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Purification and Structural Analysis of QS-inhibiting Compounds from Staphylococcus delphini
海豚葡萄球菌的QS抑制化合物的纯化和结构分析   

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Abstract

The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we present a purification protocol for molecules excreted by a group of Gram-positive zoonotic pathogen bacteria, the ‘Staphylococcus intermedius group’, that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini (S. delphini). They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. These substances can be isolated and purified from the culture supernatant using this upscalable purification method.

Materials and Reagents

  1. Staphylococcus delphini DSMZ20771 strain (DSM number: 20071 )
    Note: DSMZ stands for Deutsche Sammlung von Mikroorganismen und Zellkulturen.
  2. Tryptic Soy Broth (TSB) (Sigma-Aldrich, catalog number: T8907 )
  3. Amberilite XAD-16 resin (Sigma-Aldrich, catalog number: 1-0379 )
  4. Methanol
  5. Acetic acid (Merck KGaA)
  6. Amberilite IRC 50 cation exchange resin (SERVA Electrophoresis GmbH, catalog number: 40501 )
  7. Sodium hydroxide
  8. Ethanol
  9. 50 mM and 1 M sodium phosphate buffer
  10. SP sepharose cation exchange column (GE Healthcare, catalog number: 17-5161-01 )
  11. Sodium chloride
  12. Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: T6508 )
  13. Phosphoric acid for HPLC (Sigma-Aldrich, catalog number: 79606 )
  14. Acetonitrile for HPLC (Mallinckrodt Baker, catalog number: 9012 )

Equipment

  1. 37 °C shaking incubator (Infors AG)
  2. Centrifuge (Eppendorf)
  3. Rotary evaporator (BÜCHI Labortechnik AG)
  4. Äkta FPLC equipped with P-900, UV-900, PH/C-900 (GE Healthcare)
  5. Preparative HPLC System equipped with Bischoff HPLC compact pump QC-P 2250 and Multiwavelength detector QC-1157 (Bischoff)
  6. Nucleosil 100 C-18 (8 x 250 mm column) (MACHEREY-NAGEL, catalog number: 715332.80 )
  7. Agilent 1200 series HPLC system (Agilent)
  8. Waters XBridge C18 (5 mm, 4.6 x 150 mm column) (Waters, part number: 186003116 )

Procedure

  1. S. delphini is cultivated in 100 ml TSB at 37 °C on a shaking incubator at 150 rpm for 20 h.
  2. Cells are centrifuged at 5,000 rpm (4,500 x g) at 4 °C for 10 min and the supernatant is applied on to a column filled with 10 ml Amberilite XAD-16 resin.
  3. The column is first washed with 5 bed volumes each of milliQ water, then with 40% and 60% methanol and finally eluted with 80% methanol containing 5% acetic acid at a flow rate of 10 bed volumes per hour.
  4. The eluate is evaporated using a rotary evaporator until all methanol is removed.
  5. The eluate is resuspended with 50 ml water and the pH adjusted to 7.0 with 1 M NaOH. It is then applied on to a column filled with 10 ml Amberilite IRC-50 cation exchange resin.
  6. The column is washed first with water, then with 70% ethanol and eluted with 80% ethanol acidified with 5% acetic acid each with 5 bed volumes at a flow rate of 10 bed volumes per hour.
  7. The eluate is concentrated using a rotary evaporator until all methanol is removed.
  8. The eluate is diluted with water and adjusted to a final concentration of 50 mM sodium phosphate using 1 M sodium phosphate buffer and pH is lowered to 4.2 using 85% phosphoric acid.
  9. In the third purification step, the eluate is separated on a 5 ml SP sepharose cation exchange column with a linear 0 to 1 M NaCl gradient in 50 mM sodium phosphate buffer on an Äkta purifier FPLC at a flow rate of 3 ml/min.
  10. The final purification and desalting of each peak is carried out by reversed phase preparative HPLC (RP-PHPLC) on a nucleosil 100 C-18, 8 x 250 mm column with a linear water acetonitrile (containing 0.1% TFA) gradient of 0% to 60% in 25 min.
  11. Purified compounds are lyophilized and stored at -20 °C.
  12. Qualitative analysis is carried out on an Agilent 1200 HPLC system and a RP-HPLC Waters xBridge C18, 5 mm, 4.6 x 150 mm column. Compounds are eluted with a 15 min linear gradient of aquaeus phosphoric acid (0.1% vol/vol) to acetonitrile at a flow rate of 1.5 ml/min and detected at 210 nm.


    Figure 1. RP-HPLC profile, UV-spectrum and structures of the two QS-inhibitors purified from S. delphini. A. QS-inhibitor, N-[2-(1H-indol-3-yl)ethyl]-urea (yayurea A). B. QS-inhibitor, N-(2-phenethyl)-urea (yayurea B). RP-HPLC is carried out on an Agilent 1200 and Waters XBridge C18, 5 mm, 4.6 x 150 mm column; compounds are eluted with a 15 min linear gradient of 0.1% phosphoric acid to acetonitrile at a flow rate of 1.5 ml/min.

Acknowledgments

This work was funded by grants from the Landesgraduiertenförderung Baden Württemberg “Antimicrobial compounds” and the Deutsche Forschungsgemeinschaft SFB 766. This protocol was adapted from the original published paper Chu et al. (2013).

References

  1. Chu, Y. Y., Nega, M., Wölfle, M., Plener, L., Grond, S., Jung, K. and Götz, F. (2013). A new class of quorum quenching molecules from Staphylococcus species affects communication and growth of gram-negative bacteria. PLoS Pathog 9(9): e1003654.

简介

许多病原体依赖于被称为群体感应的细胞间通信机制的知识,开启了一种新的疾病控制策略:群体猝灭。 在这里,我们提出了一组由革兰氏阳性动物传染病原体细菌分泌的分子的纯化方案,所述细菌是抑制群体感应信号传导并抑制广谱革兰氏菌的生长的'中间体葡萄球菌 - 阴性β-和γ-变形细菌。 这些化合物从葡萄球菌delphini ( S. delphini )中分离。 它们代表一类新的具有化学式N - [2-(1H-吲哚-3-基)乙基] - 脲和N,N-二 - - (2-苯乙基) - 脲,其分别命名为yayurea A和B. 这些物质可以使用这种可扩大的纯化方法从培养物上清液中分离和纯化

材料和试剂

  1. Staphylococcus delphini DSMZ20771菌株(DSM编号:20071)
    注意:DSMZ代表Deutsche Sammlung von Mikroorganismen und Zellkulturen。
  2. 胰蛋白酶大豆肉汤(TSB)(Sigma-Aldrich,目录号:T8907)
  3. Amberilite XAD-16树脂(Sigma-Aldrich,目录号:1-0379)
  4. 甲醇
  5. 乙酸(Merck KGaA)
  6. Amberilite IRC 50阳离子交换树脂(SERVA Electrophoresis GmbH,目录号:40501)
  7. 氢氧化钠
  8. 乙醇
  9. 50mM和1M磷酸钠缓冲液
  10. SP琼脂糖阳离子交换柱(GE Healthcare,目录号:17-5161-01)
  11. 氯化钠
  12. 三氟乙酸(TFA)(Sigma-Aldrich,目录号:T6508)
  13. 用于HPLC的磷酸(Sigma-Aldrich,目录号:79606)
  14. HPLC的乙腈(Mallinckrodt Baker,目录号:9012)

设备

  1. 37℃振荡培养箱(Infors AG)
  2. 离心机(Eppendorf)
  3. 旋转蒸发器(BÜCHILabortechnik AG)
  4. 装备有P-900,UV-900,PH/C-900(GE Healthcare)的ÄktaFPLC
  5. 配有Bischoff HPLC紧凑泵QC-P 2250和多波长检测器QC-1157(Bischoff)的制备型HPLC系统
  6. Nucleosil 100 C-18(8×250mm柱)(MACHEREY-NAGEL,目录号:715332.80)
  7. Agilent 1200系列HPLC系统(Agilent)
  8. Waters XBridge C18(5mm,4.6×150mm柱)(Waters,部件号:186003116)

程序

  1. delphini 在37℃下在100ml TSB中在振荡培养箱上以150rpm培养20小时。
  2. 将细胞在4℃下以5,000rpm(4500xg)离心10分钟,将上清液施加到填充有10ml Amberilite XAD-16树脂的柱上。
  3. 首先用5倍床体积的milliQ水,然后用40%和60%甲醇洗柱,最后用含5%乙酸的80%甲醇以10床体积/小时的流速洗脱。
  4. 使用旋转蒸发器蒸发洗脱液,直到除去所有甲醇
  5. 将洗脱液用50ml水重悬,并用1M NaOH将pH调节至7.0。 然后将其施加到填充有10ml Amberilite IRC-50阳离子交换树脂的柱上。
  6. 首先用水,然后用70%乙醇洗涤柱,用5%乙酸酸化的80%乙醇洗脱,每个柱床体积为5倍,流速为每小时10床体积。
  7. 使用旋转蒸发器浓缩洗脱液,直到除去所有甲醇
  8. 洗脱液用水稀释,并用1M磷酸钠缓冲液调节至终浓度为50mM磷酸钠,用85%磷酸将pH降至4.2。
  9. 在第三纯化步骤中,洗脱液在5ml SP琼脂糖阳离子交换柱上在50mM磷酸钠缓冲液中的线性0-1M NaCl梯度在Äkta纯化器FPLC上以3ml/min的流速分离。 br />
  10. 每个峰的最终纯化和脱盐通过反相制备型HPLC(RP-PHPLC)在核苷100C-18,8×250mm柱上进行,用线性水乙腈(含0.1%TFA)梯度0% 60分钟在25分钟内。
  11. 将纯化的化合物冻干并储存在-20℃
  12. 在Agilent 1200 HPLC系统和RP-HPLC Waters xBridge C18,5mm,4.6×150mm柱上进行定性分析。化合物用15分钟线性梯度的磷酸水溶液(0.1%体积/体积)洗脱至乙腈,流速为1.5ml/min,并在210nm检测。

    图1.从S中纯化的两种QS抑制剂的RP-HPLC图,UV-光谱和结构。 delphini A.QS抑制剂,N' - [2-(1H-吲哚-3-基)乙基] - 脲(yayurea A)。 B.QS抑制剂,N' - (2-苯乙基) - 脲(乙酰脲B)。 RP-HPLC在Agilent 1200和Waters XBridge C18,5mm,4.6×150mm柱上进行;化合物用15分钟线性梯度的0.1%磷酸至乙腈以1.5ml/min的流速洗脱。

致谢

这项工作是由LandesgradüllenförderungBadenWürttemberg"抗微生物化合物"和Deutsche Forschungsgemeinschaft SFB 766的赠款资助的。 该协议改编自原始发表的论文Chu等人(2013)。

参考文献

  1. Chu,Y. Y.,Nega,M.,Wölfle,M.,Plener,L.,Grond,S.,Jung,K.andGötz, 来自葡萄球菌物种的一类新的quorum淬灭分子影响沟通和生长 的革兰氏阴性细菌。 PLoS Pathog 9(9):e1003654。
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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: © 2014 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. Chu, Y., Nega, M. and Götz, F. (2014). Purification and Structural Analysis of QS-inhibiting Compounds from Staphylococcus delphini. Bio-protocol 4(11): e1146. DOI: 10.21769/BioProtoc.1146.
  2. Chu, Y. Y., Nega, M., Wölfle, M., Plener, L., Grond, S., Jung, K. and Götz, F. (2013). A new class of quorum quenching molecules from Staphylococcus species affects communication and growth of gram-negative bacteria. PLoS Pathog 9(9): e1003654.
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