肺炎链球菌转化模型的建立与优化

建立肺炎链球菌转化模型,优化转化体系,提高转化率,以便于进一步研究其致病的分子机制。制备肺炎链球菌感受态,首先在不同菌密度下转化外源DNA,计数抗生素筛选平板上的转化菌落,比较其转化率,确定转化的最适菌密度;然后在此菌密度下比较CSP诱导不同时相的转化率,同时用RT-PCR检测感受态调控基因comE的表达。对所用血清3型菌株而言转化的最适菌密度在OD550=0.09~0.10之间;CSP-2诱导10 min后转化率最高,可达(15.6±3)%;comE的表达也在CSP-2诱导10 min后达到最高。在实验室条件下,肺炎链球菌转化受多种因素的影响,必需控制好各种因素,选择最优条件才能获得稳定、高效的转化。     

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...     

铜绿假单胞菌的双组分系统

双组分系统是存在于原核和少部分真核生物细胞中的信号转导系统,主要由组氨酸蛋白激酶和反应调节蛋白组成,通过感应外界环境信号、信号输入、磷酸基团传递、信号输出等环节调节基因表达,使细胞能更加适应环境变化。铜绿假单胞菌为条件致病菌,其双组分系统构成多样、功能复杂且参与介导耐药性产生,因此铜绿假单胞菌的双组分系统日益引起人们关注。本文对铜绿假单胞菌双组分系统的组成、信号转导机制、种类、研究方法及其临床意义进行了综述。     

肺炎链球菌疫苗的研究进展

肺炎链球菌疫苗研制对于肺炎链球菌感染的防治具有重要意义。常见的荚膜多糖疫苗存在很大缺陷,需要更为有效的疫苗来替代。本文综述肺炎链球菌疫苗研制中的一些进展,包括结合疫苗的研制、几种公认的蛋白疫苗因子的研究进展和新的蛋白疫苗因子的筛选。同时还介绍肺炎链球菌疫苗研制过程中的一些新思路（联合疫苗、活疫苗、DNA疫苗）,它们是对肺炎链球菌疫苗研制方法的丰富。     

植物的双组分信号系统

双组分系统由感受信号输入的组氨酸(His)蛋白激酶和调节信号输出的反应调控因子组成,涉及许多原核生物、真菌、黏菌和植物的各种信号转导途径.在植物中,还存在更复杂的包括杂合的His激酶、磷酸传递中间体和反应调控因子的信号系统,称为多步骤双组分系统.最近的研究表明,双组分系统在对环境刺激和生长调节剂(如乙烯、细胞分裂素、光和渗透胁迫)的反应中起重要作用.     

植物的双组分信号系统

双组分系统由感受信号输入的组氨酸(His) 蛋白激酶和调节信号输出的反应调控因子组成,涉及许多原核生物、真菌、黏菌和植物的各种信号转导途径。在植物中,还存在更复杂的包括杂合的His激酶、磷酸传递中间体和反应调控因子的信号系统,称为多步骤双组分系统。最近的研究表明,双组分系统在对环境刺激和生长调节剂(如乙烯、细胞分裂素、光和渗透胁迫)的反应中起重要作用。     

肺炎链球菌性肺炎诊断方法研究进展

肺炎链球菌是一种常见的细菌性肺炎的病原体,快速、准确的确定病原体是控制疾病的关键。本文就目前诊断肺炎链球菌性肺炎的免疫层析法、酶联免疫吸附法、胶乳颗粒凝集试验等研究进展作以简要综述。     

肺炎链球菌二元信号转导系统研究进展

肺炎链球菌是引起细菌性肺炎的主要病原菌。透彻地了解肺炎链球菌的信号转导对于系统地认识该菌的致病机制和靶标药物的设计具有重要意义。二元信号转导系统作为在病原菌中普遍存在的一种跨膜信号转导机制,在细菌响应外界环境变化并作出适应性反应的过程中起着主要作用。随着大规模基因组测序、信号标签诱变、差异荧光诱导以及DNA微阵列等技术的蓬勃发展,关于肺炎链球菌的二元信号转导系统已经取得了许多重要的研究成果。本文就肺炎链球菌二元信号转导系统的研究进展作一综述。     

儿科痰培养肺炎链球菌耐药性分析

目的了解厦门市妇幼保健院儿科患者下呼吸道感染肺炎链球菌的耐药情况。方法对厦门市妇幼保健院2007年9月至2009年12月从儿科分离的95株肺炎链球菌用K-B纸片法检测其对青霉素等5种抗菌药物的敏感度,对耐苯唑西林的菌株用E-test法检测青霉素和头孢曲松的MIC值。结果95株肺炎链球菌对红霉素、克林霉素高度耐药,耐药率分别为97.9%、96.8%,对左氧氟沙星几乎敏感,未检出耐万古霉素菌株。E-test法测试青霉素不敏感率为58.9%（56株）,头孢曲松敏感率为93.7%（89株）。ermB因介导的红霉素耐药菌株92株,占98.9%,mefE基因介导的红霉素耐药仅1株（1.1%）。结论厦门市妇幼保健院分离的肺炎链球菌耐药严重,PNSP检出率较高（58.9%）,对红霉素、克林霉素高度耐药,临床应尽量减少此类药物的经验性用药,依据药敏结果选择抗菌药物进行治疗。     

链霉菌次级代谢调控相关的双组分系统研究进展

链霉菌具有强大的次级代谢能力, 能够产生众多具有生物活性的次级代谢产物, 如目前广泛应用的抗生素、抗肿瘤药物以及免疫抑制剂等。在链霉菌中, 次级代谢产物的生物合成受到包括途径特异性、多效性以及全局性调控基因在内的多层次严格调控。关键调控基因的缺失或过表达可以显著影响次级代谢产物的生物合成, 提示对于链霉菌次级代谢重要调控基因的功能及其作用机制的研究具有巨大的潜在应用价值。其中, 作为细菌信号传导系统的双组分系统(Two-component system, TCS)一直是大家研究的关注点。越来越多的研究表明TCS在链霉菌次级代谢过程中发挥着全局性的调控功能。本文重点介绍链霉菌模式菌株——天蓝色链霉菌中TCS(包括典型TCS)、孤立的组氨酸蛋白激酶(HK)以及应答调控蛋白(RR)参与次级代谢调控的研究进展。这些TCS的功能鉴定及机制解析为工业链霉菌的定向遗传改造以提高重要次级代谢产物的含量提供了理论依据。     

Competence increases survival during stress in Streptococcus pneumoniae

Horizontal gene transfer mediated by transformation is of central importance in bacterial evolution. However, numerous questions remain about the maintenance of processes that underlie transformation. Most hypotheses for the benefits of transformation focus on what bacteria might do with DNA, but ignore the important fact that transformation is subsumed within the broader process of competence. Accordingly, the apparent benefits of transformation might rely less on recombination than on other potential benefits associated with the broader suite of traits regulated by competence. We examined the importance of this distinction in the naturally competent species Streptococcus pneumoniae, focusing specifically on predictions of the DNA-for-repair hypothesis. We confirm earlier results in other naturally competent species that transformation protects against DNA-damaging stress. In addition, we show that the stress-protection extends to non-DNA-damaging stress. More important, we find that for some forms of stress transformation is not required for cells to benefit from the induction of competence. This rejects the narrowly defined DNA-for-repair hypotheses and provides the first support for Claverys' hypothesis that competence, but not necessarily transformation, may act as a general process to relieve stress. Our results highlight the need to distinguish benefits of transformation from broader benefits of competence that do not rely on DNA uptake and recombination.     

Expression of Streptococcus pneumoniae Bacteriocins Is Induced by Antibiotics via Regulatory Interplay with the Competence System

Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have unpredictable consequences on pneumococcal colonization dynamics by activating genes that mediate intra-specific interference competition.     

Competence in Streptococcus pneumoniae Is a Response to an Increasing Mutational Burden

Competence for genetic transformation in Streptococcus pneumoniae has previously been described as a quorum-sensing trait regulated by a secreted peptide pheromone. Recently we demonstrated that competence is also activated by reduction in the accuracy of protein biosynthesis. We have now investigated whether errors upstream of translation in the form of random genomic mutations can provide a similar stimulus. Here we show that generation of a mutator phenotype in S. pneumoniae through deletions of mutX, hexA or hexB enhanced the expression of competence. Similarly, chemical mutagenesis with the nucleotide analog dPTP promoted development of competence. To investigate the relationship between mutational load and the activation of competence, replicate lineages of the mutX strain were serially passaged under conditions of relaxed selection allowing random accumulation of secondary mutations. Competence increased with propagation in these lineages but not in control lineages having wild-type mutX. Resequencing of these derived strains revealed between 1 and 9 single nucleotide polymorphisms (SNPs) per lineage, which were broadly distributed across the genome and did not involve known regulators of competence. Notably, the frequency of competence development among the sequenced strains correlated significantly with the number of nonsynonymous mutations that had been acquired. Together, these observations provide support for the hypothesis that competence in S. pneumoniae is regulated in response to the accumulated burden of coding mutations in the bacterial genome. In contrast to previously described DNA damage response systems that are activated by physical lesions in the chromosome, this pneumococcal pathway may represent a unique stress response system that monitors the coding integrity of the genome.     

Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae

An unmodified heptadecapeptide pheromone capable of eliciting competence for genetic transformation in Streptococcus pneumoniae has recently been identified and characterized. In considering possible signaltransduction mechanisms for the peptide, the previously characterized Ami oligopeptide permease and the three highly homologous oligopeptide-binding lipoproteins, AmiA. AliA, and AliB, appeared to be good candidates for receptors. We therefore compared the spontaneous transformability of Ami, AliA and AliB mutants to that of an isogenic wild-type strain and we investigated the response of the various mutants to treatment with synthetic competence-stimulating peptide (CSP). Our results clearly demonstrate that neither Ami nor any of the three highly homologous oligopeptide-binding lipoproteins identified so far in S. pneumoniae are required for competence induction following treatment with synthetic CSP. Although the existence of a fourth unidentified oligopeptide-binding lipoprotein and/or a second oligopeptide permease operon could not be completely ruled out, we favour the hypothesis that CSP signal transmission rather involves a two-component regulatory system. Although none of the single or double Ami and Ali mutants tested appeared severely affected for competence, an exceptional aliB plasmid-insertion mutation abolished competence completely. In addition, the triple AmiA-AliA-AliB mutant differed from wild type in showing no sharp peak of competence but exhibiting transformability throughout the exponential phase of growth. These and previous observations are discussed and a general hypothesis is proposed to account for the modulation of competence by peptide permease mutants in S. pneumoniae.     

Uptake of extracellular DNA: Competence induced pili in natural transformation of Streptococcus pneumoniae

Transport of DNA across bacterial membranes involves complex DNA uptake systems. In Gram‐positive bacteria, the DNA uptake machinery shares fundamental similarities with type IV pili and type II secretion systems. Although dedicated pilus structures, such as type IV pili in Gram‐negative bacteria, are necessary for efficient DNA uptake, the role of similar structures in Gram‐positive bacteria is just beginning to emerge. Recently two essentially very different pilus structures composed of the same major pilin protein ComGC were proposed to be involved in transformation of the Gram‐positive bacterium Streptococcus pneumoniae – one is a long, thin, type IV pilus‐like fiber with DNA binding capacity and the other one is a pilus structure that was thicker, much shorter and not able to bind DNA. Here we discuss how competence induced pili, either by pilus retraction or by a transient pilus‐related opening in the cell wall, may mediate DNA uptake in S. pneumoniae.     

Effects of Streptococcus pneumoniae strain background on complement resistance

Background

Immunity to infections caused by Streptococcus pneumoniae is dependent on complement. There are wide variations in sensitivity to complement between S. pneumoniae strains that could affect their ability to cause invasive infections. Although capsular serotype is one important factor causing differences in complement resistance between strains, there is also considerable other genetic variation between S. pneumoniae strains that may affect complement-mediated immunity. We have therefore investigated whether genetically distinct S. pneumoniae strains with the same capsular serotype vary in their sensitivity to complement mediated immunity.

Methodology and Principal Findings

C3b/iC3b deposition and neutrophil association were measured using flow cytometry assays for S. pneumoniae strains with different genetic backgrounds for each of eight capsular serotypes. For some capsular serotypes there was marked variation in C3b/iC3b deposition between different strains that was independent of capsule thickness and correlated closely to susceptibility to neutrophil association. C3b/iC3b deposition results also correlated weakly with the degree of IgG binding to each strain. However, the binding of C1q (the first component of the classical pathway) correlated more closely with C3b/iC3b deposition, and large differences remained in complement sensitivity between strains with the same capsular serotype in sera in which IgG had been cleaved with IdeS.

Conclusions

These data demonstrate that bacterial factors independent of the capsule and recognition by IgG have strong effects on the susceptibility of S. pneumoniae to complement, and could therefore potentially account for some of the differences in virulence between strains.     

Regulation of Streptococcus pneumoniae clp Genes and Their Role in Competence Development and Stress Survival

In vitro mariner transposon mutagenesis of Streptococcus pneumoniae chromosomal DNA was used to isolate regulatory mutants affecting expression of the comCDE operon, encoding the peptide quorum-sensing two-component signal transduction system controlling competence development. A transposon insertion leading to increased comC expression was found to lie directly upstream from the S. pneumoniae clpP gene, encoding the proteolytic subunit of the Clp ATP-dependent protease, whose expression in Bacillus subtilis is controlled by the CtsR repressor. In order to examine clp gene regulation in S. pneumoniae, a detailed analysis of the complete genome sequence was performed, indicating that there are five likely CtsR-binding sites located upstream from the clpE, clpP, and clpL genes and the ctsR-clpC and groESL operons. The S. pneumoniae ctsR gene was cloned under the control of an inducible promoter and used to demonstrate regulation of the S. pneumoniae clpP and clpE genes and the clpC and groESL operons by using B. subtilis as a heterologous host. The CtsR protein of S. pneumoniae was purified and shown to bind specifically to the clpP, clpC, clpE, and groESL regulatory regions. S. pneumoniae Delta ctsR, Delta clpP, Delta clpC, and Delta clpE mutants were constructed by gene deletion/replacement. ClpP was shown to act as a negative regulator, preventing competence gene expression under inappropriate conditions. Phenotypic analyses also indicated that ClpP and ClpE are both required for thermotolerance. Contrary to a previous report, we found that ClpC does not play a major role in competence development, autolysis, pneumolysin production, or growth at high temperature of S. pneumoniae.     

Two synthetic antigens related to Streptococcus pneumoniae type 3 capsular polysaccharide

The synthesis of the allyl beta-glycosides (8 and 20, respectively) of beta-D-GlcpA-(1----4)-D-Glcp and beta-D-Glcp-(1----3)-D-GlcpA (overlapping disaccharide fragments A and B) in the linear chain of the capsular polysaccharide (S3) from Streptococcus pneumoniae type 3 is described. Oxidation of allyl 2,3,6,2',3',4'-hexa-O-acetyl-beta-cellobioside with chromic acid and saponification of the product gave 8. The synthesis of 20 involved glycosylation of methyl 5-O-acetyl-1,2-O-isopropylidene-alpha-D-glucofuranuronate or its 3-O-trityl derivative and subsequent furanose----pyranose transformation. The derivatives 8 and 20 were each copolymerised with acrylamide. In serological tests (enzyme immunoassay and passive hemagglutination), the resulting antigens exhibited the specificity of S3. It was concluded that fragment A was a much stronger immunodeterminant than fragment B.