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

伯克霍尔德氏菌属中保守的蛋白质 O-糖基化途径对于人类细菌适应性和抗原性至关重要

基本信息

批准号：
BB/T005807/1
负责人：
Miguel Valvano
金额：
$ 58.1万
依托单位：
Queen's University Belfast
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
未结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FT005807%2F1
关键词：
conserved protein glycosylation pathway Burkholderia

项目摘要

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.

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，并与不同的宿主相关联。一些伯克霍尔德菌属物种对人类特别危险，因为它们引起播散性且通常致命的感染，如类鼻疽病（B.假鼻疽）和鼻疽（B.鼻疽），并且由于其作为生物战剂的潜在用途，也被分类为B类生物。其他伯克霍尔德氏菌属种（例如B. cenocepacia和B.多噬菌体），在囊性纤维化患者中引起使人衰弱的肺部感染。另一方面，伯克霍尔德氏菌物种对于生物修复、植物生长促进和害虫生物防治非常有用。在易感人群中预防伯克霍尔德菌感染将消除伯克霍尔德菌对人类的威胁，并使人们有可能更好地利用这些细菌的多个有益方面。我们已经表征了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。伯克霍尔德氏菌蛋白糖基化途径由所有伯克霍尔德氏菌属物种中保守的基因编码，并且由参与将糖以特定序列拼接在一起以形成寡糖分子的蛋白质组成，寡糖分子然后掺入位于细菌细胞包膜上的几种细菌蛋白质。我们正在建立至少23种伯克霍尔德氏菌蛋白质所附着的低聚糖的碳水化合物结构，并已阐明低聚糖组装和输出所需的基因。我们的研究小组还发现，来自感染伯克霍尔德氏菌的囊性纤维化患者（B. cenocepacia和B.多噬菌）、类鼻疽（B.假鼻疽）和鼻疽（B.鼻疽）具有特异性识别从B纯化的糖基化蛋白的抗体。cenocepacia，表明糖基化伯克霍尔德氏菌蛋白被人类免疫系统感知。为了测试用伯克霍尔德氏菌糖基化蛋白免疫是否刺激保护性免疫应答，用从B纯化的糖基化蛋白免疫小鼠组。cenocepacia，其还与明矾（临床上使用的疫苗抗原）混合。在用B腹膜内攻击后，保护小鼠免受感染。多噬菌体，也表明该疫苗增强了针对不同伯克霍尔德氏菌属物种的交叉保护。这一提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）解码参与伯克霍尔德氏菌蛋白糖基化途径的酶的功能，并阐明寡糖聚糖的分子结构;（ii）阐明细菌中蛋白糖基化丧失导致生理改变的机制;（iii）阐明细菌中蛋白糖基化丧失导致生理改变的机制。和（iii）确定寡糖基转移酶PglL的结构功能，以通过糖工程方法实现生物技术应用。根据BBSRC路线图，这个创新项目通过推进生物技术研究来阐明伯克霍尔德氏菌中的蛋白质糖基化系统，并利用这些知识开发广谱疫苗，从而应对寻找新方法来应对危险的机会致病菌的挑战。它也很好地符合需要找到抗生素的替代品，以控制多重耐药细菌，如伯克霍尔德氏菌，通过暴露新的方法来预防易感患者感染这些细菌。