Spatial organization of Pseudomonas aeruginosa biofilms probed by combined matrix-assisted Laser desorption ionization mass spectrometry and confocal Raman microscopy
Spatial organization of Pseudomonas aeruginosa biofilms probed by combined matrix-assisted Laser desorption ionization mass spectrometry and confocal Raman microscopy
复制标题
DOI：
10.1039/c4an00435c
复制
发表时间：
2014-11-21
期刊：
影响因子：
4.2
通讯作者：
Bohn, Paul W.
中科院分区：
文献类型：
--
作者：
Masyuko, Rachel N.;Lanni, Eric J.;Bohn, Paul W.
作为表面附着生物膜生长的细菌在几个重要特征上与浮游细胞显着不同，这些特征反映在细胞的时空组织及其分泌的细胞外聚合物中。本文结合基质辅助激光解吸电离质谱 (MALDI MS) 和共焦拉曼显微光谱 (CRM) 来探索定义这些生物膜的结构和化学特征，以表征和比较铜绿假单胞菌生物膜和浮游细胞中发现的生物分子的组成和分布。与浮游培养物相比，三天的铜绿假单胞菌生物膜在分子组成上表现出显着差异。 CRM 显示，野生型浮游细胞拉曼光谱的特征是与细胞成分相关的谱带，并且主要由 DNA 和 RNA 相关谱带的贡献组成。相反，生物膜光谱主要由糖脂-鼠李糖脂-多糖和分泌蛋白的特征谱带主导。依次应用 LDI MS 来鉴定生物膜中存在的鼠李糖脂。还使用酰基高丝氨酸内酯群体感应缺陷突变体（Delta lasl Delta rhl1）进行了实验，该突变体无法产生鼠李糖脂。 CRM 和 LDI MS 分析表明，虽然浮游群体感应缺陷细胞的分子组成与野生型浮游细胞相似，但在生物膜生长后，突变体中观察到了一些组成差异，包括完全不存在可检测到的鼠李糖脂。突变细胞的 CRM 振动光谱在浮游和生物膜生长条件下非常相似，表明在缺乏功能性群体感应机制的情况下，生物膜的形成受到极大阻碍。
Bacteria growing as surface attached biofilms differ significantly from planktonic cells in several important traits that are reflected in the spatiotemporal organization of the cells and the extracellular polymeric substances they secrete. The structural and chemical features that define these biofilms are explored here using a combination of matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and confocal Raman microspectroscopies (CRM) to characterize and compare the composition and distribution of biomolecules found in biofilms and planktonic cells of the bacterium Pseudomonas aeruginosa. Three-day old P. aeruginosa biofilms show dramatic differences in molecular composition compared to planktonic cultures. CRM reveals that wild-type planktonic cell Raman spectra are characterized by bands linked to cellular constituents and are dominated by contributions from DNA-and RNA-related bands. In contrast, biofilm spectra are dominated by bands characteristic of glycolipids - rhamnolipids - polysaccharides and by secreted proteins. LDI MS was applied in turn to identify the rhamnolipids present in the biofilm. Experiments were also conducted using an acyl homoserine lactone quorum sensing-deficient mutant (Delta lasl Delta rhl1), which is incapable of producing rhamnolipids. CRM and LDI MS analyses revealed that while molecular composition of the planktonic quorum sensing-deficient cells is similar to that of the wild-type planktonic cells, several compositional differences are observed in the mutant after biofilm growth, including complete absence of detectable rhamnolipids. CRM vibrational spectra of the mutant cells are very similar for planktonic and biofilm growth conditions, indicating that biofilm formation is greatly hindered in the absence of functioning quorum sensing machinery.