牙菌斑链球菌与牙龈卟啉单胞菌相互作用的研究

牙菌斑生物膜是牙周病最主要的致病因素。早期定植菌链球菌与晚期定植菌牙龈卟啉单胞菌(P.gingivalis)的相互作用复杂多样,而牙龈卟啉单胞菌是重要的牙周致病菌,本文就链球菌与牙龈卟啉单胞菌的相互作用作一综述。     

牙龈卟啉单胞菌分子生物学研究进展

牙龈卟啉单胞菌已公认为是牙周炎的致病菌,它的一些表面结构诸如细胞外囊泡,菌毛,外膜蛋白,凝集素介导该菌对牙周组织粘附、定植,或作为毒性因子破坏牙周组织,随着分子生物学技术的发展,已对这些结构进行了分子克隆,本文拟就牙龈卟啉单胞菌(Porphyromonasgingivalis)简称Pg)分子生物学进展作一简要综述。1　菌毛基因的分子克隆牙龈卟啉单胞菌菌毛作为该菌表面结构之一介导了该菌对牙周组织的粘附和定植。已纯化了41kda菌毛亚单位蛋白,并克隆了菌毛蛋白基因[1]。使用寡核苷酸M1和M2作为引物,采用PCR从9株Pg菌株中扩增了1.3kb的DNA片段,E…     

牙龈卟啉单胞菌蛋白酶及疫苗的研究进展

牙龈卟啉单胞菌 (P g) ,革兰阴性厌氧菌 ,是人类牙周炎的主要致病菌[1] 。动物实验表明它在小鼠、大鼠和灵长类动物的龈下定植与牙周炎的发生和进展相关。P g可以调整真核细胞信号转导途径 ,为了满足新陈代谢的需要 ,P g基因表达的调节可以控制在转录水平。证据表明 ,P g的感染会导致严重的全身系统疾病如心血管疾病和分娩早产儿。P g含有大量毒性因子[2 ] ,如菌毛 ,血凝素[3 ] ,脂多糖等 ,其中Arg、Lys样半胱氨酸蛋白酶在牙周致病作用中占据了一个重要地位 ,他们通过激活宿主前体酶 ,例如 :血纤维蛋白酶原 ,或通过暴露细胞隐位及改变血…     

牙龈卟啉单胞菌相关致病因子的研究进展

牙龈卟啉单胞菌是一种生长于口腔内的革兰阴性厌氧茵.它能够利用脂多糖、荚膜多糖、菌毛、牙龈蛋白酶等一系列致病因子,侵袭局部牙周组织并逃避宿主的免疫防御机制,是诱发牙周炎的重要因素之一,引起了学者们的广泛关注.因此,探讨牙龈卟啉单胞菌的致病因子对于牙周炎的防治具有重要意义.     

牙龈卟啉单胞菌菌毛FimA基因型在牙周病患者中的分布情况

目的：探讨牙龈卟啉单胞菌FimA基因型在牙周患者的分布情况。方法：采用PCR技术对40例牙周患者龈下菌斑中牙龈卟啉单胞菌及其基因型进行检测。结果：牙龈卟啉单胞菌在牙周患者的牙周袋内检出率为87．5％,牙龈卟啉单胞菌FimA各基因型在牙龈卟啉单胞菌携带者的检出率分别为：I型22．9％,Ⅱ型60．0％,Ⅲ型17．1％,Ⅳ37．1％．V型未检出;基因I型在牙周袋内未单独检出,与基因Ⅱ型混合感染。结论：牙龈卟啉单胞菌FimA基因Ⅱ型和Ⅵ型是牙龈卟啉单胞菌在牙周病损部位的主要定植菌,两基因型可能与牙周病的发生发展有关。     

牙龈卟啉单胞菌血凝素2氯化血红素结合位点多肽对牙龈卟啉单胞菌生长抑制作用

目的探讨牙龈卟啉单胞菌血凝素2（Porphyromonas gingivalis hemagglutinin-2,PgHA-2）的氯化血红素结合位点多肽对牙龈卟啉单胞菌（Porphyromonasgingivalis,Pg）摄取氯化血红素生长的影响。方法合成多肽DHYAVMISK（肽1）,DEYAVMISK（肽2,肽1中第2位氨基酸突变为谷氨酸）,ALHPDHYLI（肽3,HA-2结合位点不相关多肽）。将肽l、肽2、肽3分别与氯化血红素琼脂糖珠预孵育,加入Pg重组血凝素2（Porphyromonas gingivalis recombinant HA-2,PgrHA-2）,收集与氯化血红素结合的PgrHA-2,SDS—PAGE电泳,分析多肽对PgrHA-2与氯化血红素结合的抑制作用。肽1、肽2、肽3与氯化血红素预孵育后,加入到CDC液体培养基中培养Pg,测定菌液A600值,分析多肽对Pg生长的抑制作用。结果肽1浓度依赖性抑制PgrHA-2与氯化血红素结合,而肽2和肽3对PgrHA-2与氯化血红素的结合无抑制作用。在24、48和72h时间点,肽1组的A600值较肽2、肽3和PBS组明显降低（P〈0．05）。结论本研究表明PgHA-2氯化血红素结合位点多肽DHYAVMISK与Pg竞争结合氯化血红素,抑制Pg的生长,为开发新的牙周病防治方法奠定基础。     

不同fimA基因型牙龈卟啉单胞菌超微结构观察

目的 探查具有不同粘附、侵入能力的各fimA基因型P.gingivalis菌体表面结构特点.方法 选取临床培养的经PCR鉴定、筛选的fimA基因Ⅰ、Ⅱ和Ⅳ型P.gingivalis野生菌株,制备超薄切片,在透射电镜下进行菌体表面结构观察.结果 fimA基因Ⅰ、Ⅱ和Ⅳ型P.gingivalis的菌毛存在差别：Ⅰ型和Ⅱ型菌体表面有放射状菌毛,Ⅱ型略显致密,Ⅳ型表面未见明显菌毛.同时也观察到各型荚膜存在差别：Ⅰ型荚膜最厚,Ⅳ型荚膜较薄,Ⅱ型荚膜最薄.结论 fimA基因Ⅰ、Ⅱ和Ⅳ型P.gingivalis的菌毛和荚膜存在较大差别,推测其粘附、侵入或其它致病能力的差异可能不仅与菌毛有关,还与荚膜或其它因素有关.     

抗牙龈卟啉单胞菌血凝素2单克隆抗体的制备

目的制备抗牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)血凝素2(hemagglutinin-2,HA-2)的单克隆抗体(monoclonal antibody,mAb)。方法用重组HA-2(recombinant HA-2,rHA-2)免疫BALB/C小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0融合,间接ELISA方法筛选杂交瘤细胞.用ELISA方法测效价。结果获得1株能够高效识别rHA-2的mAb,命名为4F11。此单克隆抗体的免疫球蛋白亚类为IgG1,效价达1?106。结论成功制备了重组牙龈卟啉单胞菌血凝素2的单克隆抗体mAb.将进一步用于牙龈卟啉单胞菌的诊断,并为牙周疾病的治疗研究奠定基础。     

牙龈卟啉单胞菌酪氨酸激酶致病性的分子机制

目的 探讨牙龈卟啉单胞菌（Porphyromonas gingivalis,P. gingivalis）酪氨酸激酶（Ptk1)致病性的分子机制。方法 采用重组PCR技术构建P. gingivalis野生菌株ATCC 33277的Ptk1单基因缺失的突变菌株（ΔPtk1）,通过Real-time PCR技术检测并比较参与调控P. gingivalis（野生型P. gingivalis ATCC 33277与突变型ΔPtk1）细胞外多糖（extracellular polysaccharides,EPS）合成的转录因子SinR的表达情况,同时采用激光共聚焦显微镜观察Ptk1缺失的突变菌株与野生型菌株EPS的形成情况,最后通过ELISA试剂盒检测并比较Ptk1缺失的突变菌株与野生型菌株白细胞介素-1β(IL-1β)表达情况。结果 与野生菌株P. gingivalis ATCC 33277比较,Ptk1单基因缺失的突变菌株转录因子SinR的表达量没有显著变化（t=–1.572,P>0.05）;ELISA检测发现,Ptk1单基因缺失的突变菌株IL-1β的表达量较野生型菌株显著下降,差异有统...     

牙龈卟啉单胞菌血凝素2与氯化血红素结合位点分析

目的 通过竞争性ELISA方法明确牙龈卟啉单胞菌血凝素2(Porphyromonas gingivalis hemagglutinin-2,Pg HA-2)与氯化血红素结合的多肽位点,为牙周病保护性抗体的制备奠定基础.方法 人工合成疑为Pg HA-2氯化血红素结合位点的多肽片段,制备抗Pg HA-2单克隆抗体(MAb QB),通过间接竞争性ELISA,进一步分析血红素特异性结合位点.结果 Pg HA-2氯化血红素结合位点的氨基酸序列为DGFPGDHYAVMISK.MAb QB可以抑制Pg HA-2与氯化血红素结合.结论 明确了Pg HA-2氯化血红素结合位点的氨基酸序列,确定了Pg HA-2氯化血红素结合位点的位置.为今后Pg HA-2氯化血红素结合位点的鉴定、功能结构区分析和多肽疫苗的制备奠定基础.     

健康青少年口腔优势菌对牙龈卟啉单胞菌拮抗作用的实验研究

目的分析健康青少年口腔优势菌（简称S．t2003）与牙龈卟啉单胞菌的相互关系。方法运用定量混合培养技术,以血链球菌标准株和变形链球菌作为对照。结果S．t2003与血链球菌标准株都对牙龈卟啉单胞菌有很强的抑制作用,但S．t2003的抑菌强度强于血链球菌的标准株;另外S．t2003和血链球菌标准株对牙龈卟啉单胞菌的抑制作用随其相对浓度的变化而改变,显示了S．t2003和血链球菌标准株与牙龈卟啉单胞菌的相互拮抗的关系。结论S．t2003对牙龈卟啉单胞菌具有较强的拮抗作用。     

COPD患者口腔和呼吸道内牙龈卟啉单胞菌、齿垢密螺旋体和福赛坦菌的同源性分析

目的分析慢性阻塞性肺疾病(COPD)患者口腔和呼吸道内牙龈卟啉单胞菌(Pg)、齿垢密螺旋体(Td)和福赛坦菌(Tf)的同源性,探讨牙周炎与COPD之间的关系。方法收集53例急性加重期COPD患者,采集龈下菌斑及呼吸道分泌物,同时进行牙周指标的检查。通过16S r DNA序列分析,构建患者口腔及呼吸道内Pg、Td和Tf的系统进化树,进行同源性分析。结果 53例患者中,5例患者口腔和呼吸道内Pg具有同源性,6例患者Td具有同源性,3例患者Tf具有同源性。结论 COPD患者口腔和呼吸道内Pg、Td和Tf具有同源性,COPD患者呼吸道内的这3种病原体均来自于口腔。     

Purification and partial characterization of a lysine-specific protease of Porphyromonas gingivalis

Abstract A lysine-specific protease hydrolysing peptide bonds at the carboxyl side of lysine residues in Porphyromonas gingivalis was purified from culture supernatant by a combination of ion-exchange chromatography, gel filtration, and affinity chromatography. The molecular mass was 48 kDa and the p I value was 7.3. The enzyme hydrolysed the peptide bonds at the carboxyl side of lysine residues in synthetic substrates and natural proteins.     

Intra- and inter-individual comparison of Porphyromonas gingivalis genotypes

Abstract Genetic analysis of 31 clinical strains of Porphyromonas gingivalis isolated from nine subjects, 2–6 strains per subject, was performed by Southern hybridization. Chromosomal DNA was extracted by the method of Moncla et al. [1] and digested to completion with restriction endonucleases Pst I, Cla I and Bgl I. The DNA fragments were separated electrophoretically on agarose gels, transferred to nylon membranes and hybridized to the non-radioactively labelled plasmid pKK 3535 which contains the rrn B ribosomal RNA operon of the Escherichia coli chromosome. Of the three enzymes, Bgl I was the most suitable for the genetic analysis of P. gingivalis . With this enzyme, the intra-individual strains were shown to be identical in eight of the nine subjects, whereas inter-individual strains were different.     

Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis

We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability.     

Quantitative proteomics of intracellular Porphyromonas gingivalis

Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction.     

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.     

Expression of the short fimbriae of Porphyromonas gingivalis is regulated in oral bacterial consortia

The Mfa1 protein of Porphyromonas gingivalis is the structural subunit of the short fimbriae and mediates coadhesion between P. gingivalis and Streptococcus gordonii. We utilized a promoter-lacZ reporter construct to examine the regulation of mfa1 expression in consortia with common oral plaque bacteria. Promoter activity of mfa1 was inhibited by S. gordonii, Streptococcus sanguinis and Streptococcus mitis. In contrast, Streptococcus mutans, Streptococcus cristatus, Actinomyces naeslundii, Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum did not affect mfa1 expression. Expression of SspA/B, the streptococcal receptor for Mfa1, was not required for regulation of mfa1 promoter activity. Proteinaceous molecule(s) in oral streptococci may be responsible for regulation of Mfa1 expression. Porphyromonas gingivalis is capable of detecting heterologous organisms, and responds to selected organisms by specific gene regulation.     

Quantitative detection of Porphyromonas gingivalis fimA genotypes in dental plaque

We developed quantitative fimA genotype assays and applied them in a pilot study investigating the fimbrial genotype distribution of Porphyromonas gingivalis in European subjects with or without chronic periodontitis. P. gingivalis was found in 71% and 9% of the samples from patients and healthy subjects, respectively. Enumeration of total P. gingivalis cell numbers by polymerase chain reaction and immunofluorescence showed excellent correspondence (r = 0.964). 73% of positive samples contained multiple fimA genotypes, but generally one genotype predominated by one to three orders of magnitude. Genotype II predominated in 60% of the samples. Genotype IV occurred with similar prevalence (73%) as genotype II but predominated in only 20% of the samples. Genotypes I, III and V were of much lower prevalence and cell densities of the latter two remained sparse. Our results suggest marked differences among the fimA genotypes' ability to colonize host sites with high cell numbers.