Temperature-Induced Unfolding Pathway of Staphylococcal Enterotoxin B:Insights from Circular Dichroism and Molecular Dynamics Simulation

In this study,circular dichroism(CD)and molecular dynamics(MD)simulation were used to investigate the thermal unfolding pathway of staphylococcal enterotoxin B(SEB)at temperatures of 298–371 and 298–500 K,and the relationship between the experimental and simulation results were explored.Our computational findings on the secondary structure of SEB showed that at room temperature,the CD spectroscopic results were highly consistent with the MD results.Moreover,under heating conditions,the changing trends of helix,sheet and random coil obtained by CD spectral fitting were highly consistent with those obtained by MD.In order to gain a deeper understanding of the thermal stability mechanism of SEB,the MD trajectories were analyzed in terms of root mean square deviation(RMSD),secondary structure assignment(SSA),radius of gyration(R_(g)),free energy surfaces(FES),solvent-accessible surface area(SASA),hydrogen bonds and salt bridges.The results showed that at low heating temperature,domain Ⅰ without loops(omitting the mobile loop region)mainly relied on hydrophobic interaction to maintain its thermal stability,whereas the thermal stability of domain Ⅱ was mainly controlled by salt bridges and hydrogen bonds.Under high heating temperature conditions,the hydrophobic interactions in domain Ⅰ without loops were destroyed and the secondary structure was almost completely lost,while domain Ⅱ could still rely on salt bridges as molecular staples to barely maintain the stability of the secondary structure.These results help us to understand the thermodynamic and kinetic mechanisms that maintain the thermal stability of SEB at the molecular level,and provide a direction for establishing safer and more effective food sterilization processes.

Assessment of the inhibitory effects of sodium nitrite, nisin, potassium sorbate, and sodium lactate on Staphylococcus aureus growth and staphylococcal enterotoxin A production in cooked pork sausage using a predictive growth model

This study was conducted to analyze the effects of sodium nitrite,nisin,potassium sorbate,and sodium lactate against Staphylococcus aureus(S.aureus)growth and staphylococcal enterotoxins(SEs)production in cooked pork sausage by inoculating sausage samples containing preservative with an S.aureus strain producing staphylococcal enterotoxin A(SEA)and then storing them at 37℃ for 36 h.Samples were analyzed every 3 h to count the S.aureus colonies and to detect SEA.The modified Gompertz model was used to describe S.aureus growth in the samples under various conditions,and the preservatives with a significant antimicrobial effect were selected.In addition,the antimicrobial effects of the selected preservatives under various concentrations were tested.Results showed that sodium nitrite,nisin,and potassium sorbate had a weak effect against S.aureus growth and had no effect against SEA production,whereas sodium lactate could significantly inhibit S.aureus growth and SEA production.Moreover,the antimicrobial effect of sodium lactate was concentration-dependent,wherein sodium lactate concentration<12 g/kg showed no inhibitory effect,but when the concentration was increased to 24 g/kg,sodium lactate could effectively inhibit S.aureus growth and SEA production,and at 48 g/kg,sodium lactate had a significant inhibitory effect.

Effect and Mechanism of Superantigen Staphylococcal Enterotoxin Therapy for Mouse Gastric Tumor

The anti-tumor effect and mechanism of the staphylococcal enterotoxin A (SEA) were studied. The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumor ceils (MGC80-3). The experimental group was treated with SEA, and the control group was treated with normal saline. The percentage of tumor generation and tumor mass was measured. The results showed that the percentage of the tumor generation in the SEA-treated mice was lower than in the control group, but there was no significant difference (P>0. 05). However, the tumor mass in the experimental group was significantly lighter than in the control group, with the difference being very significant (P<0. 001). There were more CD4+ T cells and CD8+ T cells in the tumor of the mice treated with SEA than those of the control group. SEA has an obvious anti-tumor effect on mice gastric tumor. The mechanism might be that SEA induces the effect of superantigen-dependent cell mediated cytotoxicity to the tumor cells.

THE EFFECT OF SUPERANTIGEN STAPHYLOCOCCALENTEROTOXIN B AND D-GALACTOSAMINE ON BALB/CMOUSE HEPATOCYTES

Objective To observe the role of superantigen staphylococcal enterotoxin B(SEB) andD - galactosamine (D - GalN) on Balb/c mouse hepatocytes and its mechanism. Methods After Balb/c mice wereinjected intraperitoneally with SEB, D- GalN or both, blood samples were collected and livers were removed at 2,6, 12, 24h. Patterns of hepatocellular death were studied morphologically and biochemically, circulating cytokines(TNF, IFN-γ) were determined, and mice mortality within 24h was assessed. Results SEB could induce thetypical apoptotic changes of hepatocytes morphologically and biochemically. The mechanism is probably associatedwith the production and release of Cytokines (such as TNF, IFN- γ, etc).D - GalN could induce hepatocytesapoptosis and degeneration at the same time. Besides this, we confirmed hepatocytes of the mice which wereadministered SEB and D - GalN developing apoptosis at 2, 6h, but after 12h hepatocytes were characterized bysevere injury, the mice mortality within 24h is 50%. Conclusion SEB or D - GalN alone could induce the typicalapoptotic changes of hepatocytes. SEB+D-GalN developed hepatocytes apoptosis in the early stage and necrosisin the later. It suggests that there is some relationship between hepatic cell apoptosis and necrosis, and massivehepatocyte apoptosis is the probably initiating step of acute hepatic necrosis in mice.

The Akt Pathway Inhibitor Degeulin Prevents Staphylococcal Enterotoxin B Induced Splenocyte Proliferation and Inflammation

Staphylococcal Enterotoxin B (SEB) is considered a potential biological weapon. It is toxic by both inhalation and ingestion. Effects of ingestion include fever, vomiting and diarrhoea, while inhalation may additionally result in chest pain, dyspnoea, pulmonary oedema and respiratory failure. Severe exposure may be fatal and treatment relies on symptomatic support. At a cellular level, SEB up-regulates T-cell proliferation leading to a pathological inflammatory response. Deguelin, a rotenoid isolated from the African plant Mundulea sericea (Leguminosae), has been shown to reduce cellular proliferation by inhibiting the phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathway. Using isolated murine splenocytes, we have demonstrated that treatment with deguelin reduces SEB inducing T cell proliferation by 60%. Deguelin treatment also decreased IL-2 and CCL2 secretion by splenocytes exposed to SEB. We demonstrate that targeting cellular proliferation can significantly reduce inflammation after SEB exposure and suggest that anti-proliferatives may have a role as potential generic medical counter measures if superantigens are used as biological weapons.

Predictive Modeling for Growth and Enterotoxin Production of Staphylococcus aureus in Milk

Predictive microbiology was utilized to model Staphylococcus aureus （S. aureus） growth and staphylococcal enterotoxin A （SEA） production in milk in this study. The modifed logistic model, modifed Gompertz model and Baranyi model were applied to model growth data of S. aureus between 15℃ and 37℃. Model comparisons indicated that Baranyi model described the growth data more accurately than two others with a mean square error of 0.0129. Growth rates generated from Baranyi model matched the observed ones with a bias factor of 0.999 and an accuracy factor of 1.01, and ft a square root model with respect to temperature; other two modifed models both overestimated the observed ones. SEA amount began to be detected when the cell number reached106.4 cfu ？ mL-1, and showed the linear correlation with time. Besides, the rate of SEA production ftted an exponential relationship as a function of temperature. Predictions based on the study could be applied to indicate possible growth of S. aureus and prevent the occurrence of staphylococcal food poisoning.

A non-viral gene therapy for melanoma by staphylococcal enterotoxin A

Staphylococcal enterotoxin A(SEA)derived from Staphylococcus aureus,as a superantigen,shows potential for cancer immunotherapy,but systemic immunotoxicity restricts its clinical application.Targeted delivery of SEA to tumor site provides a promising option for reducing the systemic toxicity.Here,we constructed an iRGD peptide(H-[Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys]-NH_(2))modified nanoparticle(iDPP)to deliver plasmids encoding SEA for melanoma treatment.The iDPP/SEA nanocomplexes efficiently mediated SEA expression in B16-F10 cells in vivo and in vitro and induced the activation of lymphocytes and maturation of murine bone marrow-derived dendritic cells(BMDCs)in vitro.In the subcutaneous B16-F10 melanoma model,the iDPP/SEA nanocomplexes could effectively enhance immune response and T lymphocytes infiltration in tumor site after intravenous administration,thereby considerably decreased melanoma growth.Meanwhile,no obvious adverse effect was observed after intravenous administration of the iDPP/SEA nanocomplexes in vivo.Our findings demonstrated that gene therapy of SEA is a potential candidate for melanoma treatment.

基于荧光生物传感技术检测牛奶中金黄色葡萄球菌肠毒素B

目的利用纳米金粒子(gold nanoparticles,AuNPs)荧光猝灭性能和核酸适配体的高亲和力,构建一种简便、灵敏的纳米金“Turn-on”型荧光生物传感方法检测牛奶中金黄色葡萄球菌肠毒素B(staphylococcal enterotoxins B,SEB)的方法。方法以AuNPs作为荧光体的能量受体(猝灭剂),荧光素-单链DNA(fluorescein-ssDNA,FAM-ssDNA)作为荧光能量供体,冷冻法制备AuNPs-SEB适配体复合物,基于AuNPsSEB适配体复合物/SEB/FAM-ssDNA的竞争性结合,构建纳米金“Turn-on”型荧光生物传感检测方法。对缓冲体系pH和反应时间等条件进行优化,以牛奶为代表对方法检测性能进行验证。结果在优化好的实验条件(pH 7.5、反应时间15 min和反应温度25℃)下,在10^(-1)~10^(4)ng/mL范围内,荧光强度与SEB质量浓度之间呈现良好的线性关系,其相关系数为0.995,检出限为0.062 ng/mL。应用于牛奶样品中SEB的测定,方法回收率为91.2%~108.0%,相对标准偏差在2.6%~5.2%范围之间。结论该纳米金“Turn-on”型荧光生物传感检测技术具有简便、灵敏和准确等优点,可为食品中污染物的检测提供一种可行的新方法。

Inhibition of squamous cancer growth in a mouse model by Staphylococcal enterotoxin B-triggered Th9 cell expansion

Currently, therapy for squamous cancer （SqC） is unsatisfactory. Staphylococcal enterotoxin B （SEB） has strong immune regulatory activity. This study tests the hypothesis that SEB enforces the effect of immunotherapy on SqC growth in a mouse model. C3H/HeN mice and the SqC cell line squamous cell carcinoma VII were used to create an SqC mouse model. Immune cell assessment was performed by flow cytometry. Real-time RT-PCR and western blotting were used to evaluate target molecule expression. An apoptosis assay was used to assess the suppressive effect of T helper-9 （Th9） cells on the SqC cells. The results showed that immunotherapy consisting of SEB plus SqC antigen significantly inhibited SqC growth in the mice. The frequency of Th9 cells was markedly increased in the SqC tissue and mouse spleens after treatment. SEB markedly increased the levels of signal transducer and activator of transcription 5 phosphorylation and the expression of histone deacetylase-1 （HDAC1） and PU.1 （the transcription factor of the interleukin 9 （IL-9） gene） in CD4^＋ T cells. Exposure to SqC-specific Th9 cells markedly induced SqC cell apoptosis both in vitro and in vivo. In conclusion, the administration of SEB induces Th9 cells in SqC-bearing mice, and theseTh9 cells inhibit SqC growth.

食物中毒样品中金黄色葡萄球菌及肠毒素检测

[目的]对3个可疑食物中毒样品进行金黄色葡萄球菌及肠毒素检测。[方法]按照GB/T4789.10-2003进行金黄色葡萄球菌检测,同时设计4对引物,分别扩增金黄色葡萄球菌的耐热核酸酶基因(nuc基因)和相关肠毒素基因(SEA、SEB、SECs),建立一种快速检测金葡菌和肠毒素的方法。[结果]3个样品通过增菌接种,均检出有完全溶血环、G+葡萄球菌,血浆凝固酶阳性的金黄色葡萄球菌;Baird-Parker直接计数菌落结果分别为:9.6×107cfu/g、1.5×105cfu/g、1.7×108cfu/g;PCR检测3个样品均扩增出了金黄色葡萄球菌nuc基因(279bp)和肠毒素A(SEA)基因(288bp)。[结论]3个样品中均检出含有葡萄球菌肠毒素A的金黄色葡萄球菌,是引起该次食物中毒的主要原因。

金黄色葡萄球菌新型肠毒素I双抗夹心-酶联免疫检测方法的建立

目的:建立简便、灵敏检测金黄色葡萄球菌肠毒素I(staphylococcal enterotoxin I,SEI)的双抗夹心酶联免疫吸附方法(double-antibody sandwich-enzyme linked immunosorbent assay,DAS-ELISA)。方法:利用DAS-ELISA检测程序确定单克隆抗体、抗血清、辣根过氧化物酶(horseradish peroxidase,HRP)标记的羊抗兔Ig G(Ig G/HRP)的最佳稀释度,再通过检测不同包被缓冲液、封闭时间、抗原包被时间、Ig G/HRP作用时间以及四甲基联苯胺(3,3’,5,5’-tetramethylbenzidine,TMB)显色时间条件下的OD450 nm值对实验条件进行优化,最后用灵敏度、批内变异、批间变异和加标回收率指标对方法进行评价。结果:抗SEI单克隆抗体的最佳稀释质量浓度为2.89 mg/L,抗SEI兔血清稀释度1∶2 000,酶标二抗稀释度1∶6 000;1×磷酸缓冲盐溶液(p H 7.4)为最佳包被缓冲液;最佳封闭时间、抗原孵育时间、酶标二抗孵育时间和TMB显色时间分别为60、90、30 min和15 min。该方法的回归方程为y=0.040 9x+0.042 9,R2=0.993 3;灵敏度为0.5μg/L,精密度批内变异低于10%,批间变异低于15%,除巴氏杀菌牛乳外,对生理盐水、熟牦牛肉糜、大米饭和超高温瞬时灭菌牛乳回收率达90%以上。结论:本研究建立了一种快速检测SEI的双抗夹心方法。

葡萄球菌肠毒素A脂质体的制备及其体内分布试验

目的 制备葡萄球菌肠毒素 A(staphylococcal enterotoxin A,SEA)脂质体 ,研究其在体内的分布特性 ,为 SEA脂质体治疗肝癌提供依据。方法 用 1 2 5I标记 SEA,逆相蒸发法制备 SEA脂质体 ,考察其包封率及粒径 ,观察 1 2 5I- SEA及 SEA脂质体静脉注射后不同时间小鼠体内的分布。结果 5批 SEA脂质体样品平均粒径为5 0 5± 34 nm,平均包封率为 44 .4%± 4.8%。SEA脂质体主要分布于肝、脾 ,尤以肝脏中分布最多 ,而在血中清除加快 ,在其他组织中分布显著减少 ,与游离 SEA比较具有显著差异 (P<0 .0 5 )。结论 逆相蒸发法制备 SEA脂质体简单、重复性好 ,可获得较高的包封率和符合肝靶向要求的脂质体 ;SEA脂质体具有一定的肝靶向性 。

葡萄球菌肠毒素基因分型PCR检测技术的研究

通过生物信息学分析,设计了不同基因型肠毒素检测引物;应用含溶菌酶的碱裂解法提取葡萄球菌DNA及梯度PCR方法提高了肠毒素检出率和特异性。从国内28株葡萄球菌分离株中共检测出14种不同型的肠毒素基因(sea-see、tsst-1、seg-sei、sek-sel、sen-seo、seq-ser和seu),未检测到sej和sem基因。毒素基因携带率为16.67%,同时携带4种及以上毒素基因的菌株有11株,占39.29%,其中sek、seq和sea毒素基因在所研究菌株中分布最广,分别占到15.50%、14.30%和10.70%。结果表明不同葡萄球菌菌株间毒素型的关联度不同,sek与seq,seb和sed,sec、sei、sen和seu基因型呈现密切相关,各型肠毒素的分布还与菌株分离来源有着密切联系。所建立的PCR方法可用于金黄色葡萄球菌肠毒素基因分型和分布的研究。

超抗原SEA的基因克隆、原核表达与鉴定

目的 构建pRSET SEA重组表达载体 ,转化大肠杆菌BL2 1 (DE3)pLysS ,诱导表达超抗原葡萄球菌肠毒素A(staphylococcalenterotoxinA ,SEA) ,进行分离、纯化及westernblot鉴定。方法 采用PCR技术 ,从产SEA的葡萄球菌标准菌株FRI1 0 0基因组DNA中获得SEA全长序列 ,克隆入pUC1 9中 ,进行测序 ,构建pRSET SEA表达质粒 ,转化大肠杆菌BL2 1 (DE3)pLysS ,通过异丙基硫代 β D 半乳糖苷 (isopropyl beta D thiogalactopyranoside,IPTG)诱导表达 ,分离、纯化及westernblot鉴定。结果 PCR获得超抗原SEA基因片段 ,DNA测序结果与文献报道一致 ;构建了pRSET SEA表达质粒 ,并成功地诱导表达出32 0 0 0u的蛋白 ;Westernblot鉴定所得蛋白能够与SEA单克隆抗体特异性结合。结论 本研究成功地克隆了SEA全长 ,并进行了原核表达和分离、纯化 ,获得了SEA蛋白。

重组葡萄球菌B型肠毒素超抗原的制备及抗肿瘤活性分析

目的 采用基因工程技术制备葡萄球菌B型肠毒素 (SEB) ,并对其体内外抗肿瘤活性进行分析。方法 对SEB在大肠杆菌中进行高效表达和分离纯化 ,并对其超抗原活性和免疫学活性与天然SEB进行比较研究。观察重组SEB对人肝癌细胞的体外抑制作用 ,以及对小鼠肝癌和肉瘤的体内抑瘤效果。结果 SEB获得高效表达 ,并一步将其纯化至均质。与天然毒素相比较 ,重组SEB的免疫学活性与超抗原活性基本相似。首次证明 ,即使SEB的浓度为 10ng/L ,仍然对人肝癌细胞有显著地抑制作用 ,其对小鼠体内的肝癌和肉瘤有一定的抑制作用。结论 重组SEB在体内外均有一定肿瘤抑制作用 。

酶联免疫发光法检测金葡菌肠毒素(SE)B和SEC1方法的建立

目的:比较酶联免疫测定法(ELISA)和酶联免疫化学发光法(CLIA)在检测SEB、SEC1中的敏感性及稳定性。方法:常规法制备、纯化抗SEB和SEC1单克隆抗体(mAb),以碱性磷酸酶(AP)标记的FMMUSEB.D6和FMMUSEC1.C4mAb为检测抗体,FMMUSEB.B4和FMMUSEC1.G8mAb为包被抗体,以单磷酸酚酞(PMP)为显色底物,以LumigenAPS5为发光底物。结果:成功地建立了敏感、特异检测SEB、SEC1的ELISA和CLIA方法,CLIA较传统ELISA具有灵敏度高、线性范围宽和省时等优点。结论:CLIA法是一种比传统ELISA更优越的免疫学检测方法,在可疑SE污染标本检测、流行病学调查等领域具有良好的应用前景。

食品中金黄色葡萄球菌肠毒素的快速检测方法

采用反向被动乳胶凝集法 (RPLA)和mini VIDAS (ELFA)两种方法对 42份金黄色葡萄球菌阳性样品进行了肠毒素检测。结果RPLA法的金葡菌肠毒素检出率为 61 9% (P<0 0 5 )要高于ELFA法的检出率 5 0 0 % (P <0 0 5 ) ,但检测时间长 ( 2 0h)。在实验中我们还发现 ,血浆凝固酶阴性的葡萄球菌也可产生肠毒素 ,其原因有待进一步的研究。

金黄色葡萄球菌B型肠毒素的培养产毒与柱层析纯化

本文采用玻璃纸覆盖琼脂平板法,在37℃培养金黄色葡萄球菌48h后,收集菌体,10000×g离心15min,上清液经聚乙二醇20000浓缩。浓缩液依次经羧甲基阳离子交换纤维素CM-32层析和葡聚糖凝胶SephadexG-75过滤纯化,获得电泳纯的金黄色葡萄球菌B型肠毒素。实验证明,利用该简化的二步柱层析分离得到的金黄色葡萄球菌B型肠毒素完全能满足血清学检实验和鉴定的要求,方法简便易行,一般实验室均可使用。

金黄色葡萄球菌肠毒素A基因克隆、表达、纯化与鉴定

目的构建含SEA基因的原核表达载体,并在大肠杆菌表达系统中表达。方法应用PCR扩增SEA基因片段,与克隆载体pGEMT-easy连接,插入到表达载体pET-30a中,转化大肠杆菌BL21(DE3),经诱导表达及鉴定。结果PCR扩增约770bp的基因片段,克隆至载体后,经测序与文献报道结果一致,表达蛋白相对分子质量约31000,纯化后鉴定为SEA蛋白。结论已成功获得SEA蛋白,为其进一步研究和应用奠定基础。

超抗原SED在大肠杆菌中的表达

目的构建稳定的SED原核表达系统。方法采用PC R技术扩增葡萄球 菌D型肠毒素（SED）超抗原的成熟蛋白编码区DNA序列，构建SED DNA与6个组氨酸基因融合的表达载体pTrcHis-SED，转入E.coli DH5α，IPTG诱导后，用SDS-PAGE和免疫印迹 检测融合蛋白的表达情况。目的蛋白用Ni-NTA金属亲和层析法进行纯化，SDS-PAGE和毛细管电泳检测蛋白纯度。结果成功构建了原核表达载体pTrcHis-SED。分子量 约 为28 000 u的6His-SED融合蛋白可在E.coli DH5α中稳定表达。Ni-NTA金属亲和层析 后得到 纯度较高（>95%）的SED融合蛋白，且具有免疫学活性。结论本研究为SE D免疫识别研究奠定了实验基础。