A conserved protein O-glycosylation pathway in the Burkholderia genus essential for bacterial fitness and antigenicity in humans

伯克霍尔德氏菌属包括广泛分布在地球各地的细菌，它们可以在不同的环境中生存，并与不同的宿主相关联。有些伯克霍尔德菌对人类特别危险，因为它们会引起弥散性且往往致命的感染，如类鼻疽（B. pseudomallei）和腺病（B. mallei），由于它们可能被用作生物战剂，也被归类为B类生物。其他伯克霍尔德菌种类（例如，囊性纤维化B. cenocepacia和B. multivorans）可导致囊性纤维化患者肺部感染。另一方面，伯克霍尔德菌在生物修复、促进植物生长和害虫生物防治方面具有很高的应用价值。在易感人群中预防伯克氏菌感染将消除伯克氏菌对人类的威胁，并使更好地利用这些细菌的多种有益方面成为可能。我们已经确定了在所有伯克氏菌中保守的蛋白质糖基化途径，这使得开发通用伯克氏菌疫苗成为可能。伯克霍尔德菌蛋白糖基化途径由所有伯克霍尔德菌物种中保守的基因编码，由参与将糖以特定序列拼接在一起形成寡糖分子的蛋白质组成，然后将其结合到位于细菌细胞包膜上的几种细菌蛋白质中。我们正在建立至少23个伯克霍尔德菌蛋白上的低聚糖的碳水化合物结构，并已经阐明了低聚糖组装和输出所需的基因。我们的研究小组还发现，患有囊性纤维化（B. cenocepacia和B. multivorans），类melidosis （B. pseudomallei）和腺体炎（B. mallei）的伯克霍尔德菌感染患者的血清中具有特异性识别从B. cenocepacia中纯化的糖基化蛋白的抗体，这表明糖基化的伯克霍尔德菌蛋白可被人体免疫系统感知。为了测试伯克霍尔德氏菌糖基化蛋白免疫是否能刺激保护性免疫反应，研究人员对小鼠进行了免疫，免疫对象是一种从结核杆菌中纯化的糖基化蛋白，该蛋白还与明矾（一种临床使用的疫苗抗原）混合。小鼠在腹腔内受到多梭菌攻击后可免受感染，这也表明该疫苗可引起对不同种类伯克霍尔德菌的交叉保护。这一建议支持了微生物糖生物学、分子生物学和糖化学研究前沿的基础研究，其目标是：(i)解码伯克霍尔德菌蛋白糖基化途径中涉及的酶的功能，并阐明低聚糖的分子结构；（ii）阐明细菌中由于蛋白质糖基化丧失而引起的生理改变背后的机制；（iii）确定低聚糖转移酶pgl的结构功能，以通过糖工程方法实现生物技术应用。与BBSRC路线图一致，这一创新项目通过推进生物技术研究，阐明伯克霍尔德氏菌的蛋白糖基化系统，并利用这些知识开发广谱疫苗，迎接寻找应对危险机会性病原体的新方法的挑战。它还非常符合寻找抗生素替代品来控制多重耐药细菌（如伯克霍尔德氏菌）的需要，方法是在易感患者中发现预防这些细菌感染的新方法。

The Burkholderia genus includes bacteria widely distributed around the planet, which can survive in diverse environments and in association with diverse hosts. Some of the Burkholderia species are particularly dangerous to humans, as they cause disseminated and often lethal infections such as melioidosis (B. pseudomallei) and glanders (B. mallei), and are also classified as category B organisms due their potential use as biological warfare agents. Other Burkholderia species (e.g. B. cenocepacia and B. multivorans), cause debilitating lung infections in cystic fibrosis patients. On the other hand, Burkholderia species are highly useful for bioremediation, plant growth promotion, and pest biocontrol. Preventing Burkholderia infections in susceptible people will eliminate the threat of Burkholderia for humans and make it possible to better exploit the multiple beneficial aspects of these bacteria. We have characterised a protein glycosylation pathway conserved in all Burkholderia that allows the possibility to develop a universal Burkholderia vaccine. The Burkholderia protein glycosylation pathway is encoded by genes conserved in all Burkholderia species, and consists of proteins involved in stitching sugars together in a particular sequence to form an oligosaccharide molecule, which is then incorporated to several bacterial proteins that are located on the bacterial cell envelope. We are in the process to establishing the carbohydrate structure of the oligosaccharide attached to at least 23 Burkholderia proteins, and have elucidated the genes required for oligosaccharide assembly and export. Our research team also discovered that sera from Burkholderia-infected patients suffering from cystic fibrosis (B. cenocepacia and B. multivorans), melioidosis (B. pseudomallei) and glanders (B. mallei) have antibodies that specifically recognize a glycosylated protein purified from B. cenocepacia, indicating that glycosylated Burkholderia proteins are perceived by the human immune system. To test whether immunization with a Burkholderia glycosylated protein stimulates a protective immune response, groups of mice were immunized against a glycosylated protein purified from B. cenocepacia, which was also mixed with Alum, a clinically used vaccine antigen. Mice were protected from infection upon intraperitoneal challenge with B. multivorans, also indicating that the vaccine elicits cross-protection against different Burkholderia species. This proposal underpins fundamental studies at the forefront of microbial glycobiology, molecular biology and glycochemistry research with the goals to: (i) Decode the functions of the enzymes involved in the Burkholderia protein glycosylation pathway and elucidate the molecular structure of the oligosaccharide glycan; (ii) Elucidate the mechanism behind the physiological alterations due to loss of protein glycosylation in bacteria; and (iii) Determine the structure function of the oligosaccharyltransferase PglL to enable biotechnological applications through glycoengineering approaches. Aligned to the BBSRC roadmap, this innovative project rises to the challenges of finding novel means to deal with dangerous opportunistic pathogens by advancing biotechnological research elucidating the protein glycosylation system in Burkholderia and exploiting this knowledge to develop wide-spectrum vaccine. It also fits well with the need to find alternatives to antibiotics for the control of multidrug resistant bacteria, such as the Burkholderia, by exposing new ways to prevent infection by these bacteria in susceptible patients.