Mihooliya Kanti N, Kumar Ambrish, Heiss Christian, Kumari Alka, Boniche-Alfaro Camilla, Azadi Parastoo, Fries Bettina C
Division of Infectious Diseases, Department of Medicine, Renaissance School of Medicine, Stony Brook University, Stony Brook, USA; Veteran's Administration Medical Center, Northport, New York, USA.
Complex Carbohydrate Research Center (CCRC), University of Georgia, Athens, GA 30602, USA.
Carbohydr Polym. 2025 Jun 15;358:123531. doi: 10.1016/j.carbpol.2025.123531. Epub 2025 Mar 24.
Cell surface-associated polysaccharides in Klebsiella are major virulence determinants and crucial targets for developing vaccines. Traditionally, the purification of these cell surface polysaccharides from Klebsiella pneumoniae involves a multi-step process comprising phenol extraction, nuclease digestion, ultracentrifugation, and repeated ethanol extractions. In this study, we evaluated size exclusion chromatography for effectively eliminating nucleic acid contamination while purifying high molecular weight cell surface-associated polysaccharides. Post-initial extraction, the nucleic acid content remains significantly elevated, and kinetic analysis reveals that DNase I and RNase A digestion is neither economically viable nor effective for removing these contaminants. Employing an appropriate size exclusion resin removes over 99 % of nucleic acid contamination, as confirmed by nucleic acid content analysis and agarose gel electrophoresis. Purity and structural analysis using H 1D-NMR and 2D-NMR demonstrate that the cell surface-associated polysaccharide purified with this study is highly homogeneous and identified as antigenic O-polysaccharide. This approach streamlines the purification process by eliminating the need for nuclease digestion and additional ethanol precipitation steps.
肺炎克雷伯菌中的细胞表面相关多糖是主要的毒力决定因素，也是开发疫苗的关键靶点。传统上，从肺炎克雷伯菌中纯化这些细胞表面多糖涉及一个多步骤过程，包括苯酚提取、核酸酶消化、超速离心和反复乙醇提取。在本研究中，我们评估了尺寸排阻色谱法在纯化高分子量细胞表面相关多糖时有效去除核酸污染的能力。初始提取后，核酸含量仍显著升高，动力学分析表明，DNA酶I和RNA酶A消化在经济上既不可行，也无法有效去除这些污染物。通过核酸含量分析和琼脂糖凝胶电泳证实，使用合适的尺寸排阻树脂可去除超过99%的核酸污染。使用H 1D-NMR和2D-NMR进行的纯度和结构分析表明，本研究纯化的细胞表面相关多糖高度均一，并被鉴定为抗原性O-多糖。这种方法通过消除核酸酶消化和额外乙醇沉淀步骤的需要，简化了纯化过程。
