Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.
Glycobiology. 2012 Aug;22(8):1103-17. doi: 10.1093/glycob/cws080. Epub 2012 May 3.
Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-β-d-ManpNAc-(1 → 4)-[3-O-acetyl]-β-d-GlcpNAc-(1 → 6)-α-d-GlcpNH(2)-(1→.
炭疽杆菌 CDC684 是一种天然存在的、无毒的变体，是高度致病性炭疽杆菌 Vollum 的近亲。炭疽杆菌 CDC684 同时含有毒力质粒 pXO1 和 pXO2，但在动物模型中无致病性，这促使人们更仔细地研究其衰减的分子基础。我们使用化学和 NMR 光谱分析对炭疽杆菌 CDC684（Ba684）的次生细胞壁多糖（SCWP）进行了结构表征。SCWP 由一个与炭疽杆菌巴氏、斯特恩和艾姆斯相同的六己糖三糖骨架组成，→4）-β-d-ManpNAc-（1→4）-β-d-GlcpNAc-（1→6）-α-d-GlcpNAc-（1→。值得注意的是，尽管骨架完全聚合，但 SCWP 完全没有所有的半乳糖侧基，这一特征通常构成致病性菌株高度半乳糖化 SCWP 中糖基残基的 50%。这一观察结果突出了缺陷细胞壁组装在毒力中的作用，并表明聚合的发生独立于半乳糖侧基的附着。特别有趣的是，聚合的 Ba684 骨架保留了在正常炭疽杆菌菌株的 SCWP 中发现的亚化学计量丙酮酸缩醛、O-乙酰基和氨基修饰，免疫荧光分析证实 SCWP 表达与结合表面层同源（SLH）结构域的能力相吻合，该结构域包含可提取抗原-1 的 S 层蛋白。丙酮酸先前被证明是保守表位的一部分，介导 SLH 结构域蛋白与底层肽聚糖层的附着。我们发现，Ba684 SCWP 的远端（非还原）末端的一个重复单元在结构上发生了修饰，并且这种修饰方式在正常炭疽杆菌菌株的 SCWP 中完全相同。这些多糖以以下序列结尾：（S）-4,6-O-（1-羧乙基亚甲基）-β-d-ManpNAc-（1→4）-[3-O-乙酰基]-β-d-GlcpNAc-（1→6）-α-d-GlcpNH（2）-（1→。
