Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance.

Gram-positive bacteria surround themselves with a multi-layered macromolecular cell wall that is essential to cell survival and serves as a major target for antibiotics. The cell wall of S. aureus is composed of two major structural components, peptidoglycan (PG) and wall teichoic acid (WTA), together creating a heterogeneous and insoluble matrix that poses a challenge to quantitative compositional analysis. Here, we present 13C CPMAS solid-state NMR spectra of intact cell walls, purified PG, and purified WTA. The spectra reveal the clear molecular differences in the two polymers and enable quantification of PG and WTA in isolated cell walls, an attractive alternative to estimating teichoic acid content from a phosphate analysis of completely pyrolyzed cell walls. Furthermore, we discovered that unique PG and WTA spectral signatures could be identified in whole-cell NMR spectra and used to compare PG and WTA levels among intact bacterial cell samples. The distinguishing whole cell 13C NMR contributions associated with PG include the GlcNAc-MurNAc sugar carbons and glycyl alpha-carbons. WTA contributes carbons from the phosphoribitol backbone. Distinguishing 15N spectral signatures include glycyl amide nitrogens in PG and the esterified D-alanyl amine nitrogens in WTA. 13C NMR analysis was performed with samples at natural abundance and included ten whole-cell sample comparisons. Changes consistent with altered PG and WTA content were detected in whole-cell spectra of bacteria harvested at different growth times and in cells treated with tunicamycin. This use of whole-cell NMR provides quantitative parameters of composition in the context of whole cell activity.

革兰氏阳性菌被多层大分子细胞壁所包围，细胞壁对细胞生存至关重要，并且是抗生素的主要作用靶点。金黄色葡萄球菌的细胞壁由两种主要结构成分组成，即肽聚糖（PG）和壁磷壁酸（WTA），它们共同形成一种异质且不溶的基质，这对定量成分分析构成了挑战。在此，我们展示了完整细胞壁、纯化的肽聚糖以及纯化的壁磷壁酸的13C交叉极化魔角旋转固态核磁共振谱。这些谱图揭示了两种聚合物在分子层面的明显差异，并能够对分离的细胞壁中的肽聚糖和壁磷壁酸进行定量，这是一种比通过对完全热解的细胞壁进行磷酸盐分析来估算磷壁酸含量更具吸引力的替代方法。此外，我们发现可以在全细胞核磁共振谱中识别出独特的肽聚糖和壁磷壁酸的谱图特征，并用于比较完整细菌细胞样本中肽聚糖和壁磷壁酸的水平。与肽聚糖相关的全细胞13C核磁共振的独特贡献包括N - 乙酰葡糖胺 - N - 乙酰胞壁酸的糖碳和甘氨酰α - 碳。壁磷壁酸贡献来自磷酸核糖醇主链的碳。独特的15N谱图特征包括肽聚糖中的甘氨酰胺氮和壁磷壁酸中酯化的D - 丙氨胺氮。13C核磁共振分析是在自然丰度的样本上进行的，包括十次全细胞样本比较。在不同生长时间收获的细菌以及用衣霉素处理的细胞的全细胞谱图中，检测到了与肽聚糖和壁磷壁酸含量改变相符的变化。这种全细胞核磁共振的应用提供了全细胞活动背景下的成分定量参数。