Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, Germany.
mBio. 2021 Jun 29;12(3):e0089721. doi: 10.1128/mBio.00897-21. Epub 2021 May 26.
Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (3 and 7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from 2, 3, 7, 9, and 11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.
囊聚合物是致病性细菌的重要毒力因子，被用作糖缀合物疫苗制剂中的抗原。一些革兰氏阴性病原体表达类似于革兰氏阳性壁磷壁酸的多（糖基甘油基磷酸盐）囊聚合物，由 TagF 样囊聚合物聚合酶合成。到目前为止，由于缺乏聚合所需的供体底物之一对映纯 CDP-甘油，这些酶在疫苗开发研究中的生物技术应用受到限制。在这里，我们使用 CTP：甘油-磷酸胞苷转移酶（GCT）和 TagF 样聚合酶来合成猪病原体胸膜肺炎放线杆菌血清型 3 和 7（3 和 7）的多（糖基甘油基磷酸盐）囊聚合物主链。通过高效液相色谱法证实了 GCT 活性，并通过全面的核磁共振研究分析了聚合物。建立了固相合成方案，以允许潜在的聚合物生产扩大规模。此外，利用甘油激酶的一锅反应使我们能够从廉价、广泛可用的底物开始反应。最后，这项研究强调，通过突变活性位点残基，多结构域 TagF 样聚合酶可以转化为单作用转移酶，而转移酶反过来又可以作用于构建结构新颖的聚合物。总的来说，我们的方案从 2、3、7、9 和 11、脑膜炎奈瑟菌血清组 H 以及 Bibersteinia trehalosi 血清型 T3 和 T15 提供了对映纯、天然同型的囊聚合物主链。用于生产动物疫苗的经济合成平台可以帮助减少畜牧业中抗生素的过度和滥用，这极大地助长了抗生素耐药性的增加。在这里，我们描述了一种高度通用、易于使用的混合搭配工具箱，用于生成含有甘油磷酸的囊聚合物，这些聚合物可作为胸膜肺炎放线杆菌和 Bibersteinia trehalosi 的糖缀合物疫苗的抗原，这两种病原体在猪、羊和牛的工业中造成了相当大的经济损失。我们已经建立了可扩展的协议，用于利用具有模块化架构的多功能酶级联进行开发，从制备规模生产对映纯 CDP-甘油开始，这是许多细菌表面结构的前体。因此，我们的方法不仅允许合成囊聚合物，还可能可用于（化学）酶促合成其他含有甘油磷酸的结构，如革兰氏阳性壁磷壁酸或脂磷壁酸。
