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上的细菌，它们可以在不同的环境中生存，并与不同的宿主联系在一起。一些伯克霍尔德氏菌物种对人类特别危险，因为它们会引起传播的、往往是致命的感染，如类鼻疽病(假鼻疽)和腺体(假鼻疽)，还被归类为B类生物，因为它们可能用作生物战剂。其他伯克霍尔德氏菌(例如，盲肠杆菌和多食杆菌)会在囊性纤维化患者中引起衰弱的肺部感染。另一方面，伯克霍尔德氏菌在生物修复、促进植物生长和害虫生物防治方面具有很高的实用价值。在易感人群中预防伯克霍尔德氏菌感染将消除伯克霍尔德氏菌对人类的威胁，并使更好地利用这些细菌的多种有益方面成为可能。我们已经确定了一种在所有伯克霍尔德氏菌中保守的蛋白质糖基化途径，这使得开发通用伯克霍尔德氏菌疫苗的可能性成为可能。伯克霍尔德氏菌蛋白糖基化途径是由所有伯克霍尔德氏菌中保守的基因编码的，由参与将糖按特定序列缝合形成低聚糖分子的蛋白质组成，低聚糖分子随后被结合到位于细菌细胞膜上的几种细菌蛋白上。我们正在建立至少23种伯克霍尔德氏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，感染伯克霍尔德氏菌的患者患有囊性纤维化(盲肠弯孢杆菌和多毛杆菌)、类鼻疽病(假腮腺杆菌)和腺体(B.Mallei)的血清具有特异性识别从盲肠弯孢杆菌提纯的糖化蛋白的抗体，这表明糖基化的伯克霍尔德氏菌蛋白可被人类免疫系统感知。为了测试伯克霍尔德氏菌糖化蛋白免疫是否刺激保护性免疫反应，对一组小鼠进行了免疫，以对抗从线虫中提纯的糖化蛋白，该蛋白也与临床使用的疫苗抗原明胶混合。小鼠在腹膜腔内攻击多食杆菌后被保护免受感染，这也表明该疫苗对不同种类的伯克霍尔德氏菌具有交叉保护作用。这一建议为微生物糖生物学、分子生物学和糖化学研究的前沿基础研究奠定了基础，目的是：(1)破译伯克霍尔德氏菌蛋白糖基化途径中涉及的酶的功能，并阐明寡糖多糖的分子结构；(2)阐明细菌中蛋白质糖基化丧失引起的生理变化的机制；以及(3)确定寡糖转移酶PglL的结构功能，以便通过糖工程方法进行生物技术应用。与BBSRC路线图保持一致，这一创新项目通过推进生物技术研究，阐明Burkholderia的蛋白质糖基化系统，并利用这一知识开发广谱疫苗，从而迎接寻找处理危险的机会性病原体的新方法的挑战。它也很好地适应了寻找抗生素的替代品来控制多重耐药细菌的需要，比如伯克霍尔德氏菌，通过揭示新的方法来防止易感患者感染这些细菌。

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.