Biosynthesis of the Campylobacter jejuni phophoramidate

食源性病原体空肠弯曲杆菌是世界范围内细菌性肠胃炎的主要原因之一。与其他用碳水化合物包裹表面，然后用磷酸化结构修饰表面的细菌一样，空肠假梭菌表达复杂的荚膜多糖（CPS），这些多糖被磷酸甲酯（MeOPN）残基修饰，我们已经证明这些残基在细胞侵袭、血清敏感性、生物膜形成、噬菌体结合、昆虫和真菌杀死中起重要作用。本研究采用高分辨率魔角旋转核磁共振波谱（HR-MAS NMR）和靶向代谢组学质谱等方法研究空肠C. MeOPN生物合成过程中涉及的酶。HR-MAS NMR已经成为分析少量生物材料和获得近液体样光谱的强大技术，我们计划将这项技术扩展到表征宿主-病原体相互作用。此外，我们将研究MeOPN取代对CPS三维结构的影响。该项目将为合成一种具有多种生物学作用的关键空肠梭菌CPS修饰提供基础的生化信息。了解这部分的生物合成以及它如何影响CPS的三维结构将使我们更好地了解细菌如何逃避宿主先天免疫机制并避开它们的病毒捕食者。这些研究还将使我们能够在多价碳水化合物疫苗上设计确定的MeOPN残基，因为CPS与MeOPN偶联物已经显示出作为可能的疫苗的希望。扩大核磁共振的应用范围也将为糖生物学研究界提供新的有价值的工具。该项目将培养一名博士研究生和两名硕士研究生，并通过开发和优化最先进的细菌糖结合物研究技术，使他们在碳水化合物化学、分析和生物物理分析方面发展新的技能。

The foodborne pathogen Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis worldwide. Like other bacteria that coat their surfaces with carbohydrates and then decorate them with phosphorylated structures, C. jejuni expresses complex capsular polysaccharides (CPS) that are decorated with methyl phosphoramidate (MeOPN) residues that we have shown are important in cell invasion, serum sensitivity, biofilm formation, bacteriophage binding, insect and fungal killing. This proposal examines the enzymes involved in the biosynthesis of C. jejuni MeOPN by methods such as high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR) and targeted metabolomics mass spectrometry. HR-MAS NMR has become a powerful technique to analyze small amounts of biological materials and obtain near-liquid like spectra and we plan to extend this technology to characterize host-pathogen interactions. Furthermore, we will examine the affect of MeOPN substitution on the CPS three dimensional structure. This project will provide fundamental biochemical information on the synthesis of a key C. jejuni CPS modification which plays multiple biological roles for this prevalent pathogen. Understanding the biosynthesis of this moiety and how it affects the three-dimensional structure of the CPS will give us a better understanding about how bacteria evade host innate immune mechanisms and avoid their viral predators. These studies will also enable us to engineer defined MeOPN residues on multivalent carbohydrate vaccines since CPS conjugates with MeOPN have already shown promise as possible vaccines. Extending the applications of HR-MAS NMR will also provide new valuable tools for the glycobiology research community. This project will train a PhD student and two MSc students, and allow them to develop novel skills in carbohydrate chemistry, analytical and biophysical analyses through the development and optimization of state-of-the art techniques for the study of bacterial glycoconjugates.