感受态与口腔链球菌多种表型间关系的研究进展

链球菌是人类口腔中最为常见的细菌类群之一,在口腔微生态平衡的维持与致病中发挥了重要作用。口腔链球菌中的大多数可以进入感受态,在此生理状态下,细菌可摄取环境中的DNA并整合进入自身基因组从而获得新的遗传表型或特性。大量研究表明,口腔链球菌的感受态调控通路不是孤立的,与生物膜形成、细菌素产生、耐酸、氧应激、细胞自溶和耐药性等多个表型的调控存在紧密关系,研究这些不同表型间的相互影响对理解口腔菌群稳态及防治疾病有重要意义。本文以变异链球菌、格氏链球菌、血链球菌和肺炎链球菌4种典型的口腔链球菌为代表,对感受态与口腔链球菌多种表型间关系的研究进展做一综述。     

一种可快速检测食品中酿脓链球菌和无乳链球菌的RPA方法的建立

为了实现食品中酿脓链球菌(Streptococcus pyogenes)和无乳链球菌(S.agalactiae)快速、高效检测,本研究建立了一种同时快速检测食品中这两种细菌的方法.本研究基于重组酶聚合酶等温扩增技术(recombinase polymerase amplification,RPA)原理,选择酿脓链球菌致...     

氟对正畸儿童牙面菌斑变形链球菌的影响

目的:观察和探讨2%氟化钠对正畸患者牙面菌斑内细菌总数及变形链球菌数的影响.方法:选择34例正畸儿童,分为两组,试验组涂布2%氟化钠;对照组不做处理.分别于戴用矫治器前,戴入后1月采集上颌牙唇颊面菌斑,测定菌斑中细菌总数及变形链球菌数.结果:对照组戴用后1月,细菌总数及变形链球菌数较戴用前明显增加(P＜0.01).对照组与试验组戴用后1月相比,试验组细菌总数及变形链球菌数明显少于对照组(P＜0.05).结论:戴用固定矫治器后,牙面菌斑内细菌总数及变形链球菌数较戴用前增加,应用2%氟化钠可明显抑制正畸患者口腔内变形链球菌数,减少龋坏发生.     

壳寡聚糖对变形链球菌乳酸脱氢酶及γ谷丙转氨酶的影响

目的 研究壳寡聚糖对变形链球菌乳酸脱氢酶（LDH）及γ谷丙转氨酶（γ-GT）的影响。方法 将变形链球菌接种在含壳寡聚糖的培养基中,测定培养上清液中乳酸脱氢酶及γ谷丙转氨酶等细胞内酶的含量。结果 菌液上清中的LDH和γ-GT2种细胞内酶含量显著高于空白对照组。表明接种在含壳寡聚糖培养基中的细菌出现了明显的细胞内酶溢出,并与浓度相关。结论 壳寡聚糖对细菌的细胞壁有破坏作用,可能是壳寡聚糖具有防龋功能的原因。     

链球菌神经氨酸酶的作用机制及酶活性的测定技术

神经氨酸酶不仅存在于流感病毒,在细菌中也有分布。细菌的神经氨酸酶可裂解宿主体内糖结合物末端的神经氨酸残基,有助于细菌实现在宿主体内的定殖、穿透和扩散,是细菌重要的毒力因子之一。链球菌是自然界广泛存在的人畜共患的病原菌,在多种链球菌中均可检测出神经氨酸酶。肺炎链球菌的神经氨酸酶研究最为透彻,该菌可产生3种神经氨酸酶(NanA,NanB,NanC),NanA不但可以发挥酶的催化作用,分解唾液酸残基,暴露细菌的黏附受体,还能不依赖酶活基团,辅助细菌感染宿主细胞;NanB催化后产物可作为细菌的碳源;NanC可辅助细菌入侵脑部。在无乳链球菌和猪链球菌中,神经氨酸酶的活性一直未得到确切的验证,可能是由于它们的荚膜均含有神经氨酸,所以其神经氨酸酶的活性逐渐在进化中丧失。另外一些链球菌,例如化脓链球菌和C、G、L群链球菌,其神经氨酸酶的底物偏好相近,均对唾液类黏蛋白的催化活性较强,利于链球菌在含唾液类黏蛋白的组织中扩散。在口腔链球菌和血链球菌中,神经氨酸酶破坏血液成分中的神经氨酸链。由此可见,神经氨酸酶的特异性催化作用与链球菌在宿主体内的定植部位密切相关。此外,随着科技的发展,对神经氨酸酶的活性检测,也由早期的硫代巴比妥法,转为现在的荧光值和吸光度的测定,更为便捷和敏感。本文旨在对链球菌的神经氨酸酶的作用机制、与毒力关系及酶活测定方法等研究进展作一综述,为从事相关研究的科学工作者提供参考。     

肺炎链球菌粘附机制的研究现状

肺炎链球菌粘附宿主我肺炎链球菌侵袭、感染宿主细胞的先决条件。粘附过程是特异的,是细菌表面的粘附分子和宿主细胞膜受体相互作用的结果。英膜对肺炎链球菌的粘附无影响,而细胞壁（ＣＷ）在介导肺炎链附粘附宿主细胞过程中起重要作用;ＣＷ亚组人脂磷酸（ＬＴＡ）介导肺炎链球菌的粘附过程,并导致炎症反应;细菌表面的结构蛋白或分泌蛋白是细菌与宿主细胞连接的桥梁;肺炎链球菌能与宿主细胞外基质蛋白特异性结合,进而粘附宿主     

054表皮细胞总mRNA表达谱研究揭示化脓性链球菌属FAS调节活性与侵袭性的相关性

化脓性链球菌[A群链球菌（group Astreptococci,GAS）]是感染人类的重要革兰阳性菌。细菌对宿主黏膜表面和皮肤表皮细胞的黏附和内化是感染的关键起始步骤,但对感染宿主细胞的转录反应还了解甚少。作者采用Affymetrix人类基因组DNA芯片分别检测了HEp-2细胞感染M49化脓性链球菌的野生型细菌和△fasX型细菌的总mRNA转录,     

肺炎链球菌在复发性阿弗他溃疡中定居部位的变化

目的探讨复发性阿弗他溃疡表面细菌的定居状态.方法 分别对病例组溃疡灶及对照组唇部粘膜进行了细菌学分离对比研究.结果 病例组溃疡灶以肺炎链球菌为主,而对照组唇部粘膜以甲型溶血性链球菌为主. 结论复发性阿弗他溃疡患者中肺炎链球菌定居部位发生改变.     

早晚牙菌斑变异链球菌定植差异的定量PCR检测

目的 采用实时荧光定量PCR检测白天和晚上所形成牙菌斑生物膜中变异链球菌的数量,比较早晚牙菌斑中变异链球菌定植的差异.方法 收集30名健康成人全口口腔洁治后白天和晚上形成的龈上菌斑,提取细菌基因组DNA.合成变异链球菌特异性引物,纯化PCR产物获得目的基因连接于pTA-TA载体,克隆于大肠埃希菌DH5 α感受态细胞.选取阳性克隆测序后纯化质粒DNA,获得质粒标准品.将样本和梯度稀释的质粒标准品进行SYBR Green Ⅰ实时荧光定量PCR检测,确定标准曲线,定量样本中变异链球菌DNA的拷贝数.结果 早晚牙菌斑细菌基因组DNA样本的扩增曲线均在标准品的扩增曲线范围内.晚上所形成牙菌斑中定植的变异链球菌数量(对数值7.67 ±0.77)高于白天定植的变异链球菌数量(对数值7.25±0.62)(P =0.007).结论 牙菌斑的微生物定植存在日夜节奏变化,晚上所形成牙菌斑中变异链球菌数量多于白天.     

肺炎链球菌假想蛋白SPD0414亚细胞定位及对细菌黏附侵袭的影响

目的 为研究肺炎链球菌假想蛋白SPD0414在肺炎链球菌（S.pn）的亚细胞定位,并初步研究其在S.pn黏附和定植中的作用.方法 通过分别在SPD0414的N端和C端融合绿色荧光蛋白（GFP）,共聚焦荧光显微镜观察定位情况.用203野生菌和203△spd0414缺陷菌对鼻咽癌细胞CNE和肺腺癌细胞A549细胞的黏附侵袭实验.体内实验中用203和203△spd0414滴鼻感染BALB/c,在6 h和12 h观察细菌在鼻腔和肺中的载量.结果 无论N端或C端融合的GFP都显示绿色荧光,且整个菌体都显示荧光.与203野生菌相比,203△spd0414缺陷菌对CNE和A549的黏附和侵袭能力均显著下降（P＜0.05）.滴鼻感染BALB/c小鼠,在6 h和12 h,203△spd0414在鼻腔和肺中的细菌载量也明显低于203野生菌（P＜0.05）.结论 肺炎链球菌假想蛋白SPD0414定位于细菌的胞质.该蛋白在细菌的定植和侵袭中发挥了重要的作用.     

Epidemiological characterization of serotype group B Streptococci neonatal infections associated with interleukin-6 level as a sensitive parameter for the early diagnosis

Group B streptococcal infection (Streptococcus agalactiae) is one of the leading causes of life-threatening disease in the early neonatal period, resulting in sepsis, pneumonia, and meningitis. During invasive infections, an excessive release of pro-inflammatory cytokine, such as interleukin-6 (IL-6), thus IL-6 gene is significant, as a diagnostic marker of systemic infection of the newborns. The present study aimed to describe the epidemiology diagnostic of GBS disease in neonatal by phenotypic and genotypic methods. Nine hundred and ninety-six samples were taken at Maternity and Children Hospital, Jeddah, Saudi Arabia for a period of one year (2011–2012). Results indicated that out of 217 infected samples, twenty (9.23.0%) were positive for group B Streptococci bacteria. This study also shows that female infants are more susceptible than males. The level of IL-6 was higher in mothers above 30 years. Twenty positive Streptococci group B isolates showed bands with the cylE gene primers in the border between 228 bp, 267 bp and 50 bp. Molecular detection by Real time polymerase chain reaction was also done to detect the target (Sip gene) encoding the Sip surface immunogenic protein. Specific primers and TaqMan probe were chosen for this purpose. A Real-time PCR method targeting the sip gene of GBS in neonates after delivery has been evaluated.     

Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.     

Opacity factor from group A streptococci is an apoproteinase

Opacity factor (OF) is an enzyme, elaborated by certain serotypes of group A streptococci, which produces opalescence in mammalian sera. OF has been designated a lipoproteinase. Lipoproteins are complex structures and many enzymes are involved in their catalysis. We therefore set out to establish which of the many enzymes OF could be. Results showed that OF rendered high density lipoprotein (HDL) insoluble, accounting for the opalescence in serum, and altered its electrophoretic mobility. Electron microscopy revealed that OF caused an aggregation of HDL and an alteration in molecule shape. OF specifically split apoprotein AI of HDL into two fragments demonstrable by SDS-PAGE. We therefore designate OF as an apoproteinase.     

革兰氏阴性菌脂蛋白Lol系统转运蛋白的功能及表面展示分泌机制

细菌脂蛋白是细胞膜的重要组成成分,在革兰氏阴性菌的生理及致病性中扮演着重要的角色。革兰氏阴性菌中已知负责胞内脂蛋白转运的是Lol(Localization of lipoprotein)系统。该系统识别成熟脂蛋白的分泌信号,将外膜脂蛋白转运并定位于细胞外膜内侧。近年来的研究发现,跨细胞外膜进行表面展示的脂蛋白实际上在革兰氏阴性菌中广泛存在,其分泌机制开始成为研究热点。为了对革兰氏阴性菌中脂蛋白分泌机制的研究现状有一个系统全面的了解,本文概述了脂蛋白转运过程中Lol系统5个转运蛋白的功能与保守性、不同细菌中脂蛋白分泌信号的差异以及表面展示脂蛋白可能的分泌机制。     

Elucidation of lipoprotein particles structure by proteomic analysis

Lipoproteins are responsible for lipid packaging and transport through the bloodstream, and for their delivery to target tissues. Their participation in process, such as inflammation and innate immunity has also been suggested recently. Lipoprotein particles have very complex biochemical structures, which result from intricate processes involving coordinated mechanisms of protein and lipid synthesis, intracellular assembling and trafficking, and intra- and extracellular metabolism. Alterations in these mechanisms cause several negative effects on human health. The ability of current proteomic approaches to dissect the dynamic nature of complex particles revealing protein composition and post-translational modifications is shedding further light on lipoprotein structures and functions. This review summarizes lipoprotein classification, biogenesis and metabolism as well as discussing how the results of 20 proteomics-based reports integrate our knowledge on both their biochemical composition and their effects on target cells, thus contributing to reveal the possible functions.     

Small-Molecule Inhibitors of Gram-Negative Lipoprotein Trafficking Discovered by Phenotypic Screening

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.     

Biophysical characterization and stabilization of detergent-solubilized lipoprotein N-acyl transferase from P. aeruginosa and E. coli

Lipoproteins are important for bacterial growth and virulence and interest in them as targets for antibiotic development is growing. Lipoprotein N-acyl transferase (Lnt) catalyzes the final step in the lipoprotein posttranslational processing pathway. The mature lipoprotein can remain in the inner membrane or be trafficked to the outer membrane in the case of diderm prokaryotes. With a view to obtaining high-resolution crystal structures of membrane integral Lnt for use in drug discovery a program was undertaken to generate milligram quantities of stable, homogenous and functional protein. This involved screening across bacterial species for suitable orthologues and optimization at the level of protein expression, solubilization and stability. Combining biophysical and functional characterization, orthologous Lnt from Escherichia coli and the opportunistic human pathogen Pseudomonas aeruginosa was identified as suitable for the proposed structure determination campaign that ultimately yielded crystal structures. The rational approaches taken that eventually provided structure-quality protein are presented in this report.     

A new ABC transporter mediating the detachment of lipid-modified proteins from membranes

Lipoproteins in Escherichia coli are anchored to the periplasmic side of either the inner or the outer membrane by a lipid moiety that is covalently attached to the amino-terminal cysteine residue. Membrane specificity depends on a sorting signal at position 2 of the lipoprotein. Lipoproteins directed to the outer membrane are released from the inner membrane in an ATP-dependent manner through the formation of a complex with LolA, a periplasmic chaperone. However, the ATPase involved in this reaction has not been identified. Here we show, using reconstituted proteoliposomes, that a new complex, LolCDE, belonging to the ATP-binding cassette (ABC) transporter family, catalyses the release of lipoproteins in LolA- and sorting-signal-dependent manners. The LolCDE complex differs mechanistically from all other ABC transporters as it is not involved in the transmembrane transport of substrates. This new mechanism is evolutionarily conserved in other gram-negative bacteria.     

Dissecting the complete lipoprotein biogenesis pathway in Streptomyces scabies

Following translocation, bacterial lipoproteins are lipidated by lipoprotein diacylglycerol transferase (Lgt) and cleaved of their signal peptides by lipoprotein signal peptidase (Lsp). In Gram-negative bacteria and mycobacteria, lipoproteins are further lipidated by lipoprotein N-acyl transferase (Lnt), to give triacylated lipoproteins. Streptomyces are unusual amongst Gram-positive bacteria because they export large numbers of lipoproteins via the twin arginine protein transport (Tat) pathway. Furthermore, some Streptomyces species encode two Lgt homologues and all Streptomyces species encode two homologues of Lnt. Here we characterize lipoprotein biogenesis in the plant pathogen Streptomyces scabies and report that lgt and lsp mutants are defective in growth and development while only moderately affected in virulence. Lipoproteins are lost from the membrane in an S. scabies lgt mutant but restored by expression of Streptomyces coelicolor lgt1 or lgt2 confirming that both encode functional Lgt enzymes. Furthermore, lipoproteins are N-acylated in Streptomyces with efficient N-acylation dependent on Lnt1 and Lnt2. However, deletion of lnt1 and lnt2 has no effect on growth, development or virulence. We thus present a detailed study of lipoprotein biogenesis in Streptomyces, the first study of Lnt function in a monoderm bacterium and the first study of bacterial lipoproteins as virulence factors in a plant pathogen.