Department of Structural Biology, School of Medicine, University of Pittsburgh, Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15261, United States.
Department of Biological Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, United States.
J Magn Reson. 2023 Dec;357:107587. doi: 10.1016/j.jmr.2023.107587. Epub 2023 Nov 10.
We present a multidimensional magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize native Pseudomonas fluorescens colony biofilms at natural abundance without isotope-labelling. By using a high-resolution INEPT-based 2D H-C ssNMR spectrum and thorough peak deconvolution at the 1D ssNMR spectra, approximately 80/134 (in 1D/2D) distinct biofilm chemical sites were identified. We compared CP and INEPT C ssNMR spectra to differentiate signals originating from the mobile and rigid fractions of the biofilm, and qualitatively determined dynamical changes by comparing CP buildup behaviors. Protein and polysaccharide signals were differentiated and identified by utilizing FapC protein signals as a template, a biofilm forming functional amyloid from Pseudomonas. We identified several biofilm polysaccharide species such as glucose, mannan, galactose, heptose, rhamnan, fucose and N-acylated mannuronic acid by using H and C chemical shifts obtained from the 2D spectrum. To our knowledge, this study marks the first high-resolution multidimensional ssNMR characterization of a native bacterial biofilm. Our experimental pipeline can be readily applied to other in vitro biofilm model systems and natural biofilms and holds the promise of making a substantial impact on biofilm research, fostering new ideas and breakthroughs to aid in the development of strategic approaches to combat infections caused by biofilm-forming bacteria.
我们提出了一项多维魔角旋转（MAS）固态 NMR（ssNMR）研究，以在不进行同位素标记的情况下，对天然荧光假单胞菌菌落生物膜进行特征描述。通过使用高分辨率基于 INEPT 的 2D H-C ssNMR 光谱和在 1D ssNMR 光谱上进行彻底的峰分解，我们鉴定了大约 80/134 个（在 1D/2D 中）不同的生物膜化学位点。我们比较了 CP 和 INEPT C ssNMR 光谱以区分来自生物膜可移动和刚性部分的信号，并通过比较 CP 积累行为定性地确定动态变化。通过利用 FapC 蛋白信号作为模板（一种来自假单胞菌的生物膜形成功能淀粉样蛋白），我们区分和鉴定了蛋白质和多糖信号。我们通过使用从 2D 光谱获得的 H 和 C 化学位移来鉴定几种生物膜多糖，如葡萄糖、甘露聚糖、半乳糖、庚糖、鼠李聚糖、岩藻糖和 N-酰化甘露糖酸。据我们所知，这项研究标志着首次对天然细菌生物膜进行高分辨率多维 ssNMR 表征。我们的实验方案可以很容易地应用于其他体外生物膜模型系统和天然生物膜，并有望对生物膜研究产生重大影响，为对抗生物膜形成细菌引起的感染提供新的思路和突破。
