Bacterial oligosaccharyltransferase for glycoengineering and vaccine development

用于糖工程和疫苗开发的细菌寡糖转移酶

• 批准号: BB/F009496/1
• 负责人: Dennis Linton
• 金额: $ 37.36万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF009496%2F1
• 关键词: Bacterial; oligosaccharyltransferase; glycoengineering; vaccine; development

Vaccination has been incredibly successful in reducing the burden of infectious diseases. Examples of successful vaccines include those against the deadly bacteria Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. The basis for these vaccines is a complex sugar structure, known as the capsule, which covers bacterial cells protecting them from immune attack. In order to evoke an appropriate immune response, vaccines against these bacteria consist of capsule linked to a protein carrier forming a glycoprotein or glycoconjugate. However, despite the success of these glycoconjugate vaccines they have major drawbacks in terms of the technical difficulties in purifying the capsule material from bacterial cells and then conjugating the capsule to carrier proteins. Additionally, capsules are often highly variable, and the specific immunity elicited by immunisation with one type of capsule will not protect against bacteria with different capsule structures. Thus as new disease strains emerge (e.g. from selective pressure by large scale vaccination regimes) the existing vaccines become ineffective. An inexpensive, rapid and flexible method for glycoconjugate vaccine production would enable a more effective response to the emergence of new pathogenic bacterial strains with different capsule structures. One such approach is to produce glycoconjugate vaccines in the genetically tractable bacterium Escherichia coli. E. coli is already used as a 'cellular factory' to produce large amounts of proteins; however, until recently it has not been possible to generate glycoproteins in this bacterium. That could now change. We have recently identified and characterised a gene cluster (pgl) which is responsible for the synthesis of glycoproteins in the bacterial food-borne pathogen, Campylobacter jejuni. We have been able to transfer the segment of DNA containing the pgl genes into E. coli to produce recombinant glycoproteins, thus opening up the field of glycoengineering. The key enzyme in the C. jejuni pathway that couples proteins to sugars is the oligosaccharyltransferase protein termed CjPglB. Although CjPglB can transfer many sugar structures unfortunately there are many structures from various pathogens that it cannot. Essentially, the end of the glycostructure that is attached to the protein by CjPglB, must have a sugar unit with a specific configuration - an acetamido group at the C-2 position of the sugar at the reducing end of the glycan. This severely limits the potential applications of this technology. Indeed many capsules of pathogenic bacteria do not have this configuration and therefore CjPglB could not be used to produce glycoconjugate vaccines for protection against these bacteria. In this study we propose a number of strategies to overcome this problem. We will seek to identify or engineer alternative PglB proteins that will have a modified specificity for different glycostructures. We will use a dual approach of seeking alternative PglBs from other bacteria that may naturally have a different specificity to the original CjPglB, and also a mutagenesis approach to alter the enzymatic specificity of CjPglB. To ascertain if the specificity of the natural and mutated PglBs have been altered we will test separate capsular polysaccharides from the important pathogens Streptococcus pneumoniae and Burkholderia pseudomallei to determine if the respective capsules can now be coupled/conjugated to an appropriate carrier protein. The new recombinant glycoconjugates in E. coli will be ideal vaccine candidates that can be readily purified and tested. The glycoengineering principles to be pioneered in this study could be applied generically to the design of other glycoconjugate and combination vaccines. Irrespective of vaccine development, this new and emerging technology will be of direct importance to scientists interested in basic research and in applied research in glyco-biotechnology.

疫苗接种在减轻传染病负担方面取得了令人难以置信的成功。成功疫苗的例子包括针对致命细菌流感嗜血杆菌、脑膜炎奈瑟菌和肺炎链球菌的疫苗。这些疫苗的基础是一种被称为胶囊的复杂糖结构，它覆盖着保护它们免受免疫攻击的细菌细胞。为了引起适当的免疫反应，针对这些细菌的疫苗包括连接到形成糖蛋白或糖偶联物的蛋白质载体的胶囊。然而，尽管这些糖结合疫苗取得了成功，但它们在从细菌细胞中提纯胶囊材料然后将胶囊与载体蛋白结合方面存在技术困难。此外，胶囊往往是高度可变的，用一种类型的胶囊免疫所产生的特异性免疫不能保护不同胶囊结构的细菌。因此，随着新的疾病株的出现(例如，来自大规模疫苗接种制度的选择性压力)，现有疫苗变得无效。一种廉价、快速和灵活的糖结合疫苗生产方法将使人们能够更有效地应对不同衣壳结构的新病原菌株的出现。一种这样的方法是在基因易驯化的大肠杆菌中生产糖结合疫苗。大肠杆菌已经被用作生产大量蛋白质的‘细胞工厂’；然而，直到最近，在这种细菌中还不可能产生糖蛋白。现在，这种情况可能会改变。我们最近鉴定了一个基因簇(PGL)，该基因簇负责合成食源性细菌空肠弯曲菌中的糖蛋白。我们已经能够将含有PGL基因的DNA片段转移到大肠杆菌中生产重组糖蛋白，从而打开了糖工程的领域。空肠弯曲菌途径中将蛋白质与糖偶联的关键酶是寡糖转移酶蛋白，称为CjPglB。虽然CjPglB可以转移许多糖结构，但不幸的是，有许多来自各种病原体的结构是不能转移的。本质上，通过CjPglB连接到蛋白质上的糖结构的末端必须有一个具有特定构型的糖单元--糖链还原末端糖的C-2位置上的乙酰氨基基。这严重限制了这项技术的潜在应用。事实上，许多致病细菌的胶囊没有这种构型，因此CjPglB不能用于生产保护这些细菌的糖结合疫苗。在这项研究中，我们提出了一些克服这一问题的策略。我们将寻求识别或设计替代的PglB蛋白，这些蛋白将对不同的糖结构具有修饰的特异性。我们将使用一种双重的方法，从其他细菌中寻找替代的PglB，这些细菌可能自然地对原始CjPglB具有不同的特异性，以及一种突变的方法来改变CjPglB的酶特异性。为了确定天然和突变的PGlB的特异性是否已经改变，我们将测试从重要病原体肺炎链球菌和假鼻疽伯克霍尔德氏菌分离出的胶囊多糖，以确定各自的胶囊现在是否可以偶联/结合到适当的载体蛋白上。在大肠杆菌中新的重组糖结合物将是理想的候选疫苗，可以很容易地提纯和测试。本研究中率先提出的糖工程原理可普遍应用于其他糖结合疫苗和联合疫苗的设计。无论疫苗开发如何，这项新兴的新技术将对对糖类生物技术基础研究和应用研究感兴趣的科学家具有直接的重要性。