肺炎链球菌血清群33和35的荚膜多糖抗原的结构特征与遗传基础

Streptococcus pneumoniae is a human pathogen responsible for invasive pneumococcal diseases such as pneumonia, meningitis and septicemia. Capsular polysccharide (CPS) on the cell surface is a major virulence factor, but also is a target for host antibodies and the basis for effective vaccines. 93 individual serotypes are recognized by their immunogenic diversity and classified into 46 serogroups. CPS structures of the serotypes most commonly associated with invasive disease have been identified, and polyvalent polysaccharide vaccines that target these serotypes are available. However, many CPS serotypes have not been characterized and the genetic basis of CPS diversity has not been elucidated. Moreover, with the broad introduction of heptavalent polysaccharide-conjugated vaccine (PCV7), some non-vaccine serotypes as well as new serotypes have emerged that cause serious infection. Therefore, it is necessary to investigate the structures and genetic basis of S. pneumoniae capsular polysaccharides. Strains of serogroups 33 and 35 have emerged as non-PCV7 vaccine serotypes causing meningitis and septicemia, and some of these are highly resistent to penicillin. In this proposal, we plan to study serogroup 33 and 35 capsular polysccharides. Key genes for their CPS biosynthesis will be predicted by bioinformatics method, and will be identified by carbohydrate-engineering techinique. CPS structures will be identified by NMR and GC-MS. Furthermore, by using immunology methods, the epitopes on capsular polysaccharides will be analyzed. The final goal of this project is to reveal the structural, genetic and antigenic characterization of capsular polysaccharide serogroup 33 and 35, which is expected to provide a theoretical basis for development of improved pneumococcal vaccine.

肺炎链球菌是引起侵袭性疾病如肺炎、脑膜炎和败血症等的致病菌。其表面的荚膜多糖是关键的致病因子和抗原。根据荚膜多糖抗原性的差异将肺炎链球菌分为93个血清型46个血清群。虽然感染力强的荚膜多糖结构已得到解析，以此为基础开发的多价糖疫苗在临床上得到应用，但是大多数血清型的结构和遗传基础并不清楚。同时，随着疫苗的广泛使用，近年还出现了血清型替换和新的感染力强的血清型。因此，全面解析肺炎链球菌荚膜多糖的结构和遗传基础迫在眉睫。肺炎链球菌血清群33和35可引起脑膜炎和败血症，并对青霉素产生高抗性，目前还没有相应的疫苗。本项目拟以肺炎链球菌血清群33和35为研究对象，通过生物信息学方法预测荚膜多糖合成基因，并通过糖工程技术确定关键基因的功能；用NMR、GC-MS鉴定多糖的结构，并确定荚膜多糖的抗原决定簇，从而揭示血清群33和35荚膜多糖的结构和抗原特征及其生物合成的遗传基础，为新疫苗的设计提供理论基础。

肺炎链球菌是引起侵袭性疾病如肺炎、脑膜炎和败血症等的致病菌。其表面的荚膜多糖（CPS）是最重要的致病因子、抗原和疫苗成分。 荚膜多糖的结构是揭示其抗原特征和遗传基础的相互关系以及疫苗多糖合成的关键。血清组35和33是新出现的引起脑膜炎和败血症的非多糖疫苗（PCV7）血清型，并对青霉素产生高抗性，目前还没有相应的疫苗。血清组35含有四个血清型（35F, 35A, 35B和35C），其中35F和35C的荚膜多糖结构未知。我们首先用NMR和GC-MS鉴定了它们的结构。CPS35F的结构是[-6Galf-(2-OAc)-β1-3Galα1-2ribitol-5-PO4--3Galfβ1-3Galβ1-]n，CPS35C的结构是 [-6Galf-(2-OAc)-β1-1mannitol-6-PO4--3Gal-(α1-2-Glc)-β1-3Galf-(5,6-OAc)-β1-3Glcβ1-]n。 比较发现，CPS35F与血清组35中的其他血清型的结构不同，而是与CPS34和CPS47F的结构相似。CPS35C和CPS35A的结构非常相似，与CPS42的结构一样，只是CPS42的Galf失去O-乙酰基团（2-OAc）。同时发现，血清组35与血清型20、29、34、42和47F的部分结构相似是它们有血清学交叉反应的原因。其次，通过敲除关键基因、免疫杂交、免疫扩散并结合NMR分析突变多糖的结构，我们鉴定了CPS35C多糖的抗原决定簇及其相应的遗传基础。结果揭示O-乙酰基转移酶WciG负责合成Galf-2-OAc，是抗血清因子35a识别的抗原决定簇；另一个O-乙酰基转移酶WcjE负责合成Galf-5,6-OAc，是抗血清因子20b识别的抗原决定簇；WcrK负责转移支链葡萄糖到主链上合成Gal-(α1-2-Glc)，是抗血清因子42a识别的靶位点。而敲除其他糖基转移酶基因，细菌不能合成荚膜多糖。另外，根据研究进展，我们还拓宽了研究内容，进一步检测比较了野生菌株和突变菌株对巨噬细胞的侵染，发现荚膜多糖极大地阻止了巨噬细胞识别黏附肺炎链球菌，相反，荚膜多糖的O-乙酰修饰帮助细菌被巨噬细胞黏附。这些结果为深入研究肺炎链球菌引起脑膜炎的机制奠定了基础，对脑膜炎的防治有重要意义，也为新疫苗的设计提供理论指导。