Laabei Maisem, Jamieson William D, Lewis Simon E, Diggle Stephen P, Jenkins A Tobias A
Department of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
Appl Microbiol Biotechnol. 2014 Aug;98(16):7199-209. doi: 10.1007/s00253-014-5904-3. Epub 2014 Jun 29.
Rhamnolipids (RLs) are heterogeneous glycolipid molecules that are composed of one or two L-rhamnose sugars and one or two β-hydroxy fatty acids, which can vary in their length and branch size. They are biosurfactants, predominantly produced by Pseudomonas aeruginosa and are important virulence factors, playing a major role in P. aeruginosa pathogenesis. Therefore, a fast, accurate and high-throughput method of detecting such molecules is of real importance. Here, we illustrate the ability to detect RL-producing P. aeruginosa strains with high sensitivity, based on an assay involving phospholipid vesicles encapsulated with a fluorescent dye. This vesicle-lysis assay is confirmed to be solely sensitive to RLs. We illustrate a half maximum concentration for vesicle lysis (EC50) of 40 μM (23.2 μg/mL) using pure commercial RLs and highlight the ability to semi-quantify RLs directly from the culture supernatant, requiring no extra extraction or processing steps or technical expertise. We show that this method is consistent with results from thin-layer chromatography detection and dry weight analysis of RLs but find that the widely used orcinol colorimetric test significantly underestimated RL quantity. Finally, we apply this methodology to compare RL production among strains isolated from either chronic or acute infections. We confirm a positive association between RL production and acute infection isolates (p = 0.0008), highlighting the role of RLs in certain infections.
鼠李糖脂（RLs）是由一个或两个L-鼠李糖和一个或两个β-羟基脂肪酸组成的异质糖脂分子，其长度和支链大小各不相同。它们是生物表面活性剂，主要由铜绿假单胞菌产生，并且是重要的毒力因子，在铜绿假单胞菌的致病过程中起主要作用。因此，一种快速、准确且高通量的检测此类分子的方法具有重要意义。在此，我们展示了基于一种涉及用荧光染料包封的磷脂囊泡的检测方法，能够高灵敏度地检测产生RL的铜绿假单胞菌菌株。这种囊泡裂解检测方法被证实仅对鼠李糖脂敏感。我们使用纯商业鼠李糖脂说明了囊泡裂解的半数最大浓度（EC50）为40 μM（23.2 μg/mL），并强调了直接从培养上清液中半定量鼠李糖脂的能力，无需额外的提取或处理步骤或专业技术知识。我们表明该方法与鼠李糖脂的薄层色谱检测和干重分析结果一致，但发现广泛使用的间苯二酚比色法显著低估了鼠李糖脂的含量。最后，我们应用该方法比较从慢性或急性感染中分离出的菌株之间的鼠李糖脂产量。我们证实了鼠李糖脂产量与急性感染分离株之间存在正相关（p = 0.0008），突出了鼠李糖脂在某些感染中的作用。
