链球菌脂蛋白的功能作用及其作为疫苗候选蛋白和抗菌药物靶标的研究进展

脂蛋白是一类广泛存在于革兰氏阴性细菌和革兰氏阳性细菌中的细胞膜锚定蛋白,具有多种生物学功能.脂蛋白不仅作为细菌的毒力因子,而且能够识别和激活宿主的免疫系统,是当前预防和治疗细菌感染的热门靶点之一,本文对链球菌中脂蛋白的主要功能及其在疫苗和抗菌药物研究中的进展进行了综述和展望,为今后链球菌中脂蛋白的深入研究拓宽了思路.     

Versatility of choline metabolism and choline-binding proteins in Streptococcus pneumoniae and commensal streptococci

The pneumococcal choline-containing teichoic acids are targeted by choline-binding proteins (CBPs), major surface components implicated in the interaction with host cells and bacterial cell physiology. CBPs also occur in closely related commensal species, Streptococcus oralis and Streptococcus mitis , and many strains of these species contain choline in their cell wall. Physiologically relevant CBPs including cell wall lytic enzymes are highly conserved between Streptococcus pneumoniae and S. mitis . In contrast, the virulence-associated CBPs, CbpA, PspA and PcpA, are S. pneumoniae specific and are thus relevant for the characteristic properties of this species.     

Platelet aggregation by oral streptococci

One proposed mechanism in the pathogenesis of infective endocarditis is the direct aggregation of platelets by the bacteria causing the disease. Some, but not all, strains of Streptococcus sanguis have been reported to aggregate platelets but the taxonomy of this and related taxa has changed recently. The ability to aggregate platelets by 24 genetically grouped laboratory stock strains was studied along with 8 recent isolates from cases of endocarditis. Strains belonging to S. sanguis could aggregate platelets, but not S. gordonii, "S. parasanguis", S. mitis, S. oralis or related taxa. Also, preliminary data indicate that certain biotypes of S. sanguis lack the ability to aggregate platelets. Of the recent clinical isolates, only 4 aggregated platelets and each of these showed phenotypes typical of S. sanguis. These data suggest that the ability to aggregate platelets is not essential for an organism to be able to cause endocarditis, although it may be a significant virulence factor.     

变异链球菌和血链球菌全代谢途径比对分析

为了比较变异链球菌和血链球菌全代谢途径,依据KEGG数据库(http://www.genome.ad.jp/kegg)对变异链球菌和血链球菌的全部代谢途径作逐项比对。结果显示,二者参与了85个代谢途径,包括多数以相同的酶参与的中央代谢途径,即糖酵解、三羧酸循环、磷酸戊糖途径等,和多数以不同的酶参与的双组分感应系统等。通过变异链球菌和血链球菌整体代谢网络对比,了解了变异链球菌和血链球菌理论上的全部代谢途径,为全面揭示二者代谢交流研究奠定了基础。     

Comparative evaluation of Strepto B ID chromogenic medium and Granada media for the detection of Group B streptococcus from vaginal samples of pregnant women

Two types of selective media, the chromogenic medium Strepto B ID® and two non-chromogenic media Strepto B agar® and the Granada® medium, were tested and compared to blood agar plates (BAP) for screening of Group B streptococcus vaginal colonization in pregnant women. All tested media were comparable in terms of sensitivity however, their use in routine laboratories may markedly facilitate the rapid detection of GBS in vaginal samples.     

Streptococcal beta-hemolysins: genetics and role in disease pathogenesis

A zone of β-hemolysis surrounding colonies on blood-agar media is a hallmark phenotypic feature of the pathogens group A Streptococcus (GAS) and group B Streptococcus (GBS). In each case, lysis of red blood cells reflects the action of a potent protein exotoxin. Although these toxins have been the subjects of numerous investigations over the years, their purification and molecular identification have proven elusive. These difficulties reflect the instability of hemolytic activity, as both toxins function only in the context of the bacterial surface or certain high molecular weight ‘stabilizer’ molecules. This review highlights the recent discoveries of two markedly distinct genetic loci, necessary and sufficient for the β-hemolytic phenotypes of GAS and GBS, respectively. The generation of isogenic GAS and GBS β-hemolysin-deficient mutants and their analysis using in vitro and in vivo model systems has shown that both toxins function as virulence factors in the pathogenesis of invasive infections.     

Protoplast transformation of group N streptococci with cryptic plasmids

Abstract By using an extension to group N streptococci of a contransformation procedure we have introduced 4 different-sized cryptic plasmids for Streptococcus lactis into the plasmid-free S. lactis IL1403. A mixture of 4 cryptic plasmids with an indicator plasmid (pHV1301) conferring erythromycin resistance was used for IL1403 protoplast transformation. Under such conditions, 41.5% of the erythromycin-resistant transformants were contransformed with one of the cryptic plasmids in addition to pHV1301. Indicator plasmid pHV1301 was later spontaneously segregated from doubly transformed cells. This protocol should be very useful for constructing lactic streptococcal strains bearing any phenotypically cryptic plasmid.     

Heterologous expression of an endoglucanase gene (endA) from the ruminai anaerobe Ruminococcus flavefaciens 17 in Streptococcus bovis and Streptococcus sanguis

Abstract The heterologous expression of a cloned endoglucanase gene ( endA ) from the ruminai bacterium Ruminococcus flavefaciens 17 was demonstrated in the Streptococcus species S. bovis JB1 and S. sanguis DLL The endA gene was introduced into S. bovis and S. sanguis using the Escherichia coli/Streptococcus shuttle vector pVA838. Expression of the gene was detected by clearing zones around the recombinant colonies on agar plates containing carboxymethylcellulose stained with Congo red. S. bovis JB1 containing the endA gene was capable of utilizing cellotetraose at a faster rate than the parent strain. This is the first demonstration that Streptococcus species can express a gene from a Ruminococcus flavefaciens strain.     

Clinically acceptable restorations may be a hotbed for cariogenic microbes

doi: 10.1111/j.1741‐2358.2011.00571.x Clinically acceptable restorations may be a hotbed for cariogenic microbes Objective: The aim of this study was to investigate the cross‐sectional association of dental restorations with salivary cariogenic pathogens among the elderly to establish effective parameters of caries risk for this population. Materials and methods: Stimulated whole saliva was collected from 289 community‐dwelling older adults (66.2 ± 3.9 years old) who had 20 or more teeth. Salivary levels of three cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus and lactobacilli) were estimated using quantitative polymerase chain reaction (real‐time PCR) method. Results: The mean number of residual teeth was 26.4, and restored teeth with crowns, inlays and composite resin were 7.35, 3.88 and 0.68, respectively. The number of crowns correlated positively with salivary S. mutans, S. sobrinus and lactobacilli. Multiple linear regression analyses showed that the number of restored teeth with crowns was independently associated with salivary S. mutans, S. sobrinus and lactobacilli after controlling for age, gender, number of residual teeth and salivary flow rate. Salivary flow rate was independently associated with salivary S. mutans and lactobacilli. Conclusion: The number of crowns had an association with salivary levels of cariogenic bacteria, suggesting that this parameter may be a caries risk indicator for the elderly population.     

The influence of saliva on the hydrophobic surface properties of bacteria isolated from oral sites of macaque monkeys

Abstract The surface hydrophobicity of 64 bacterial strains isolated from discrete, intra-oral sites of monkeys ( Macaca fascicularis ) was determined by measuring their affinity for hexadecane. Bacteria were also exposed to monkey saliva which either increased or reduced the surface hydrophobicity of the cells. After exposure to saliva those bacteria isolated solely from the mucosal surfaces were significantly more hydrophobic than bacteria ( Streptococcus mutans and Actinomyces spp.) whose major habitat was the dentition. Streptococcus sanguis strains isolated from all intra-oral sites and among the early plaque formers were as hydrophobic as the organisms isolated only from the mucosal surfaces.     

Phenotypic characteristics of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus)

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis , respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges–Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.     

ELISA检测奶牛乳房炎疫苗效果方法的建立

建立一种检测抗乳房炎疫苗抗体的酶联免疫吸附试验(ELISA)方法,用于免疫后具有保护性效果的抗体水平。通过对细胞抗原制备、最佳抗原包被浓度的确定、抗原包被和封闭最佳条件确定、酶标抗体的最佳工作浓度及作用时间等反应体系的筛选和确定,建立了检测抗乳房炎疫苗抗体的间接ELISA方法。结果表明,金黄色葡萄球菌(Staphylococcus aureus)、无乳链球菌(Streptococcus agalactiae)、停乳链球菌(Streptococcus dysgalactiae)抗原最佳包被质量浓度分别为5 mg.L-1、10 mg.L-1、10 mg.L-1;包被条件为4℃过夜;用2%人血白蛋白作为封闭剂最佳封闭条件是37℃封闭30 min;酶标抗体最佳作用时间为30 min。结论攻毒试验表明,疫苗免疫后,血清中抗体滴度达到或超过免疫前2倍时,奶牛即具有较好的乳腺保护效力。这一指标可作为疫苗效力检验的标准。     

Infection of Streptococcus oralis NCTC 11427 by pneumococcal phages

We have found a group of pneumococcal bacteriophages (Cp-1, Cp-7) that can successfully infect and replicate in Streptococcus oralis, whereas Dp-1 was unable to infect this species. We have also developed conditions that allowed transfection of S. oralis using Dp-1 DNA. Our results support the direct involvement of the phage-coded lysins in the liberation of the phage progeny from infected S. oralis cells. Since S. oralis and S. pneumoniae are bacteria that share the same ecological niche in humans, the availability of the system described here should allow to extend our current studies on the modular organization of the lytic enzymes and might serve as a tool to study the evolutionary relationships between host and parasite.     

Species-specific PCR method for identification of Streptococcus downei

AIMS: To establish a rapid method to differentiate Streptococcus downei and S. sobrinus by multiplex PCR. METHODS AND RESULTS: A PCR primer pair specific to S. downei was designed on the basis of the nucleotide sequence of the dextranase gene of S. downei NCTC 11391T. The primer pair specifically detected S. downei, but none of the other mutans streptococci (16 strains of six species). The PCR procedure was capable of detecting 1 pg of genomic DNA purified from S. downei NCTC 11391 and as few as 14 CFU of S. downei cells. The mixture of primer pairs specific to each S. downei (this study) and S. sobrinus (Igarashi et al. 2000) detected only the strains of these two species among all the mutans streptococcal strains, and concomitantly differentiated the two species by species-specific amplicons of different lengths. CONCLUSIONS: The present PCR method is highly specific to S. downei and is useful for detection and identification of S. downei. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiplex PCR using dextranase gene primers is a useful method for simultaneous detection and differentiation of S. downei and S. sobrinus.     

Tea extracts differentially inhibit Streptococcus mutans and Streptococcus sobrinus biofilm colonization depending on the steeping temperature

Abstract

This study aimed to evaluate the effects of tea extracts on oral biofilm colonization depending on steeping temperature. S. mutans and S. sobrinus were cultured and treated with green or black tea extracts prepared under different steeping conditions. Biofilm formation, glucosyltransferase (GTF) levels, bacterial growth, and acidogenicity were evaluated. Biofilms were also assessed by gas chromatography-mass spectrometry and confocal laser scanning microscopy. All extracts with hot steeping showed higher inhibitory effects on biofilm formation and cell viability and lower GTF levels compared with those with cold steeping (p?<?0.05). Hot steeping significantly reduced bacterial growth (p?<?0.05) and maintained the pH. Catechins were only identified from hot steeping extracts. Within the limits of this study, extracts with cold steeping showed lower inhibitory effects on oral biofilms. The different effects between steeping extracts may be attributed to the difference in catechins released from tea extracts under the different steep conditions.     

De novo glucan synthesis by mutans streptococcal glucosyltransferases present in pellicle promotes firm binding of Streptococcus gordonii to tooth surfaces

Adherence of 3H-labelled cells of Streptococcus gordonii and Streptococcus milleri to artificial pellicles prepared from saliva supplemented with glucosyltransferases from mutants streptococci was examined using a new assay for sucrose-dependent cell-to-pellicle attachment. Results indicate that S. gordonii, but not S. milleri, could attach tightly to hydroxylapatite surfaces through de novo glucan synthesis by mutants streptococcal glucosyltransferases present in the experimental salivary pellicles.     

人母乳细菌Streptococcus salivarius F286和S. parasanguinis F278对HT29细胞的细胞毒性

为了筛选到具有抗炎特性的有益菌,研究者通常将待测细菌的发酵液上清和热致死菌体与TNF-α刺激下的人类结肠腺癌细胞HT29共孵育,并测量细菌是否能够减少HT29细胞分泌的炎症因子。该测试的前提之一是待测细菌的发酵液上清或菌体不杀死或杀死<10%的HT29细胞。在前期的工作中,我们从人母乳中分离得到Streptococcus salivarius F286和S.parasanguinis F278两株菌。在研究这两株菌的抗炎能力之前,我们利用MTT法摸索不同浓度的S.salivarius F286和S.parasanguinis F278的发酵液上清和热致死菌体对HT29细胞的细胞毒性。实验表明,两株菌的发酵液上清的原液和稀释液对HT29细胞均没有细胞毒性;浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体、浓度5×10^5~2.5×10^6cfu/mL的F278热致死菌体对HT29细胞的细胞毒性低于10%,而浓度1×10^8cfu/mL的热致死F286和F278菌体分别杀死(23±5.3)%和(22±5.3)%的HT29细胞。因此,S.salivarius F286和S.parasanguinis F278的发酵液上清原液、以及浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体和5×10^5-2.5×10^6cfu/mL的F278热致死菌体可在HT29细胞模型中进行抗炎能力测试。本研究的方法可用于确定其他细菌在HT29细胞模型中进行抗炎能力测试的合理浓度范围。