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Immunologie und Infektion

薄层色谱分析分枝杆菌的脂质成分

Published: April 16, 2021
doi:
10.3791/62368
, ,
1Departament de Genètica i de Microbiologia, Facultat de Biociències,Universitat Autònoma de Barcelona

Summary

提出一个协议，以提取各种分枝杆菌细胞壁的总脂质含量。此外，还显示了不同类型的肌酸的萃取和分析协议。还提供了一个薄层色谱协议来监测这些分形化合物。

Abstract

分枝杆菌物种在生长速度、色素递迫存在、固体介质上显示的群落形态以及其他表型特征方面可能各不相同。然而，它们都有一个共同的分枝杆菌最相关的特征：它独特的高度疏水细胞壁。分枝杆菌物种含有一种膜-共价连结复合物，包括阿拉比诺甘丹、多肽和长链的支肠酸，其类型不同于分枝杆菌物种。此外， 肌菌还可以在其细胞表面产生位于非共价链接的脂质，如邻苯二甲酸二酯（PDIM）、酚甘油脂（PGL）、甘油脂（GPL）、乙酰三聚醇（AT）或磷脂二恶英（PIM）等。其中一些被认为是致病性分枝杆菌的毒性因素，或宿主-分枝杆菌相互作用中的关键抗原脂。由于这些原因，对支点血脂的研究有着极大的兴趣，因为这种脂质在几个领域得到应用，从了解它们在分枝杆菌感染致病性中的作用，到可能暗示它们是治疗传染病和其他疾病（如癌症）的免疫调节剂。在这里，提出了一个简单的方法来提取和分析总脂质含量和用有机溶剂混合物生长在固体介质中的分枝杆菌细胞的肌酸成分。获得脂质提取物后，将执行薄层色谱 （TLC） 来监测提取的化合物。该示例实验采用四种不同的分枝杆菌进行：环境快速增长的肌 杆菌布鲁马和支原体杆菌， 生长缓慢的 肌杆菌博维斯 杆菌Calmette-Guérin（BCG）和机会型病原体快速生长的分 枝杆菌脓肿， 表明本协议中显示的方法可用于广泛的分枝杆菌。

Introduction

Mycobacterium 属包括致病性物种和非致病性物种，其特点是具有由其特有的脂质形成的高度疏水性和不透水性细胞壁。具体来说，分枝杆菌细胞壁含有霉菌酸， 这是α-烷基和β-羟基脂肪酸，其中α分支是恒定的所有肌酸（长度除外）和β链，称为中分子链，是一个长的碱性链，可能包含不同的功能化学组描述与文献（α，α’-，甲氧基， [-，环氧树脂，卡盒-和+-1-甲氧-肌酸），因此产生七种类型的肌酸（I-VII）1.此外，其他具有毫无疑问重要性的脂质也存在于分枝杆菌物种的细胞壁中。致病物种，如 Mycobacterium tuberculosis, 结核病的致病剂2 产生特定的脂基毒性因子，如邻苯二甲酸二甲酸酯 （PDIM）、酚类糖脂 （PGL）、二、三和五氯乙酰丙烯酸酯 （DAT、TAT 和 PAT） 或硫化物等3.它们在分不育表面的存在与改变宿主免疫反应的能力有关，因此，宿主内部的分不分母的进化和持久性4.例如，三乙基甘油 （TAG） 的存在与血统 2- 北京子谱的超病毒表型有关 M. tuberculosis, 可能是因为它能够减弱宿主的免疫反应5,6.其他相关的脂质是结块状和非结管分枝杆菌中的脂糖（LOSs）。在 Mycobacterium marinum，LOS在其细胞壁的存在与滑动运动和形成生物膜的能力有关，并干扰巨噬细胞模式识别受体的识别，影响宿主噬细胞吸收和消灭细菌7,8.此外，一些脂质的缺失或存在允许同一物种的成员在与宿主细胞相互作用时，被分为具有毒性或衰减型的不同体型。例如，在粗糙的太平间类型中缺少糖镜（GPL） Mycobacterium abscessus 已与诱导噬细胞内酸化的能力有关，因此细胞凋亡9，不像光滑的太平型，拥有GPL在他们的表面。此外，分机细胞壁的脂质含量与改变宿主免疫反应的能力有关。这在使用一些分枝杆菌触发针对不同病理的保护性免疫特征的背景下是相关的10,11,12,13. 例如，已经证明这一点 Mycolicibacterium vaccae，一种作为结核病免疫治疗疫苗进行第三阶段临床试验的沙氏杆菌，显示两种殖民的死型。虽然光滑的表型，其中含有聚酯在其表面，触发Th2反应，粗糙的表型没有聚酯可以诱导Th1配置文件时，它与宿主免疫细胞相互作用14.分枝杆菌细胞中存在的脂质的种类不仅取决于分枝杆菌物种，还取决于分枝杆菌培养的条件：孵化时间15,16 或文化媒介的构成17,18.事实上，培养媒介成分的变化影响着抗肿瘤和免疫刺激活性的 M. bovis BCG 和 Mycolicibacterium brumae in vitro17.此外，保护性免疫剖面触发 M. bovis BCG 反对 M. tuberculosis 小鼠模型的挑战也取决于文化媒介 M. bovis BCG 增长17.然后，这些可能与每个培养条件下的分枝杆菌的脂质组成有关。由于所有这些原因，对分枝杆菌脂质含量的研究是相关的。提出了提取和分析支点细胞壁脂质成分的视觉程序。

Protocol

1. 从分枝杆菌中提取总非同价相关脂质（图1） 从固体介质中划出 0.2 克分枝杆菌，并加入带多氟乙烯 （PTFE） 衬里螺丝帽的玻璃管。添加由 5 mL 氯仿和 10 mL 甲醇（氯仿：甲醇，1：2）组成的溶液。注：使用有机溶剂时，只应使用玻璃接收器。不允许使用塑料容器。此外，需要为瓶子的PTFE衬里螺丝帽。警告：氯仿是一种潜在的有毒和极其危险的物质。它必须使?…

Representative Results

为了显示不同分枝杆菌物种中的各种脂质 ，M. bovis BCG 被选中，因为它是粗糙和生长缓慢的分枝杆菌。在手术中增加了粗糙和快速增长的 M.偶然 和 M.布鲁马 ，最后，也包括 了M.脓肿 的光滑的死型。这四个物种使我们能够可视化广泛的分枝杆菌衍生脂质，如乙酰链球菌（AT）、GPL、PDIM、PGL、PIM、TDM和TMM。此外，所有四个物种都有不同的肌酸模式。 在…

Discussion

提出了一个简单的协议，被认为是从分不育细胞壁中提取非高血脂化合物的黄金标准方法。显示从四种不同分枝杆菌提取的脂质中，一维和二维 TLC 的进一步可视化。

氯仿和甲醇连续两次结合混合物来恢复支核细胞的脂质含量，是使用最广泛的溶剂混合物23、24、25、26、27、28、29。</su…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

这项研究由西班牙科学、创新和大学部（RTI2018-098777-B-I00）、联邦基金和加泰罗尼亚总务局（2017SGR-229）资助。桑德拉·瓜拉尔-加里多是加泰罗尼亚总务部的博士学位合同（FI）的接受者。

Materials

Acetic Acid Merck 100063 CAUTION. Anhydrous for analysis EMSURE® ACS,ISO,Reag. Ph Eur
Acetone Carlo Erba 400971N CAUTION. ACETONE RPE-ACS-ISO FOR ANALYS ml 1000
Anthrone Merck 8014610010 Anthrone for synthesis.
Benzene Carlo Erba 426113 CAUTION. Benzene RPE – For analysis – ACS 2.5 l
Capillary glass tube Merck BR708709 BRAND® disposable BLAUBRAND® micropipettes, intraMark
Chloroform Carlo Erba 412653 CAUTION. Chloroform RS – For HPLC – Isocratic grade – Stabilized with ethanol 2.5 L
Dry block heater J.P. Selecta 7471200
Dicloromethane Carlo Erba 412622 CAUTION. Dichloromethane RS – For HPLC – Isocratic grade – Stabilized with amylene 2.5 L
Diethyl ether Carlo Erba 412672 CAUTION. Diethyl ether RS – For HPLC – Isocratic grade – Not stabilized 2.5 L
Ethyl Acetate Panreac 1313181211 CAUTION. Ethyl acetate (Reag. USP, Ph. Eur.) for analysis, ACS, ISO
Ethyl Alcohol Absolute Carlo Erba 4146072 CAUTION. Ethanol absolute anhydrous RPE – For analysis – ACS – Reag. Ph.Eur. – Reag. USP 1 L
Glass funnel VidraFOC DURA.2133148 1217/1
Glass tube VidraFOC VFOC.45066A-16125 Glass tube with PTFE recovered cap
Methanol Carlo Erba 412722 CAUTION. Methanol RS – For HPLC – GOLD – Ultragradient grade 2.5 L
Molybdatophosphoric acid hydrate Merck 51429-74-4 CAUTION.
Molybdenum Blue Spray Reagent, 1.3% Sigma M1942-100ML CAUTION.
n-hexane Carlo Erba 446903 CAUTION. n-Hexane 99% RS – ATRASOL – For traces analysis 2.5 L
n-nitroso-n-methylurea Sigma N4766 CAUTION
Orbital shaking platform DDBiolab 995018 NB-205L benchtop shaking incubator
Petroleum ether (60-80ºC) Carlo Erba 427003 CAUTION. Petroleum ether 60 – 80°C RPE – For analysis 2.5 L
Sprayer VidraFOC 712/1
Sodium sulphate anhydrous Merck 238597
Sulfuric acid 95-97% Merck 1007311000 CAUTION. Sulfuric acid 95-97%
TLC chamber Merck Z204226-1EA Rectangular TLC developing tanks, complete L × H × W 22 cm × 22 cm × 10 cm
TLC plate Merck 1057210001 TLC SilicaGel 60- 20×20 cm x 25 u
TLC Plate Heater CAMAG 223306 CAMAG TLC Plat Heater III
Toluene Carlo Erba 488551 CAUTION. Toluene RPE – For analysis – ISO – ACS – Reag.Ph.Eur. – Reag.USP 1 L
Vortex Fisher Scientific 10132562 IKA Agitador IKA vórtex 3
1-naphthol Sigma-Aldrich 102269427 CAUTION.
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Tags

Keywords: MycobacteriaLipidsThin Layer ChromatographyExtractionChloroformMethanolMycolic AcidN-hexaneTLC
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Guallar-Garrido, S., Luquin, M., Julián, E. Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography. J. Vis. Exp. (170), e62368, doi:10.3791/62368 (2021).
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