2011)27(4):374-376.
[17]Huamao Du LfX, Xiaoling Wang, Xuemei Li, Changyun Ye, Jianguo XuBiological prof iles of recombinant Suilysin. Acta Microbiologica Sinica (2009)49(6):792-798.
[18]SHIGUI LIU RT, SHANNON McCLURE, GARTH STYBA, QINWEI SHI,andGEORGE JACKOWSKI Removal of Endotoxin from Recombinant ProteinPreparations. Clinical Biochemistry(1997)Vol.30, No.6:455–463,.
[19]Henne DG TK, Lowry SF, et al. Cytokine appearance in human endotoxaemiaand primate bacteremia. Surg Gynecol Obstet (1988)i66:147-153.
[20]Tracey KJ LS, Fahey TJ Ill, etal. Cachectin/tumour necrosis factor induces lethalshock and stress hormone responses in the dog.Surg Gynecol Obstet (1987)164:415-422.
[21]Evans Cytokines in Disease. CLIN.CHEM(1990)36/7;:1269-1281.
[22]Lingfeng Xu BH, Huamao Du, Xuejun C. Zhang, Jianguo Xu, Xuemei Li, ZiheRao. Crystal structure of cytotoxin protein suilysin from Streptococcus suis. Protein&Cell(2010)1(1):96–105.
[23]Giddings KS ZJ, Sims PJ, Tweten RK. Human CD59is a receptor for thecholesterol-dependent cytolysin intermedilysin. NAT Struct Mol Biol(2004)11,:1173~1178.
[24]Han J LJ-D, Bibbs L, Ulevitch RJ. A MAP kinase targeted by endotoxin andhyperosmolarity in mammalian cells. Science(1994)265,:808.
[25]Hambleton J WS, Lem L, DeFranco AL.Activation of c-Jun N-terminal kinase inbacterial lipopolysaccharide-stimulated macrophages. Proc Natl Acad Sci USA(1996)93,:2774.
[26]Wei Zhu JSD, Yingqiu Li, Jiahuai Han Co-ordinated regulation of TNFexpression by multiple MAP kinase pathways. Journal of Endotoxin Research,(1999)Vol.5, No.4:239-243.
[27]Masayuki Ii NM, Kaoru Hazeki, Kazuyo Nakamura, KatsunoriTakashima,Tsukasa Seya, Osamu Hazeki, Tomoyuki Kitazaki, and Yuji IizawaAnovelcyclohexenederivative,Ethyl(6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate(TAK-242), Selectively Inhibits Toll-Like Receptor4-Mediated Cytokine Production through Suppression of Intracellular Signaling. MolPharmacol(2006)69:1288–1295.
[28]COOPERSTOCK' MS Inactivation of Endotoxin by Polymyxin B.ANTMMICROUAL AGzmNs mAD CHMOnrPY (1974)Vol.6, No.4:422-425.
[29]MARIELA SEGURA JS, AND MARCELO GOTTSCHALK Heat-KilledStreptococcus suis Capsular Type2Strains Stimulate Tumor Necrosis Factor Alphaand Interleukin-6Production by Murine Macrophages. INFECTION ANDIMMUNITY,(1999)Vol.67, No.9:4646–4654.
[30]D. AL-NUMANI MS, M. DORé&M. GOTTSCHALK Up-regulation ofICAM-1, CD11a/CD18and CD11c/CD18on human THP-1monocytes stimulated byStreptococcus suis serotype2. Clinical and experimental immunology(2003)l133:67–77.
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