Gannesen Andrei V, Zdorovenko Evelina L, Botchkova Ekaterina A, Hardouin Julie, Massier Sebastien, Kopitsyn Dmitry S, Gorbachevskii Maxim V, Kadykova Alexandra A, Shashkov Alexander S, Zhurina Marina V, Netrusov Alexander I, Knirel Yuriy A, Plakunov Vladimir K, Feuilloley Marc G J
Winogradsky Institute of Microbiology, Federal Research Centre «Fundamentals of Biotechnology», Russian Academy of Sciences, Moscow, Russia.
Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Front Microbiol. 2019 Jun 21;10:1284. doi: 10.3389/fmicb.2019.01284. eCollection 2019.
In skin, (former ) can behave as an opportunistic pathogen, depending on the strain and environmental conditions. Acneic strains of form biofilms inside skin-gland hollows, inducing inflammation and skin disorders. The essential exogenous products of accumulate in the extracellular matrix of the biofilm, conferring essential bacterial functions to this structure. However, little is known about the actual composition of the biofilm matrix of . Here, we developed a new technique for the extraction of the biofilm matrix of Gram-positive bacteria without the use of chemical or enzymatic digestion, known to be a source of artifacts. Our method is based on the physical separation of the cells and matrix of sonicated biofilms by ultracentrifugation through a CsCl gradient. Biofilms were grown on the surface of cellulose acetate filters, and the biomass was collected without contamination by the growth medium. The biofilm matrix of the acneic RT5 strain appears to consist mainly of polysaccharides. The following is the ratio of the main matrix components: 62.6% polysaccharides, 9.6% proteins, 4.0% DNA, and 23.8% other compounds (porphyrins precursors and other). The chemical structure of the major polysaccharide was determined using a nuclear magnetic resonance technique, the formula being →6)-α-D-Gal-(1→4)-β-D-ManNAc3NAcA-(1→6)-α-D-Glc-(1→4)-β-D-ManNAc3NAcA-(1→3)-β-GalNAc-(1→. We detected 447 proteins in the matrix, of which the most abundant were the chaperonin GroL, the elongation factors EF-Tu and EF-G, several enzymes of glycolysis, and proteins of unknown function. The matrix also contained more than 20 hydrolases of various substrata, pathogenicity factors, and many intracellular proteins and enzymes. We also performed surface-enhanced Raman spectroscopy analysis of the RT5 matrix for the first time, providing the surface-enhanced Raman scattering (SERS) profiles of the RT5 biofilm matrix and biofilm biomass. The difference between the matrix and biofilm biomass spectra showed successful matrix extraction rather than simply the presence of cell debris after sonication. These data show the complexity of the biofilm matrix composition and should be essential for the development of new anti- biofilms and potential antibiofilm drugs.
在皮肤中，（前者）可作为机会致病菌，这取决于菌株和环境条件。痤疮丙酸杆菌菌株在皮肤腺腔内形成生物膜，引发炎症和皮肤疾病。痤疮丙酸杆菌的必需外源性产物积聚在生物膜的细胞外基质中，赋予该结构重要的细菌功能。然而，对于痤疮丙酸杆菌生物膜基质的实际组成了解甚少。在此，我们开发了一种无需使用化学或酶消化来提取革兰氏阳性菌生物膜基质的新技术，已知化学或酶消化是产生假象的一个来源。我们的方法基于通过氯化铯梯度超速离心对超声处理后的生物膜的细胞和基质进行物理分离。生物膜在醋酸纤维素滤膜表面生长，收集生物量时未被生长培养基污染。痤疮丙酸杆菌RT5菌株的生物膜基质似乎主要由多糖组成。主要基质成分的比例如下：62.6%为多糖，9.6%为蛋白质，4.0%为DNA，23.8%为其他化合物（卟啉前体及其他）。使用核磁共振技术确定了主要多糖的化学结构，其化学式为→6)-α-D-半乳糖-(1→4)-β-D-甘露糖胺3NAcA-(1→6)-α-D-葡萄糖-(1→4)-β-D-甘露糖胺3NAcA-(1→3)-β-氨基半乳糖-(1→。我们在基质中检测到447种蛋白质，其中含量最丰富的是伴侣蛋白GroL、延伸因子EF-Tu和EF-G、几种糖酵解酶以及功能未知的蛋白质。基质中还含有20多种针对各种底物的水解酶、致病因子以及许多细胞内蛋白质和酶。我们还首次对痤疮丙酸杆菌RT5基质进行了表面增强拉曼光谱分析，提供了痤疮丙酸杆菌RT5生物膜基质和生物膜生物量的表面增强拉曼散射（SERS）图谱。基质和生物膜生物量光谱之间的差异表明成功提取了基质，而不仅仅是超声处理后存在细胞碎片。这些数据显示了生物膜基质组成的复杂性，对于开发新型抗痤疮丙酸杆菌生物膜和潜在的抗生物膜药物至关重要。
