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Electron Paramagnetic Resonance (EPR) Spectroscopy to Detect Reactive Oxygen Species in Staphylococcus aureus
电子顺磁共振(EPR)光谱法检测金黄色葡萄球菌中的活性氧类物质   

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Abstract

Under aerobic conditions, Staphylococcus aureus (S. aureus) primarily metabolizes glucose to acetic acid. Although normally S. aureus is able to re-utilize acetate as a carbon source following glucose exhaustion, significantly high levels of acetate in the culture media may not only be growth inhibitory but also potentiates cell death in stationary phase cultures by a mechanism dependent on cytoplasmic acidification. One consequence of acetic acid toxicity is the production of reactive oxygen species (ROS). The present protocol describes the detection of ROS in S. aureus undergoing cell death by electron paramagnetic resonance (EPR) spectroscopy. Using 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) as a cell permeable spin probe, we demonstrate the detection of various oxygen radicals generated by bacteria. Although standardized for S. aureus, the methods described here should be easily adapted for other bacterial species. This protocol is adapted from Thomas et al. (2014) and Thomas et al. (2010).

Keywords: EPR(EPR), Staphylococcus aureus(金黄色葡萄球菌), Reactive oxygen species(活性氧), Spectroscopy(光谱)

Materials and Reagents

  1. Staphylococcus aureus
  2. Bacto tryptic soy broth without dextrose (TSB) (BD Diagnostic Systems, catalog number: DF0862178 )
  3. Glucose (Sigma-Aldrich, catalog number: G8270 ).
  4. 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) (Noxygen Science Transfer & Diagnostics GmbH, catalog number: NOX-2.2-100mg )
  5. Superoxide dismutase (SOD) (Sigma-Aldrich, catalog number: S7571 )
  6. Dimethyl thiourea (DMTU) (Sigma-Aldrich, catalog number: D188700 )
  7. Critoseal (Thermo Fisher Scientific, catalog number: 0267620 )
  8. 5 µM DETC (Noxygen, Catalog number: NOX-10.1 )
  9. 25 µM deferoxamine (Noxygen, Catalog number: NOX-10.1)
  10. Culture flask (250 ml)
  11. Culture tubes
  12. 1.5 ml Eppendorf tubes
  13. Krebs-HEPES buffer (KDD buffer) (see Recipes)

Equipment

  1. 37 °C shaker-incubator (250 rpm per min)
  2. Leica Biosystems Critoseal capillary tube sealant (Leica Microsystems, catalog number: MS215003A )
  3. Bruker e-Scan EPR Spectrometer and Noxygen Temperature Controller Bio-I (Bruker, model: NOX-E.11-ESR )
  4. Micropipettes (50 µl, EPR tubes) (Noxygen Science Transfer & Diagnostics GmbH, catalog number: MS215003A )
  5. Spectrophotometer
  6. Vortex-Genie 2

Software

  1. Bruker WinEPR Data Processing software

Procedure

  1. To obtain starter culture, inoculate a single colony of S. aureus into 3 ml sterile TSB supplemented with 14 mM glucose and incubate overnight in a 37 °C shaker-incubator adjusted to 250 rpm.
  2. Inoculate 25 ml sterile TSB (suppl. with 35 mM glucose) in a 250 ml flask with starter culture to a final OD600 of 0.06 and incubate in a 37 °C shaker-incubator adjusted to 250 rpm for a period of 72 h.
  3. Following the 72 h incubation period, 10 OD600 units (~7 x 109 cfu/ml) is harvested in a 1.5 ml Eppendorf tube and suspended in 1 ml of ice cold KDD buffer. The bacterial suspension is placed on ice until further use.
  4. Prior to EPR measurements, the ROS sensitive spin probe CMH (working stock ~4 mM prepared in KDD buffer) is added to a final concentration of 200 µM in 1 ml bacterial suspension (step 3), briefly vortexed (2 sec) and allowed to stand at room temperature for 15 min.
  5. The bacterial suspension (50 µl) is then transferred into micropipettes by capillary action and sealed at the distal end using Critoseal, immediately prior to EPR analysis.
  6. EPR acquisition parameters are as follows: Field sweep width, 60 gauss; microwave frequency, 9.75 kHz; microwave power, 21.9 mW; modulation amplitude, 2.37 gauss; conversion time, 10.24 ms; time constant, 40.96 ms.
  7. Record 2-D spectra and average 10 scans for each sample to reduce background noise. Quantitation of EPR spectra and baseline correction can be accomplished using Bruker WinEPR Data Processing software.
  8. To identify the contribution of superoxide (O2−) and hydroxyl radicals (OH˙) to the overall EPR spectra, SOD (400 units), an O2− scavenging antioxidant enzyme and/or DMTU (20 mM), a OH˙ scavenger, may be added to the bacterial suspension (step 3) prior to the addition of CMH and incubated at room temperature for 15 min. Continue with step 4 of this protocol.
    Note: It is crucial to have a negative control (step 4; KDD buffer containing 200 µM CMH) when acquiring EPR signals. This control will report the degree of auto-oxidation CMH has undergone over the period of the experiment.

Representative data


A                                                                     B

Figure 1. EPR detection of S. aureus ROS production. A. Following inoculation of S. aureus to an initial OD600 of 0.06, cultures were incubated aerobically (flask: volume ratio= 10:1, 250 rpm). Culture samples for EPR analysis were withdrawn at 3 h, 24 h and 72 h of bacterial growth. B. S. aureus culture samples (72 h) were treated with either 400 units of SOD, 20 mM DMTU, or vehicle (wild-type, WT) before addition of the spin probe, CMH. The basal extent of CMH autoxidation in KDD buffer (blue line) was determined as control.

Recipes

  1. Krebs-HEPES buffer (KDD buffer) (pH 7.4)
    99 mM NaCl
    4.69 mM KCl
    2.5 mM CaCl2
    1.2 mM MgSO4
    25 mM NaHCO3
    1.03 mM KH2PO4
    5.6 mM D-glucose
    20 mM HEPES
    5 µM DETC
    25 µM deferoxamine

Acknowledgements

This work was funded by NIH grant nos. R01-A1038901 (KWB), PO1-AI083211 (KWB), R01- HL103942 (MCZ), and American Heart Association postdoctoral fellowship 12POST12080155 (VCT). The EPR spectroscopy core is supported, in part, by a NIH Center of Biomedical Research Excellence (COBRE) grant (1P30GM103335-01) awarded to the University of Nebraska's Redox Biology Center. This protocol is adapted from Thomas et al. (2014) and Thomas et al. (2010).

References

  1. Thomas, V. C., Kinkead, L. C., Janssen, A., Schaeffer, C. R., Woods, K. M., Lindgren, J. K., Peaster, J. M., Chaudhari, S. S., Sadykov, M., Jones, J., AbdelGhani, S. M., Zimmerman, M. C., Bayles, K. W., Somerville, G. A. and Fey, P. D. (2013). A dysfunctional tricarboxylic acid cycle enhances fitness of Staphylococcus epidermidis during beta-lactam stress. MBio 4(4).
  2. Thomas, V. C., Sadykov, M. R., Chaudhari, S. S., Jones, J., Endres, J. L., Widhelm, T. J., Ahn, J. S., Jawa, R. S., Zimmerman, M. C. and Bayles, K. W. (2014). A central role for carbon-overflow pathways in the modulation of bacterial cell death. PLoS Pathog 10(6): e1004205.

简介

在有氧条件下,金黄色葡萄球菌(金黄色葡萄球菌)主要将葡萄糖代谢为乙酸。虽然通常是。金黄色葡萄球菌能够在葡萄糖耗竭之后再利用乙酸盐作为碳源,培养基中显着高水平的乙酸盐不仅可以是生长抑制性的,而且还可以通过依赖于细胞质的机制增强稳定期培养物中的细胞死亡酸化。乙酸毒性的一个后果是活性氧(ROS)的产生。本协议描述了ROS的检测。金黄色葡萄球菌通过电子顺磁共振(EPR)光谱法进行细胞死亡。使用1-羟基-3-甲氧基羰基-2,2,5,5-四甲基吡咯烷(CMH)作为细胞可渗透的自旋探针,我们证明检测由细菌产生的各种氧自由基。虽然标准化了 s。 aureus ,这里描述的方法应该很容易适应其他细菌物种。该协议改编自托马斯(Thomas)等人(2014)和托马斯(Thomas)等人(2010)。

关键字:EPR, 金黄色葡萄球菌, 活性氧, 光谱

材料和试剂

  1. 金黄色葡萄球菌
  2. 不含右旋糖的细菌胰蛋白酶大豆肉汤(TSB)(BD Diagnostic Systems,目录号:DF0862178)
  3. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  4. 1-羟基-3-甲氧基羰基-2,2,5,5-四甲基吡咯烷(CMH)(Noxygen Science Transfer& Diagnostics GmbH,目录号:NOX-2.2-100mg)
  5. 超氧化物歧化酶(SOD)(Sigma-Aldrich,目录号:S7571)
  6. 二甲基硫脲(DMTU)(Sigma-Aldrich,目录号:D188700)
  7. Critoseal(Thermo Fisher Scientific,目录号:0267620)
  8. 5μMDETC(Noxygen,目录号:NOX-10.1)
  9. 25μM去铁胺(Noxygen,目录号:NOX-10.1)
  10. 培养瓶(250ml)
  11. 文化管
  12. 1.5 ml Eppendorf管
  13. Krebs-HEPES缓冲液(KDD缓冲液)(见配方)

设备

  1. 37℃摇床培养箱(250rpm/min)
  2. Leica Biosystems Critoseal毛细管密封胶(Leica Microsystems,目录号:MS215003A)
  3. Bruker e-Scan EPR光谱仪和Noxygen温度控制器Bio-I(Bruker,型号:NOX-E.11-ESR)
  4. 微量移液器(50μl,EPR管)(Noxygen Science Transfer& Diagnostics GmbH,目录号:MS215003A)
  5. 分光光度计
  6. Vortex-Genie 2

软件

  1. Bruker WinEPR数据处理软件

程序

  1. 为了获得起子培养物,接种单个集落的S.金黄色葡萄球菌注射到补充有14mM葡萄糖的3ml无菌TSB中,并在调节至250rpm的37℃摇床培养箱中孵育过夜。
  2. 在具有起始培养物的250ml烧瓶中接种25ml无菌TSB(用35mM葡萄糖补充)至0.06的最终OD 600,并在37℃摇床培养箱中孵育,调节至250rpm, 72小时。
  3. 72小时孵育期后,在1.5ml Eppendorf管中收获10OD 600单位(〜7×10 9 supu cfu/ml),并悬浮于1ml冰中冷KDD缓冲液。将细菌悬浮液置于冰上直至进一步使用
  4. 在EPR测量之前,将ROS敏感的自旋探针CMH(在KDD缓冲液中制备的工作母液〜4mM)在1ml细菌悬浮液(步骤3)中添加至200μM的最终浓度,短暂涡旋(2秒),并允许在室温下放置15分钟
  5. 然后通过毛细管作用将细菌悬浮液(50μl)转移到微量移液管中,并在EPR分析前立即使用Critoseal在末端密封。
  6. EPR采集参数如下:场扫描宽度,60高斯;微波频率,9.75kHz;微波功率,21.9mW;调制幅度,2.37高斯;转换时间,10.24 ms;时间常数,40.96 ms。
  7. 记录2-D光谱和每个样品的平均10次扫描,以减少背景噪声。 EPR光谱和基线校正的定量可以使用Bruker WinEPR数据处理软件实现
  8. 为了鉴定超氧化物(O 2 2-)和羟基自由基(OH - )对总体EPR光谱的贡献,SOD(400单位),O 2 2-清除抗氧化剂可在加入CMH之前向细菌悬浮液(步骤3)中加入酶和/或DMTU(20mM),OH·清除剂,并在室温下温育15分钟。继续执行本协议的第4步。
    注意:当获取EPR信号时,具有阴性对照(步骤4;含有200μMCMH的KDD缓冲液)是至关重要的。该控制将报告在实验期间CMH已经经历的自氧化程度。

代表数据


A                           ;                         ;                    B

图1. S的EPR检测。 aureus ROS的产生。 A.接种。金黄色葡萄球菌到0.06的初始OD 600,培养物有氧培养(培养瓶:体积比= 10:1,250rpm)。在细菌生长的3小时,24小时和72小时撤回用于EPR分析的培养物样品。 B. 在加入自旋探针CMH之前用400单位的SOD,20mM DMTU或载体(野生型,WT)处理金黄色葡萄球菌培养物样品(72小时)。测定KDD缓冲液(蓝线)中CMH自动氧化的基础程度作为对照。

食谱

  1. Krebs-HEPES缓冲液(KDD缓冲液)(pH7.4) 99 mM NaCl
    4.69mM KCl
    2.5mM CaCl 2·h/v 1.2mM MgSO 4 25mM NaHCO 3/v/v 1.03mM KH 2 PO 4 sub/
    5.6mM D-葡萄糖 20 mM HEPES
    5μMDETC
    25μM去铁胺

致谢

这项工作由NIH拨款资助。 R01-A1038901(KWB),PO1-AI083211(KWB),R01- HL103942(MCZ)和美国心脏协会博士后团契12POST12080155(VCT)。 EPR光谱学核心部分地由授予内布拉斯加大学氧化还原生物学中心的NIH生物医学研究优秀中心(COBRE)资助(1P30GM103335-01)支持。 该协议改编自托马斯(Thomas)等人(2014)和托马斯(Thomas)等人(2010)。

参考文献

  1. Thomas,VC,Kinkead,LC,Janssen,A.,Schaeffer,CR,Woods,KM,Lindgren,JK,Peaster,JM,Chaudhari,SS,Sadykov,M.,Jones,J.,AbdelGhani,SM,Zimmerman,MC ,Bayles,KW,Somerville,GA 和Fey,P.D。(2013)。 功能障碍的三羧酸循环增强β-内酰胺表皮葡萄球菌的适应性 压力。 MBio 4(4)。
  2. Thomas,V.C.,Sadykov,M.R.,Chaudhari,S.S.,Jones,J.,Endres,J.L.,Widhelm,T.J.,Ahn,J.S.Jawa,R.S.,Zimmerman,M.C.and Bayles,K.W。 碳溢出途径在调节细菌细胞死亡中的核心作用 < em> PLoS Pathog 10(6):e1004205。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Thomas, V. C., Chaudhari, S. S., Jones, J., Zimmerman, M. C. and Bayles, K. W. (2015). Electron Paramagnetic Resonance (EPR) Spectroscopy to Detect Reactive Oxygen Species in Staphylococcus aureus. Bio-protocol 5(17): e1586. DOI: 10.21769/BioProtoc.1586.
  2. Thomas, V. C., Sadykov, M. R., Chaudhari, S. S., Jones, J., Endres, J. L., Widhelm, T. J., Ahn, J. S., Jawa, R. S., Zimmerman, M. C. and Bayles, K. W. (2014). A central role for carbon-overflow pathways in the modulation of bacterial cell death. PLoS Pathog 10(6): e1004205.
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