Bacterial oligosaccharyltransferase for glycoengineering and vaccine development

疫苗接种在减少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复杂的糖结构，称为胶囊，它覆盖着细菌细胞，保护它们免受免疫攻击。为了引起适当的免疫应答，针对这些细菌的疫苗由与蛋白质载体连接的胶囊组成，形成糖蛋白或糖缀合物。然而，尽管这些糖缀合物疫苗取得了成功，但它们在从细菌细胞中纯化胶囊材料然后将胶囊缀合至载体蛋白的技术困难方面具有主要缺点。此外，胶囊通常是高度可变的，并且通过用一种类型的胶囊免疫引起的特异性免疫将不能保护免受具有不同胶囊结构的细菌的侵害。因此，随着新的疾病菌株出现（例如，来自大规模疫苗接种方案的选择性压力），现有的疫苗变得无效。一种廉价、快速和灵活的糖缀合物疫苗生产方法将能够更有效地应对具有不同胶囊结构的新致病菌株的出现。一种这样的方法是在遗传上易处理的细菌大肠杆菌中生产糖缀合物疫苗。e.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。但现在这种情况可能会改变。我们最近已经确定并表征了一个基因簇（pgl），它是负责合成的糖蛋白的细菌性食源性病原体，空肠弯曲菌。我们已经能够将含有pgl基因的DNA片段转移到E.大肠杆菌表达重组糖蛋白，为糖工程开辟了新的领域。C.在空肠途径中，将蛋白质与糖偶联的蛋白质是被称为CjPglB的寡糖基转移酶蛋白。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它不能转移的。基本上，通过CjPglB连接到蛋白质的糖结构的末端必须具有具有特定构型的糖单元-在聚糖的还原末端的糖的C-2位置处的乙酰胺基。这严重限制了这项技术的潜在应用。事实上，许多病原菌的荚膜不具有这种构型，因此CjPglB不能用于生产用于保护免受这些细菌的糖缀合物疫苗。在这项研究中，我们提出了一些策略来克服这个问题。我们将寻求鉴定或工程化替代PglB蛋白，其将对不同糖结构具有修饰的特异性。我们将使用从其他细菌中寻找替代PglB的双重方法，这些细菌可能天然地具有与原始CjPglB不同的特异性，并且还使用诱变方法来改变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，我们将测试来自重要病原体肺炎链球菌和类鼻疽伯克霍尔德氏菌的单独的荚膜多糖，以确定相应的荚膜现在是否可以偶联/缀合至适当的载体蛋白。在E.大肠杆菌将是理想的候选疫苗，可以很容易地纯化和测试。本研究中开创的糖工程原理可普遍应用于其他糖缀合物和组合疫苗的设计。无论疫苗的发展，这一新的和新兴的技术将直接重要的科学家感兴趣的基础研究和应用研究中的糖生物技术。

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.