Biosynthesis of the Campylobacter jejuni phophoramidate

食源性空肠弯曲杆菌是世界范围内细菌性胃肠炎的主要原因之一。像其他细菌一样，空肠弯曲菌的表面覆盖有碳水化合物，然后用磷酸化结构装饰它们，空肠弯曲菌表达的复杂的胶囊多糖(CP)是由甲基磷酰胺(MeOPN)残基装饰的，我们已经证明，这些残基在细胞入侵、血清敏感性、生物膜形成、噬菌体结合、昆虫和真菌杀灭方面都是重要的。本研究采用高分辨率魔角旋转核磁共振波谱(HR-MAS核磁共振)和靶向代谢组质谱等方法对空肠弯曲菌MeOPN生物合成过程中涉及的酶进行了研究。HR-MAS核磁共振已经成为分析少量生物材料和获得近液体类光谱的强大技术，我们计划将这一技术扩展到表征宿主和病原体的相互作用。此外，我们还将考察MeOPN取代对CPS三维结构的影响。该项目将为合成一种关键的空肠弯曲菌CPS修饰物提供基本的生化信息，该修饰物对这种流行的病原体具有多种生物学作用。了解这一部分的生物合成及其如何影响CPS的三维结构将使我们更好地了解细菌如何逃避宿主的先天性免疫机制，并避免它们的病毒捕食者。这些研究还将使我们能够在多价碳水化合物疫苗上设计定义的MeOPN残留物，因为CPS与MeOPN的结合已经显示出作为可能的疫苗的前景。扩展HR-MAS核磁共振的应用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生和两名硕士研究生，并允许他们通过开发和优化最先进的细菌糖结合物研究技术，发展碳水化合物化学、分析和生物物理分析方面的新技能。

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.