W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
Laboratory of Bacterial PSs, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
Carbohydr Polym. 2022 Sep 1;291:119547. doi: 10.1016/j.carbpol.2022.119547. Epub 2022 Apr 29.
Microbial polysaccharide characterization requires purification that often involves detergent precipitation and lyophilization. Here we examined physicochemical changes following lyophilization of Cryptococcus neoformans exopolysaccharide (EPS). Solution H Nuclear Magnetic Resonance (NMR) reveals significant anomeric signal attenuation following lyophilization of native EPS while H solid-state Nuclear Magnetic Resonance (ssNMR) shows few changes, suggesting diminished molecular motion and consequent broadening of H NMR polysaccharide resonances. C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide bind native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.
微生物多糖的特性分析需要进行纯化，通常包括去污剂沉淀和冷冻干燥。在此，我们研究了新型隐球菌胞外多糖（EPS）冷冻干燥后的理化变化。溶液 H 核磁共振（NMR）显示，天然 EPS 冷冻干燥后，端基信号显著衰减，而 H 固体核磁共振（ssNMR）显示变化较小，表明分子运动减弱，继而导致 H NMR 多糖共振峰变宽。C ssNMR、动态光散射和透射电子显微镜显示，尽管天然 EPS 具有刚性的分子特性，且含有较小、松散的多糖组装体，但冷冻干燥和再悬浮的 EPS 是无序的，且含有较大的致密聚集体，表明多糖组装体内部的结构水分子在剧烈的冷冻干燥过程中被除去。重要的是，针对新型隐球菌多糖的 mAb 与天然 EPS 的结合能力强于与冷冻干燥 EPS 的结合能力。综上所述，这些观察结果表明，在解释已冻干至干燥状态的多糖的生物学和免疫学特性时应谨慎。
