海豚葡萄球菌的QS抑制化合物的纯化和结构分析

Ya-Yun  Chu; Mulugeta  Nega; Friedrich  Götz

doi:10.21769/BioProtoc.1146

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Peer-reviewed

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

YC Ya-Yun Chu

MN Mulugeta Nega

FG Friedrich Götz email

发布: 2014年06月05日第4卷第11期 DOI: 10.21769/BioProtoc.1146 浏览次数: 8070

评审: Fanglian HeAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Sep 2013

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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

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

Equipment

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

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

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.
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.