We present a high-resolution magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize nontuberculous mycobacteria (NTM). We studied two different NTM strains, Mycobacterium smegmatis, a model, non-pathogenic strain, and Mycobacterium abscessus, an emerging and important human pathogen. Hydrated NTM samples were studied at natural abundance without isotope-labelling, as whole-cells versus cell envelope isolates, and native versus fixed sample preparations. We utilized 1D13C and 2D 1H-13C ssNMR spectra and peak deconvolution to identify NTM cell-wall chemical sites. More than ∼100 distinct 13C signals were identified in the ssNMR spectra. We provide tentative assignments for ∼30 polysaccharides by using well resolved 1H/13C chemical shifts from the 2D INEPT-based 1H-13C ssNMR spectrum. The signals originating from both the flexible and rigid fractions of the whole-cell bacteria samples were selectively analyzed by utilizing either CP or INEPT based 13C ssNMR spectra. CP buildup curves provide insights into the dynamical similarity of the cell-wall components for NTM strains. Signals from peptidoglycan, arabinogalactan and mycolic acid were identified. The majority of the 13C signals were not affected by fixation of the whole cell samples. The isolated cell envelope NMR spectrum overlap with the whole-cell spectrum to a large extent, where the latter has more signals. As an orthogonal way of characterizing these bacteria, electron microscopy (EM) was used to provide spatial information. ssNMR and EM data suggest that the M. abscessus cell-wall is composed of a smaller peptidoglycan layer which is more flexible compared to M. smegmatis, which may be related to its higher pathogenicity. Here in this work, we used high-resolution 2D ssNMR first time to characterize NTM strains and identify chemical sites. These results will aid the development of structure-based approaches to combat NTM infections.

中文翻译：

---

高分辨率 2D 固态 NMR 提供对非结核分枝杆菌的深入了解


我们提出了一项高分辨率魔角旋转 (MAS) 固态核磁共振 (ssNMR) 研究来表征非结核分枝杆菌 (NTM)。我们研究了两种不同的 NTM 菌株：耻垢分枝杆菌（一种模型非致病菌株）和脓肿分枝杆菌（一种新兴的重要人类病原体）。水合 NTM 样品在天然丰度下进行研究，无需同位素标记，包括全细胞与细胞包膜分离物，以及天然样品与固定样品制备。我们利用 1D13C 和 2D 1H-13C ssNMR 谱和峰解卷积来识别 NTM 细胞壁化学位点。在 ssNMR 谱中识别出超过 100 个不同的 13C 信号。我们通过使用基于 2D INEPT 的 1H-13C ssNMR 谱中解析良好的 1H/13C 化学位移，对 ∼30 种多糖进行了初步分配。利用基于 CP 或 INEPT 的 13C ssNMR 光谱选择性地分析来自全细胞细菌样品的柔性和刚性部分的信号。 CP 构建曲线提供了对 NTM 菌株细胞壁成分动力学相似性的深入了解。鉴定了来自肽聚糖、阿拉伯半乳聚糖和分枝菌酸的信号。大多数 13C 信号不受全细胞样品固定的影响。孤立细胞包膜核磁共振谱与全细胞谱有很大程度的重叠，后者信号较多。作为表征这些细菌的正交方法，电子显微镜 (EM) 用于提供空间信息。 ssNMR和EM数据表明，脓肿分枝杆菌细胞壁由较小的肽聚糖层组成，与耻垢分枝杆菌相比，其更加柔韧，这可能与其较高的致病性有关。 在这项工作中，我们首次使用高分辨率 2D ssNMR 来表征 NTM 菌株并识别化学位点。这些结果将有助于开发基于结构的方法来对抗 NTM 感染。