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Cytotoxicity Assay for Detection of Cereulide Produced by Emetic Bacillus cereus
采用细胞毒性分析法检测蜡样芽孢杆菌呕吐毒素Cereulide   

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Abstract

The emetic subgroup of Bacillus cereus strains produces cereulide, a dodecadepsipeptide (1.2 kDa), which is the causative agent of food poisonings. Cereulide is synthesized by a nonribosomal peptide synthetase (NRPS), the Ces-NRPS (Ehling-Schulz et al., 2006). Cereulide is a cyclic and lipophilic potassium ionophor structurally related to the macrolide antibiotic valinomycin. Both substances act on mitochondria by depolarization and uncoupling of ATP synthesis, and this effect (mitochondrial swelling) is used to quantify cereulide in a HEp-2 cell based viability assay.
In this protocol, which was modified from an assay published by Finlay et al. (1999), valinomycin is used as a reference standard for ionophor–induced cytotoxicity, because purified cereulide is not commercially available yet. This assay has been used to quantify cereulide amounts from different B. cereus mutants (Lücking et al., 2009; Frenzel et al., 2012) and to estimate cereulide levels extracted from foods (artificially) contaminated with B. cereus (Frenzel et al., 2011).

Keywords: Cereulide(呕吐毒素), Bacillus cereus(蜡样芽孢杆菌), Cytotoxicity assay(细胞毒性测试), Cell culture(细胞培养), Food poisoning(食物中毒)

Materials and Reagents

  1. Bacillus cereus F4810/72 (or other cereulide producing, emetic B. cereus strain)
  2. Tryptone
  3. Yeast extract
  4. NaCl
  5. HEp-2 cell line (Frenzel et al., 2011)
  6. MEM Earle′s medium (with 2.2 g/L NaHCO3, with stable glutamine) (Biochrom, catalog number: FG0325 )
  7. Penicillin-streptomycin solution (10,000 μg/ml; Biochrom, catalog number: A2212/3 )
  8. Sodium pyruvate solution (Biochrom, catalog number: L0473 )
  9. Fetal bovine serum (FBS) (Biochrom, catalog number: S0113 )
  10. Phosphate Buffered Saline (PBS) (Biochrom, catalog number: L1825 )
  11. 99% Ethanol (highest purity grade available)
  12. WST-1 cell proliferation reagent (Roche, catalog number: 0 5015944001 )
  13. Valinomycin (reference standard) (Sigma-Aldrich, catalog number: 94675 )
  14. 10x Trypsin/EDTA solution (0.5 %/0.2 %) (Biochrom, catalog number: L2153 )
  15. LB-Miller (see Recipes)
  16. MEM-Earle HEp-2 medium (see Recipes)
  17. MEM-Earle HEp-2 medium with ethanol (see Recipes)

Equipment

  1. 30 °C incubator with aeration (for liquid bacterial cultures)
  2. 37 °C incubator with 5% CO2 (for HEp-2 cell cultures)
  3. Spectrophotometer
  4. Table top centrifuge
  5. Microplate shaker
  6. Microscope and counting chamber for cell culture
  7. Microscope and counting chamber for bacteria (e.g. Helber counting chamber; depth: 0.02 mm; small square area: 0.0025 mm2)
  8. Multichannel pipet (8 channels, manual or electronic; e.g. Transferpette®, BrandTech Scientific 10-200 μl; catalog number: 2705410 )
  9. Microplate reader (multiwell scanning spectrophotometer)
  10. 500 ml baffled flask
  11. Tissue culture flasks with filter caps, 75 cm2 (Biochrom catalog number: P90076 )
  12. Safe seal tubes (e.g. Eppendorf, catalog number: 0030120.094 ) 96-well cell culture plates, polystyrene (Biochrom, catalog number: P92696 )

Procedure

  1. Cereulide sample preparation (B. cereus culture and growth conditions)
    1. Inoculate 3 ml LB medium with B. cereus and incubate at 30 °C, 150 rpm for 16 h (pre-culture).
    2. After 16 h, determine the cell count (CFU/ml) of the pre-culture with a counting chamber appropriate for bacteria (e.g. Helber counting chamber). Inoculate 100 ml LB in 500 ml baffled flask with 103 CFU/ml (approx. 1:10,000 dilution; i.e. add approx. 100 μl of 1:100 dilution of bacteria to 100 ml LB). Incubate at 30 °C, 150 rpm for 24 h.
    3. Cereulide accumulates to stable levels after 24 h of cultivation at 30 °C (Frenzel et al., 2011). Withdraw 1-2 ml samples of B. cereus cultures and autoclave in safe seal tubes (17 min at 121 °C). This step will denature heat-labile enterotoxins and proteases, whereas cereulide remains stable. Store samples at -20 °C.
    4. The remaining part of the B. cereus culture is autoclaved (17 min at 121 °C) and disposed.
  1. HEp-2 cell based cereulide cytotoxicity assay


    Figure 1: Example for setup of cereulide cytotoxicity 96-well assay plate. MEM = MEM-Earle HEp-2 medium with ethanol; val = valinomycin (5 μg/ml); green= background control wells; orange = 100% cell viability wells without test substance.

    1. Maintain HEp-2 cells as monolayers in 75 cm2 tissue culture flasks at 37 °C in a 5% CO2 atmosphere. Routinely grow HEp-2 cells in 25 ml MEM-Earle HEp-2 medium and sub-culture cells 1:4 approximately every 2-3 days when they reach 70-80% confluence. To detach adherent cells for sub-culturing, add 5 ml of trypsin/EDTA solution and incubate for 5-10 min at 37 °C. Add 5 ml of MEM-Earle HEp-2 medium and resuspend detached HEp-2 cells. Centrifuge cells (5 min, 150 x g, room temperature) and resuspend pellet in 10 ml MEM-Earle HEp-2 medium. Split cells 1:4 in new tissue culture flask containing 25 ml MEM-Earle HEp-2 medium.
    2. If cells from point 2a) reach 70-80% confluence, cytotoxicity assay can be performed. Add 90 μl of MEM-Earle HEp-2 medium with ethanol to wells A1 to H1 of a 96-well cell culture plate (see Figure 1). Fill the remaining wells with 50 μl MEM-Earle HEp-2 medium with ethanol.
    3. Dilute valinomycin stock solution (50 μg/ml) 1:10 with MEM-Earle HEp-2 medium with ethanol. Add 10 μl in wells B1 and C1 (final concentration is 500 ng/ml).
    4. Two additional samples (prepared as described in point 1 and stored at -20 °C) can be tested on this plate: add 10 μl of sample 1 to wells D1 and E1; add 10 μl of sample 2 to wells F1 and G1. Rows A and H remain free of samples (background control wells).
    5. Mix samples and medium thoroughly by repeated pipetting using a multichannel pipet. Remove 50 μl of the wells and perform serial dilutions (1:1) of the samples to column A10-H10. Columns 11 and 12 remain free of sample (100% cell viability control wells).
    6. Remove growth medium from a HEp-2 culture flask containing a 70-80% confluent monolayer. Wash the cells with 5 ml PBS. Add 5 ml of trypsin/EDTA solution and incubate for 5-10 min at 37 °C.
    7. Add 5 ml of MEM-Earle HEp-2 medium and resuspend detached HEp-2 cells.
    8. Centrifuge cells (5 min, 150 x g, room temperature) and resuspend pellet in 10 ml MEM-Earle HEp-2 medium.
    9. Determine cell count with counting chamber. Adjust the cells count to 3.4 x105 cells/ml.
    10. Dispense 150 μl of cells per well (~5 x 104 cells/well). Incubate plate for 48 h at 37 °C in a 5% CO2 atmosphere.
    11. To save WST-1 reagent, discard 100 μl of culture medium from each well and add 10 μl of WST-1 reagent to the remaining culture to detect mitochondrial activity of HEp-2 cells. Rows A and H remain free of WST-1 for calculation of the background value. Shake plate to distribute proliferation reagent uniformly and incubate plate for 20 min at 37 °C in a 5% CO2 atmosphere.
    12. Shake plate thoroughly and measure absorbance in a microplate reader at 450 nm.
  2. Calculation of cereulide amounts
    1. Import raw data from absorbance measurement to calculation/statistics program (e.g. Microsoft Excel).
    2. Perform background subtraction on all wells with the mean of the background control wells (Rows A and H; see Figure 2). Calculate dose-response curve for the internal standard valinomycin [Percent viability inhibition = (mean absorbance test wells /mean absorbance 100% cell viability control wells) x 100]. Determine the 50% inhibitory concentration for valinomycin by linear regression.
    3. Calculate dose-response curves for samples as given above. Determine the 50% inhibitory concentration for samples (presented by the reciprocal value of the sample dilution that resulted in 50% loss of mitochondrial activity). Calculate cereulide amounts as valinomycin equivalents (VE) by extrapolating the absorbance value at 50% cell viability inhibition to the dose response curve for valinomycin.



      Figure 2. Example for calculation of cereulide concentrations (given in valinomycin equivalents) from dose-response-curves plotted in Microsoft Excel

Recipes

  1. LB-Miller
    10 g tryptone
    5 g yeast extract
    10 g NaCl
    Add dH2O to 1 L
    Adjust pH to 7.0 ± 0.2. Autoclave 121 °C, 15 min
  2. MEM-Earle HEp-2 medium
    488 ml MEM Earle′s medium (with 2.2 g/L NaHCO3, with stable glutamine)
    2 ml penicillin-streptomycin solution (10,000 μg/ml)
    5 ml sodium pyruvate solution
    5 ml FBS
  3. MEM-Earle HEp-2 medium with ethanol
    478 ml MEM Earle′s medium (with 2.2 g/L NaHCO3, with stable glutamine)
    2 ml penicillin-streptomycin solution (10,000 μg/ml)
    5 ml sodium pyruvate solution
    5 ml FBS
    10 ml 99% ethanol (solvent for cereulide); highest purity grade available

Acknowledgments

This protocol was adapted from previously published papers: Finlay et al. (1999); Lücking et al. (2009); and Frenzel et al. (2011). This research project was supported by the German Ministry of Economics and Technology (via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn), projects AiF 15186 N and 16845 N. We thank Prof. Dr. Dr. E. Märtlbauer and Dr. R. Dietrich (Ludwig-Maximilians-University Munich, Germany) for kindly providing the HEp-2 cell line.

References

  1. Ehling-Schulz M., Vukov, N., Schulz, A., Shaheen, R., Andersson, M., Martlbauer, E., and Scherer, S. (2005). Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus. Appl Environ Microbiol 71, 105-113.
  2. Finlay, W. J., Logan, N. A., Sutherland, A. D. (1999). Semiautomated metabolic staining assay for Bacillus cereus emetic toxin. Appl Environ Microbiol 65, 181-1812.
  3. Frenzel, E., Doll, V., Pauthner, M., Lücking, G., Scherer, S., Ehling-Schulz, M. (2012). CodY orchestrates the expression of virulence determinants in emetic Bacillus cereus by impacting key regulatory circuits. Mol Mic 85 (1), 67-88.
  4. Frenzel E., Letzel, T., Scherer, S., Ehling-Schulz, M. (2011). Inhibition of cereulide toxin synthesis by emetic Bacillus cereus via long-chain poly-phosphates. Appl Environ Microbiol 77, 1475-1482.
  5. Lücking, G., Dommel, M. K., Scherer, S., Fouet, A., Ehling-Schulz, M. (2009). Cereulide synthesis in emetic Bacillus cereus is controlled by the transition state regulator AbrB, but not by the virulence regulator PlcR. Microbiology 155, 922-931. 
  6. The HEp-2 cell line is a permanent human larynx carcinoma cell line. The cells were kindly provided by Prof. E. Martlbauer and Dr. R. Dietrich (Ludwig Maximilians University Munich, Germany). See Nielsen, C., Casteel, M., Didier, A., Dietrich, R., Martlbauer, E. (2009). Trichothecene-induced cytotoxicity on human cell lines. Mycotox Res 25, 77-84.

简介

蜡状芽孢杆菌菌株的催吐亚组产生作为食物中毒的致病因子的十二缩酚酸肽(1.2kDa)的cereulide。谷胱甘肽通过非核糖体肽合成酶(NRPS),Ces-NRPS(Ehling-Schulz等人,2006)合成。脑苷脂是与大环内酯类抗生素缬氨霉素结构相关的环状和亲脂性钾离子体。这两种物质通过ATP合成的去极化和解偶联作用于线粒体,并且该效应(线粒体肿胀)用于在基于HEp-2细胞的活力测定中量化脑苷脂。
在该方案中,根据Finlay等人(1999)发表的试验进行了修改,缬氨霉素被用作离子体诱导的细胞毒性的参考标准,因为纯化的脑苷脂不是商业上的可用。该测定法已经用于定量来自不同B的脑梗的量。 cereus突变体(Lücking等人,2009; Frenzel等人,2012),并且估计从人类(人工)污染的食物中提取的脑苷脂水平> B。 cereus (Frenzel ,2011)。

关键字:呕吐毒素, 蜡样芽孢杆菌, 细胞毒性测试, 细胞培养, 食物中毒

材料和试剂

  1. 蜡状芽孢杆菌 F4810/72(或其他产生肉芽肿的,催吐的蜡状芽孢杆菌菌株)
  2. 胰蛋白酶
  3. 酵母提取物
  4. NaCl
  5. HEp-2细胞系(Frenzel等人,2011)
  6. MEM Earle's培养基(具有2.2g/L NaHCO 3,具有稳定的谷氨酰胺)(Biochrom,目录号:FG0325)
  7. 青霉素 - 链霉素溶液(10,000μg/ml; Biochrom,目录号:A2212/3)
  8. 丙酮酸钠溶液(Biochrom,目录号:L0473)
  9. 胎牛血清(FBS)(Biochrom,目录号:S0113)
  10. 磷酸盐缓冲盐水(PBS)(Biochrom,目录号:L1825)
  11. 99%乙醇(最高纯度级别)
  12. WST-1细胞增殖试剂(Roche,目录号:05015944001)
  13. 维拉霉素(参考标准)(Sigma-Aldrich,目录号:94675)
  14. 10x胰蛋白酶/EDTA溶液(0.5%/0.2%)(Biochrom,目录号:L2153)
  15. LB-Miller(参见食谱)
  16. MEM-Earle HEp-2培养基(参见配方)
  17. MEM-Earle HEp-2培养基与乙醇(参见配方)

设备

  1. 30℃培养箱通气(用于液体细菌培养)
  2. 37℃具有5%CO 2(对于HEp-2细胞培养物)的培养箱
  3. 分光光度计
  4. 台式离心机
  5. 微量板振荡器
  6. 显微镜和细胞培养的计数室
  7. 用于细菌的显微镜和计数室(例如Helber计数室;深度:0.02mm;小正方形面积:0.0025mm 2)
  8. 多通道移液器(8个通道,手动或电子;例如Transferpette ®,BrandTech Scientific10-200μl;目录号:2705410)
  9. 微孔板读数器(多孔扫描分光光度计)
  10. 500ml带挡板的烧瓶中
  11. 具有过滤器盖的组织培养瓶,75cm 2(Biochrom目录号:P90076)
  12. 96孔细胞培养板,聚苯乙烯(Biochrom,目录号:P92696)安全密封管(例如Eppendorf,目录号:0030120.094)

程序

  1. 糖浆样品制备(葡萄芽孢杆菌培养和生长条件)
    1. 用B接种3ml LB培养基。并在30℃,150rpm温育16小时(预培养)。
    2. 16小时后,用适于细菌的计数室(例如Helber计数室)确定预培养的细胞计数(CFU/ml)。在具有10 3 CFU/ml(大约1:10,000稀释度;即加入大约100μl1:100稀释的细菌)的500ml带挡板的烧瓶中接种100ml LB至100ml LB)。在30℃,150rpm孵育24小时
    3. 在30℃下培养24小时后,肉豆蔻累积至稳定的水平(Frenzel等人,2011)。取出1-2ml的样品B. cereus培养物和高压灭菌器在安全密封管中(在121℃下17分钟)。该步骤将使热不稳定的肠毒素和蛋白酶变性,而脑苷保持稳定。将样品储存在-20°C。
    4. B的剩余部分。 cereus 培养物在121℃下高压灭菌(17分钟)并处理
  1. 基于HEp-2细胞的cereulide细胞毒性测定


    图1:设置cereulide细胞毒性96孔测定板的实施例。MEM =具有乙醇的MEM-Earle HEp-2培养基; val =缬氨霉素(5μg/ml);   绿色=背景对照孔; 橙色= 100%细胞活力孔 无试验物质
    1. 在37℃,5%CO 2气氛中,在75cm 2组织培养瓶中将HEp-2细胞保持为单层。 在25ml MEM-Earle HEp-2培养基和亚培养细胞中常规生长HEp-2细胞 1:4大约每2-3天,当它们达到70-80%汇合时。为了分离粘附细胞用于亚培养,加入5ml胰蛋白酶/EDTA溶液并在37℃温育5-10分钟。加入5ml的MEM-Earle HEp-2培养基并重悬浮脱离的HEp-2细胞。离心细胞(5分钟,150×g,室温),并将沉淀重悬于10ml MEM-Earle HEp-2培养基中。在含有25ml MEM-Earle HEp-2培养基的新组织培养瓶中分裂细胞1:4
    2. 如果来自点2a)的细胞达到70-80%汇合,可以进行细胞毒性测定。向96孔细胞培养板的孔A1至H1中加入90μl含乙醇的MEM-Earle HEp-2培养基(见图1)。填充其余的孔与50微升MEM-Earle HEp-2培养基与乙醇。
    3. 用MEM-Earle HEp-2培养基和乙醇稀释缬氨霉素储备液(50μg/ml)1:10。加入10μl在孔B1和C1(最终浓度为500 ng/ml)。
    4. 可以在该板上测试两个另外的样品(如第1点中所述制备并储存在-20℃下):向孔D1和E1中加入10μl样品1;将10μl样品2加入孔F1和G1。行A和H没有样品(背景控制孔)。
    5. 通过使用多通道移液器反复吸取,彻底混合样品和培养基。取出50μl的孔,并对A10-H10柱进行连续稀释(1:1)样品。柱11和12保持不含样品(100%细胞活力对照孔)。
    6. 从含有70-80%汇合单层的HEp-2培养瓶中取出生长培养基。用5ml PBS洗涤细胞。加入5ml胰蛋白酶/EDTA溶液,并在37℃下孵育5-10分钟
    7. 加入5ml的MEM-Earle HEp-2培养基并重悬浮脱离的HEp-2细胞。
    8. 离心细胞(5分钟,150×g,室温),并在10ml MEM-Earle HEp-2培养基中重悬沉淀。
    9. 用计数室确定细胞计数。将细胞计数调节至3.4×10 5个细胞/ml。
    10. 分配每孔150μl细胞(〜5×10 4个细胞/孔)。在37℃,5%CO 2气氛中孵育板48小时
    11. 为了节省WST-1试剂,从每个孔中丢弃100微升培养基,并添加10微升的WST-1试剂到剩余的培养物,以检测线粒体活性的HEp-2细胞。行A和H保持不含WST-1以计算背景值。摇动板以均匀地分布增殖试剂,并在37℃,5%CO 2气氛中孵育板20分钟。
    12. 充分振荡板,在450nm的酶标仪上测量吸光度
  2. 脑量的计算
    1. 将原始数据从吸光度测量导入计算/统计程序(例如Microsoft Excel)。
    2. 使用背景对照孔的平均值对所有孔进行背景扣除(A行和H行;参见图2)。计算内标缬氨霉素的剂量 - 反应曲线[生存力百分比抑制=(平均吸光度测试孔/平均吸光度100%细胞生存力对照孔)×100]。通过线性回归确定缬氨霉素的50%抑制浓度。
    3. 计算上面给出的样品的剂量 - 反应曲线。确定样品的50%抑制浓度(由导致50%线粒体活性丧失的样品稀释度的倒数值表示)。通过将50%细胞活力抑制的吸光度值外推至缬氨霉素的剂量反应曲线来计算作为缬氨霉素等价物(VE)的脑苷量。



      图2.从Microsoft Excel 中绘制的剂量 - 反应曲线计算脑钠肽浓度(以缬氨霉素等价物计)的示例

食谱

  1. LB-Miller
    10g胰蛋白胨
    5g酵母提取物
    10克NaCl
    将dH <2> O添加到1 L
    调节pH至7.0±0.2。 高压灭菌器121°C,15分钟
  2. MEM-Earle HEp-2培养基
    488ml MEM Earle's培养基(具有2.2g/L NaHCO 3,具有稳定的谷氨酰胺)
    2ml青霉素 - 链霉素溶液(10,000μg/ml) 5ml丙酮酸钠溶液 5ml FBS
  3. MEM-Earle HEp-2培养基,含有乙醇 478ml MEM Earle's培养基(具有2.2g/L NaHCO 3,具有稳定的谷氨酰胺)
    2ml青霉素 - 链霉素溶液(10,000μg/ml) 5ml丙酮酸钠溶液 5ml FBS
    10ml 99%乙醇(脑苷脂溶剂); 最高纯度等级

致谢

该方案改编自以前发表的论文:Finlay等人(1999); Lücking(2009);和Frenzel 等人(2011)。这个研究项目得到德国经济技术部(通过AiF)和FEI(波恩Forschungskreis derErnährungsindustrieeV,项目AiF 15186 N和16845 N)的支持。我们感谢Dr.Märtlbauer博士和Dr 。Dietrich(Ludwig-Maximilians-University Munich,Germany)用于提供HEp-2细胞系。

参考文献

  1. Ehling-Schulz M.,Vukov,N.,Schulz,A.,Shaheen,R.,Andersson,M.,Martlbauer,E.,and Scherer,S。(2005)。 鉴定和部分表征负责在催吐蜡状芽孢杆菌中的脑梗产生的非核糖体肽合成酶基因 。 Appl Environ Microbiol   71,105-113。
  2. Finlay,W.J.,Logan,N.A.,Sutherland,A.D。(1999)。 蜡样芽孢杆菌呼吸毒素的半自动代谢染色测定法。 Appl Environ Microbiol 65,181-1812。
  3. Frenzel,E.,Doll,V.,Pauthner,M.,Lücking,G.,Scherer,S.,Ehling-Schulz,M.(2012)。 Cody通过影响关键的调节回路来调节在蜡状芽孢杆菌中的毒力决定簇的表达。 Mol Mic   85(1),67-88。
  4. Frenzel E.,Letzel,T.,Scherer,S.,Ehling-Schulz,M.(2011)。通过长链多磷酸酯通过催吐的蜡状芽孢杆菌抑制脑苷脂毒素合成。 Appl Environ Microbiol</em> 77,1475-1482。 />
  5. Lücking,G.,Dommel,M.K.,Scherer,S.,Fouet,A.,Ehling-Schulz,M。(2009)。 催吐蜡状芽孢杆菌中的脑胶质合成受过渡状态调节器AbrB控制,而不是通过毒力调节剂PlcR 。 微生物学 155,922-931。
  6. HEp-2细胞系是永久性人类喉癌细胞系。细胞由Prof.E. Martlbauer和Dr.D.Disrich(Ludwig Maximilians University Munich Munich,Germany)友好地提供。参见Nielsen,C.,Casteel,M.,Didier,A.,Dietrich,R.,Martlbauer,E。(2009)。 三尖射二醇对人细胞系的细胞毒性。 Mycotox Res 25,77-84。

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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Frenzel, E. and Ehling-Schulz, M. (2013). Cytotoxicity Assay for Detection of Cereulide Produced by Emetic Bacillus cereus. Bio-protocol 3(2): e318. DOI: 10.21769/BioProtoc.318.
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