合成肺炎链球菌荚膜多糖基因的研究进展

肺炎链球菌是导致婴幼儿和老年人罹患肺炎、脑膜炎、中耳炎等疾病的主要病原体之一,其致病力与位于细菌表面的荚膜多糖密切相关,而荚膜多糖层的薄厚和多糖结构是影响致病力的主要因素。在分子水平探索参与荚膜多糖合成的相关基因,不仅有助于进一步理解肺炎链球菌的致病机理,而且可从基因水平选育高表达荚膜多糖的肺炎链球菌菌株用于多糖疫苗的研发。鉴于此,现就合成肺炎链球菌荚膜多糖基因的作用机制和研究方法作一综述。     

不同剂量肺炎链球菌荚膜多糖与蛋白结合物的免疫原性比较

用肺炎链球菌19F型、23F型的荚膜多糖(C-PS)分别和破伤风类毒素(TT)蛋白结合,制备了两个型别的多糖-蛋白结合物(19F-TT,23F-TT)。为探讨结合物的最适免疫剂量,以10μg多糖和含1μg、3μg、9μg、27μg多糖的结合物经腹腔免疫NIH小鼠,ELISA方法检测小鼠血清中多糖和含不同多糖的结合物所产生的特异性IgG抗体。结果在不同的剂量免疫小鼠后,3μg剂量组在免疫三针后可诱生高浓度的抗体,但随着免疫剂量的增加,9μg与27μg诱生的抗体浓度较3μg诱生的抗体浓度之间并无显著性差异。含多糖3μg的19F型和23F型多糖蛋白结合物剂量组可诱生高浓度的特异性IgG抗体。     

~1H-NMR用于肺炎链球菌荚膜多糖质量控制的尝试

纯化的6B、18C血清型肺炎链球菌荚膜多糖用生化试验和免疫学试验检测分析后再用一维氢谱核磁共振波谱(1H-NMR)法分析。生化检测其相应多糖的主要化学基团含量是否合格,免疫学检测旨在了解多糖纯化工艺是否影响了多糖的抗原活性,并间接佐证纯化多糖的生化特性是否正确。在此基础上进行1H-NMR分析,可以对纯化多糖的特性有进一步的了解。结果表明,常规的生化检测试验和免疫学检测试验并联合应用1H-NMR分析法后可更好地控制纯化肺炎链球菌荚膜多糖的质量。     

肺炎链球菌荚膜多糖纯化及质量控制方法的研究现状

肺炎链球菌(简称肺炎球菌)荚膜多糖(capsular polysaccharide,CPS)位于肺炎球菌的最外层,是肺炎球菌主要的毒力因子之一,也是有效的保护性抗原.获得高质量的肺炎球菌CPS,是肺炎球菌多糖疫苗(pneumococcal pol-ysaccharide vaccine,PPV)和肺炎球菌结合疫苗(pn...     

6群肺炎链球菌荚膜多糖合成相关基因的分子生物学研究

目的利用分子生物学方法,对25株6群肺炎链球菌荚膜多糖合成相关基因(cps loci)进行研究。方法采用多位点序列分型(multilocus sequence typing,MLST)技术,对25株6群肺炎链球菌进行分型;依据Gen Bank中收录的6群肺炎链球菌cps loci设计合成引物,以25株肺炎链球菌基因组DNA作为模板进行PCR扩增,并对扩增产物进行基因测定及分析;采用相邻合并分析(neighbour-joining analysis),根据MLST的7个管家基因和wciP、wzy、wzx这三个cps loci的代表基因分别绘制出系统发育树。结果根据MLST技术分析发现7株新的ST型菌株。25株6群肺炎链球菌cps loci的G+C含量为35.4%~35.5%,均属于I类;所有6C型wzy基因均有6个碱基的缺失。根据MLST的7个管家基因绘制的系统发育树,并根据cps loci的3个代表基因wciP、wzy和wzx绘制的系统发育树,差异很大。3个代表基因绘制的系统发育树,6C型为进化距离比较远的分枝,6A型和6B型的分枝进化距离相对较近;6D型与6B型在同一分枝内。结论获得了25株6群肺炎链球菌ST型和全长cps loci,完善了6群肺炎链球菌的菌种档案。     

硫酸-咔唑微孔板法检测肺炎链球菌荚膜多糖中糖醛酸含量

目的利用微孔板检测肺炎链球菌荚膜多糖中糖醛酸含量,并对该方法进行验证及初步应用。方法以微孔板为反应容器,对常规硫酸-咔唑法的咔唑质量浓度和加热时间进行优化;并对建立的方法进行线性范围、精密度以及准确度的验证及初步应用。结果最佳的咔唑质量浓度为0.10 mg/m L,加热时间为20 min。标准品D-葡萄糖醛酸在4~40μg/m L范围内,其质量浓度与校正后吸光值呈良好的线性关系,r2>0.99;批内及批间CV值分别为1.54%~3.02%及4.53%~7.75%;加入5μg/m L、10μg/m L、20μg/m LD-葡萄糖醛酸的8-160502、9V-160102、22F-160103荚膜多糖,D-葡萄糖醛酸的回收率为92.21%~110.47%。微孔板法校正前与常规方法在测定肺炎链球菌荚膜多糖中糖醛酸含量时差异无统计学意义(P>0.05),与校正后结果差异有统计学意义(P<0.05)。微孔板法测定分子质量分布与常规方法有较好的一致性。结论硫酸-咔唑微孔板法可有效检测肺炎球菌荚膜多糖中糖醛酸含量,操作简便快捷,精密度和准确度良好,可用于肺炎链球菌荚膜多糖疫苗的质量控制。     

肺炎链球菌相关毒力因子及其作用的研究进展

肺炎链球菌(Streptococcus pneumonia,S.pn)是导致黏膜疾病(如中耳炎、肺炎)和系统性疾病(包括败血症和脑膜炎)等严重疾病的主要病原体之一。肺炎链球菌毒力因子分为荚膜多糖、S.pn相关蛋白、细胞壁及细胞壁多糖三大类,其中荚膜多糖为最主要的毒力因子,也是疫苗中最有效的成分。毒力因子在S.pn入侵机体及引发疾病的过程中起着重要的作用。因此,毒力因子及作用的研究,不仅对预防和治疗肺炎链球菌的感染有着重要的意义,并为研发安全、有效的多糖疫苗提供一定的技术支持。现就肺炎链球菌毒力因子及其作用作一综述。     

一种溶菌酶样蛋白对肺炎链球菌毒力及荚膜多糖合成的影响

摘要：【目的】为了研究肺炎链球菌（Streptococcus pneumoniae, S.pn）的一种假想的溶菌酶样蛋白在细菌生物学性状及其致病中的作用。【方法】利用长臂同源PCR对该基因进行敲出,并同时构建带有拯救质粒的缺失菌株,观察D39野生菌、缺失菌与带有拯救质粒的缺失菌株在相关生物学性状及其致病力改变,从而鉴定这种假想溶菌酶样蛋白的功能。【结果】缺失菌与野生菌相比,细菌生长减缓,毒力下降,荚膜多糖合成明显减少。而将拯救质粒转入缺失菌株后,该溶菌酶样蛋白的mRNA表达水平较野生菌高,其毒力及荚膜合成     

肺炎链球菌疫苗的研究进展

肺炎链球菌是引起全球所有年龄组高发病率和病死率的主要病原菌。目前投入使用的有23价荚膜多糖疫苗和7价多糖蛋白结合疫苗。荚膜多糖疫苗有许多局限性,蛋白结合疫苗是对其改进,但也不能完全取代它。两者都具有一定效力和良好的安全性,其中蛋白结合疫苗具有更好的免疫原性。此外,其他多价结合疫苗也已进入临床试验阶段。蛋白疫苗、DNA疫苗、联合疫苗、活疫苗也在研制和开发中。     

肺炎克雷伯菌RcsAB蛋白对荚膜多糖相关基因wcaJ、wcaG和magA的调控关系

荚膜多糖(CPS)是肺炎克雷伯菌(Klebssiella pneumoniae)重要的毒力因子.Rcs系统是肠杆菌科(En-terobacteriacese)中重要的双组分信号转导系统,RcsAB是其中主要的转录调控蛋白.本研究选取了肺炎克雷伯菌NTUH-K2044 cps基因簇上的3个基因(wcaJ,wcaG和magA),研究RcsAB对上述3个基因可能存在的调控关系.通过实时荧光定量PCR(RT-qPCR)技术和LacZ报告基因融合试验来确认RcsAB是否能够调控上述3个基因的转录表达.进而用凝胶阻滞试验(EMSA)验证RcsAB能否直接结合在靶基因的启动子区,对靶基因进行直接调控.结果表明RcsAB能够正向调控上述3个基因的转录表达,EMSA的结果提示RcsAB对这3个基因可能是通过间接的方式进行调控.RcsAB蛋白可能通过间接调控荚膜多糖基因wcaJ、wcaG和magA的转录,从而影响菌株的荚膜多糖表型.     

Synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 14

Synthesis of the regular branched polysaccharide [-6(Gal beta 1-4)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-]n structurally corresponding to capsular polysaccharide of Streptococcus pneumoniae type 14 involves blockwise synthesis of a tritylated 1,2-O-(1-cyano)ethylidene tetrasaccharide derivative from lactosamine and lactose precursors followed by stereospecific polycondensation of the tetrasaccharide monomer.     

Mechanism of type 3 capsular polysaccharide synthesis in Streptococcus pneumoniae

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n)) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.     

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 5

The structure of the capsular polysaccharide (S5) elaborated by Streptococcus pneumoniae type 5 has been investigated by using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the polysaccharide is composed of pentasaccharide repeating-units having the following structure: (Formula: see text) In this structure, L-PneNAc stands for 2-acetamido-2,6-dideoxy-L-talose (pneumosamine) and D-Sug for 2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose. The latter sugar accounts for the lability of S5 towards alkali. N.m.r. spectra indicate heterogeneity in S5, most probably associated with the hexosyl-4-ulose residue.     

The specific capsular polysaccharide of Streptococcus pneumoniae type 9V

The specific capsular polysaccharide produced by Streptococcus pneumoniae type 9V (American type 68) is composed of D-glucuronic acid (1 part), D-galactose (1 part), 2-acetamido-2-deoxy-D-mannose (1 part), D-glucose (2 parts), and O-acetyl (1.6 parts). Methylation, periodate oxidation, optical rotation, and nuclear magnetic resonance studies, and partial hydrolysis showed that the polysaccharide is an unbranched high molecular weight linear polymer of a partially O-acetylated pentasaccharide repeating unit having the structure indicated below. (Formula: see text).     

Characterization and quantification of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharide preparations

Purified capsular polysaccharide preparations from Streptococcus pneumoniae that are used for vaccine production typically contain residual levels of C-polysaccharide (C-Ps). Residual C-Ps is typically found in one of two forms, either chemically linked to the capsular polysaccharide (bound) or present by itself (free). Two analytical methods have been developed and applied to determine the relative percentages of the two C-Ps forms present in various capsular polysaccharide preparations. Both methods differentiate the two forms of C-Ps according to the difference of their hydrodynamic sizes. One method is based on labeling C-Ps with a fluorescent tag and separating the two forms of C-Ps by high-performance size exclusion chromatography with on-line refractive index and fluorescence detection, and the other method is based on measuring self-diffusion rates of the two forms of C-Ps by nuclear magnetic resonance (NMR) and quantifying each form with deconvolution. Both methods were evaluated for relative accuracy, precision, and ease of application, and they were found to provide comparable results for a large number of pneumococcal polysaccharide preparations. These analyses, combined with other quantitative NMR measurement of total C-Ps in the polysaccharide powder, provide a more refined means of evaluating the amount of each form of C-Ps in polysaccharide preparations targeted for vaccine production.     

Transport of Streptococcus pneumoniae capsular polysaccharide in MHC Class II tubules

Bacterial capsular polysaccharides are virulence factors and are considered T cell-independent antigens. However, the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4(+) T cells in a major histocompatibility complex (MHC) class II-dependent manner. The mechanism of carbohydrate presentation to CD4(+) T cells is unknown. We show in live murine dendritic cells (DCs) that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice, retrograde transport of Sp1/MHCII complexes resulting in T cell-dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore, retrograde transport requires the functional role of DM in self peptide-carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens.     

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 18A

The structure of the capsular polysaccharide (S18A) elaborated by Streptococcus pneumoniae type 18A has been investigated by using methylation analysis and n.m.r. spectroscopy. It is concluded that the polysaccharide is composed of pentasaccharide repeating-units having the following structure. (formula; see text) In this structure, the absolute configuration of the glycerol 1-phosphate moiety has not been determined but is assumed to be D from biosynthesis considerations. The structure of S18A is, as expected, closely similar to those determined for S18F and S18C.     

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 7A

Application of methylation analysis, specific degradations, and n.m.r. spectroscopy to the capsular polysaccharide elaborated by Streptococcus pneumoniae type 7A indicates a hexasaccharide repeating-unit with the structure (Formula; see text).