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。     

细菌荚膜多糖的化学结构

荚膜是一些细菌所具有的表层结构,与多种疾病有着密切联系。细菌荚膜多糖不仅结构复杂,而且在免疫活性方面发挥着重要的作用。同一种细菌根据其荚膜多糖的抗原性不同可分为不同的血清型,不同血清型细菌荚膜多糖的化学结构也存在差异。以荚膜多糖为基础的疫苗正在积极研究开发当中,对不同致病细菌荚膜多糖具体化学结构的掌握是疫苗得到许可的必备条件之一。本文对致病细菌荚膜多糖的化学结构进行了归纳和总结,以期为荚膜多糖的化学结构研究和疫苗开发提供参考。     

革兰氏阳性菌荚膜多糖研究进展*

细菌的荚膜多糖是生物膜的重要组成部分,在细菌的生长分裂、维持细胞壁形态、抵御外界环境以及免疫反应等方面都起到重要作用。在致病菌中,荚膜多糖常作为一种毒力因子发挥作用。在革兰氏阳性菌中,荚膜多糖的化学结构、生物合成过程及功能应用越来越受到关注。讨论了革兰氏阳性菌中部分致病菌的荚膜多糖与非致病菌表面多糖的分布位置、化学组成及其结构特异性。重点讨论三种具有代表性的革兰氏阳性致病菌及非致病菌株:肺炎链球菌(Streptococcus pneumonia)、金黄色葡萄球菌(Staphylococcus aureus)及乳酸乳球菌(Lactococcus lactis)。综述革兰氏阳性菌中荚膜多糖生物合成的三种方式:Wzx/Wzy-依赖通路、ABC转运蛋白(ABC transporter)途径及合酶依赖途径,并举例解释了相应多糖的合成过程及相关基因。介绍了革兰氏阳性菌荚膜多糖及表面多糖的生理功能,如屏障保护功能、胞间黏附功能以及参与宿主细胞的免疫反应等。结合荚膜多糖的生物学功能,概述其当前主要研究进展,如构建高耐受工程菌疫苗研制等。结合细菌荚膜多糖的特征差异,对其在医药与工业生产领域的广阔前景提出展望和建议。     

猪链球菌荚膜多糖合成相关基因簇保守区的克隆与分析

【目的】为了解猪链球菌各血清型荚膜多糖合成相关基因保守区的功能与基因进化关系,【方法】在分析已知的猪链球菌1、2、7、9型荚膜多糖合成相关基因簇序列,及其各orf与猪链球菌33个血清型基因组DNA杂交结果的基础上,提出猪链球菌荚膜多糖合成相关基因簇具有与肺炎链球菌相似的盒样结构的假设。并采用PCR、测序和Southern印迹杂交等方法验证这些假设。【结果】结果显示,猪链球菌的荚膜多糖合成相关基因簇确存在与肺炎链球菌相似的盒样结构,5’端的前4个调节相关基因同源性极高,基因簇两端都有保守的侧翼基因,且在3’端的侧翼序列中找到了适于扩增荚膜多糖合成相关基因簇中血清型特异性区域的下游引物所在基因(aroA)。分析发现,各血清型的orfY、orfX、cpsA、cpsB、cpsC、cpsD和aroA的亲缘关系较近。     

植物病原细菌hrp基因研究进展

hrp基因决定植物病原细菌对寄主植物致病性和诱导非寄主及抗病寄主过敏性反应 ,hrp基因在植物和动物病原细菌中具有同源性 ,编码产物具有HR的激发子、调节和组成Ⅲ型泌出系统等功能 ,hrpM是P. syringae合成 β (1 ,2 ) 葡聚糖必需的 ,除了Avr蛋白和harpins外致病性或毒性蛋白也可从Hrp系统泌出 ,表明hrp基因在病原细菌致病性和寄主范围等方面具有潜在作用。     

植物病原细菌hrp基因研究进展

hrp基因决定植物病原细菌对寄主植物致病性和诱导非寄主及抗病寄主过敏性反应,hrp基因在植物和动物病原细菌中具有同源性,编码产物具有HR的激发子、调节和组成Ⅲ型泌出系统等功能,hrpM是P．sying合成β-(1,2)—葡聚糖必需的,除了Avr蛋白和harpins外致病性或毒性蛋白也可从Hrp系统泌出,表明hrp基因在病原细菌致病性和寄主范围等方面具有潜在作用。     

细菌抗氧化系统-oxyR调节子研究进展

细菌抗氧化系统是细菌抵抗呼吸作用及环境因素导致的氧化损伤的一套防卫系统.oxyR调节子是最早发现的具有抗氧化作用的系统之一,由OxyR调节蛋白的编码基因oxyR及其调控的基因和操纵子所构成.oxyR调节子参与了细菌的抗氧化作用、抑制自发突变、致病性、铁代谢及外膜蛋白相变等多种生理代谢作用,这些发现促进了该调节子在细菌耐药性以及致突变物质筛查等方面的研究应用.作者主要从细菌oxyR调节子的结构组成、参与的生理代谢作用、OxyR调控转录的分子机制及影响因素等方面结合最新研究成果展开了介绍,以期对开展细菌抗药性研究及致突变物质的筛查等提供参考.     

金葡菌荚膜多糖血清型的研究进展

金葡菌是引起奶牛乳腺炎的一种主要的致病菌,致病性金葡菌多数含有荚膜成分,金葡菌荚膜多糖有11种血清型。从不同血清型荚膜多糖的结构,基因构成,抗吞噬作用,致病性等方面作了介绍,并简要介绍了金葡菌荚膜多糖血清型的国内外研究现状。     

肺炎链球菌荚膜多糖O-乙酰化修饰的研究进展

肺炎链球菌表面覆盖着一层荚膜,由多糖组成,是肺炎链球菌关键的毒力因子和重要的抗原,也是细菌分型的依据。强毒血清型的荚膜多糖被制成糖疫苗在抗感染方面发挥了巨大作用。荚膜多糖结构复杂,经常被O-乙酰化修饰,这些多变的化学修饰扮演着重要的生物学角色。本文对肺炎链球菌荚膜多糖O-乙酰化修饰的研究进展进行了介绍,包括荚膜多糖的遗传基础、合成途径和血清学特征,荚膜多糖的O-乙酰化修饰的化学结构及其相应的O-乙酰基转移酶,O-乙酰化修饰的化学鉴定和生物学功能。同时,我们也总结了多糖O-乙酰化修饰在肺炎链球菌微进化中的作用和对糖疫苗的影响,并对今后的研究进行了展望。本综述旨在为研究荚膜多糖的O-乙酰化修饰的致病机制奠定基础,也为糖疫苗的设计提供指导。     

多胺与细菌病原的致病性

多胺是一类具有两个以上氨基的脂肪族化合物,它们在生物体内含量的动态平衡受合成、转运、降解及互换等过程的影响,多胺及其合成和转运系统与病原菌的致病性相关.本文综述了多胺在细菌毒力因子的转录和翻译、细菌生物膜的合成、细菌对抗生素的抗性、细菌对抗宿主的酸性胁迫和氧化胁迫、细菌对抗宿主的先天免疫防御机制、细菌致病性生物分子的合成等方面的重要作用.     

艰难梭菌荚膜致病作用的初步研究

本文在我室先前研究的基础上,以3株艰难梭菌的荚膜多糖提取物、全菌悬液以及全菌免疫血清分别在小鼠中进行半数致死量和保护试验等动物实验,以探讨艰难梭菌荚膜的致病作用。结果测得其中一株艰难梭菌的荚膜多糖对小鼠有致死毒性,其LD50为2.83mg,特异性抗血清保护试验证实艰难梭菌免疫血清对动物有保护作用。实验结果可能为进一步研究艰难梭菌荚膜的致病作用提供了参考资料。     

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

The rkp-3 gene region of Sinorhizobium meliloti Rm41 contains strain-specific genes that determine K antigen structure.

The rkp-3 region is indispensable for capsular polysaccharide (K antigen) synthesis in Sinorhizobium meliloti Rm41. Strain Rm41 produces a K antigen of strain-specific structure, designated as the KR5 antigen. The data in this report show that the rkp-3 gene region comprises 10 open reading frames involved in bacterial polysaccharide synthesis and export. The predicted amino acid sequences for the rkpL-Q gene products are homologous to enzymes involved in the production of specific sugar moieties, while the putative products of the rkpRST genes show a high degree of similarity to proteins required for transporting polysaccharides to the cell surface. Southern analysis experiments using gene-specific probes suggest that genes involved in the synthesis of the precursor sugars are unique in strain Rm41, whereas sequences coding for export proteins are widely distributed among Sinorhizobium species. Mutations in the rkpL-Q genes result in a modified K antigen pattern and impaired symbiotic capabilities. On this basis, we suggest that these genes are required for the production of the KR5 antigen that is necessary for S. meliloti Rm41 exoB (AK631)-alfalfa (Medicago sativa) symbiosis.     

Effect of flavonoids on the composition of surface glycopolymers of Azospirillum lipoferum Sp59b

Cultivation of the type strain Azospirillum lipoferum Sp59b in the presence of flavonoid quercetin induced modification of the structure of the bacterial lipopolysaccharide. Cultivation in the presence of the flavonoid was shown to result in altered serological characteristics of the bacteria, increased heterogeneity of the outer membrane lipopolysaccharide pool, as well as in modified composition and fatty acid ratio of lipid A. The flavonoid was shown to induce the synthesis of the O-specific polysaccharide with the repeating structure represented by a tetrasaccharide consisting of a linear trisaccharide fragment of α-L-Rhap residues in the main chain and the terminal β-D-Glcp residue. The structure of this O-specific polysaccharide was identical to the previously determined structure of the capsular polysaccharide of these bacteria grown without quercetin. Modifications in the structural composition of the capsular polysaccharide induced by cultivation in the presence of quercetin were revealed.     

肺炎链球菌pcsB组成型表达的yycF缺陷菌株的构建及其致病能力

【背景】YycFG双组分系统是肺炎链球菌(Streptococcus pneumoniae,S. pn)应对外界环境的重要信息传递系统,其中表达反应调节子YycF的编码基因是肺炎链球菌生长的必需基因,但其是否调控细菌毒力尚不清楚。【目的】构建肺炎链球菌pcsB组成型表达及yycF缺陷菌,分析YycF对肺炎链球菌生物学特征和毒力的影响。【方法】采用Janus cassette (JC)反选的方法构建pcsB组成型表达菌株(Pc-PcsB~+),从该菌株出发用替代失活的方法构建yycF缺陷菌株(Pc-PcsB~+DyycF),比较野生株D39rpsl41、pcsB组成型表达株及yycF缺陷株的生长特性、荚膜多糖(capsular polysaccharide,CPS)含量、粘附侵袭能力和致病性的差异。【结果】成功构建pcsB组成型表达的yycF缺陷菌株(Pc-PcsB+DyycF);yycF缺陷导致细菌生长缓慢、分裂异常、胞内荚膜多糖和小分子荚膜多糖增多;体外实验结果显示,yycF缺陷菌株粘附能力较Pc-PcsB~+菌株减弱(P=0.006)。体内毒力实验显示,感染野生菌的小鼠全部死亡,感染Pc-PcsB+和Pc-PcsB~+DyycF菌株的小鼠死亡率分别为91.7%、75%,二者没有统计学差异(P=0.183),但Pc-PcsB~+DyycF菌株感染组有降低趋势;定殖结果显示,yycF缺陷菌株感染组的肺匀浆菌载量显著低于对照组(P=0.033)。【结论】成功构建yycF缺陷菌株,并初步证明yycF基因会影响肺炎链球菌的生物性状和致病能力,为后续探讨YycFG双组分系统对肺炎链球菌致病能力调控机制的研究奠定了基础。