Kataoka K, Amano A, Kawabata S, Nagata H, Hamada S, Shizukuishi S
Departments of Preventive Dentistry, Osaka University Faculty of Dentistry, Suita-Osaka, Japan.
Infect Immun. 1999 Aug;67(8):3780-5. doi: 10.1128/IAI.67.8.3780-3785.1999.
Porphyromonas gingivalis, a putative periodontopathogen, can bind to human salivary components with its fimbriae. We have previously shown that fimbriae specifically bind to a peptide domain shared by a major salivary component, i.e., proline-rich (glyco)proteins (PRPs). The synthetic domain peptide PRP-C (pPRP-C) significantly inhibits the fimbrial binding to PRPs. In this study, a recombinant strain of Streptococcus gordonii secreting pPRP-C was generated as a model of a possible approach to prevent the oral colonization by the pathogen. A duplicate DNA fragment (prpC) encoding pPRP-C was obtained by self-complementary annealing of synthetic oligonucleotides. prpC was connected downstream to a promoter and a gene encoding a signal peptide of Streptococcus downei glucosyltransferase I in frame. The linked fragments were inserted into the plasmid pMNK-4 derived from pVA838. The constructed plasmid was inserted to produce the transformant S. gordonii G9B, which then successfully secreted recombinant pPRP-C (r-pPRP-C) of the expected size. The concentrated bacterial culture supernatant containing r-pPRP-C inhibited the binding of P. gingivalis cells and fimbriae to PRP1 in a dose-dependent manner up to 72 and 77%, respectively. The r-pPRP-C concentrate also inhibited the coaggregation of P. gingivalis with various streptococcal strains as effectively as synthetic pPRP-C in a dose-dependent manner. Collectively, pPRP-C was found to be able to prevent P. gingivalis adherence to salivary receptor protein and plaque-forming bacteria. These results suggest that this recombination approach with a nonperiodontopathic bacterium may be suitable for the therapeutic prevention of P. gingivalis adherence to the oral cavity.
牙龈卟啉单胞菌是一种公认的牙周病原体，其菌毛可与人唾液成分结合。我们之前已经表明，菌毛能特异性结合主要唾液成分（即富含脯氨酸的（糖）蛋白，PRPs）所共有的一个肽结构域。合成结构域肽PRP-C（pPRP-C）能显著抑制菌毛与PRPs的结合。在本研究中，构建了一株分泌pPRP-C的重组戈登链球菌，作为预防该病原体口腔定植的一种可能方法的模型。通过合成寡核苷酸的自互补退火获得了编码pPRP-C的重复DNA片段（prpC）。prpC与一个启动子以及编码唐氏链球菌葡糖基转移酶I信号肽的基因框内连接，置于下游。连接后的片段插入到源自pVA838的质粒pMNK-4中。将构建好的质粒插入后产生转化体戈登链球菌G9B，其随后成功分泌出预期大小的重组pPRP-C（r-pPRP-C）。含有r-pPRP-C的浓缩细菌培养上清液分别以剂量依赖方式抑制牙龈卟啉单胞菌细胞和菌毛与PRP1的结合，抑制率分别高达72%和77%。r-pPRP-C浓缩物还能以剂量依赖方式有效抑制牙龈卟啉单胞菌与各种链球菌菌株的共聚，效果与合成的pPRP-C相当。总体而言，发现pPRP-C能够预防牙龈卟啉单胞菌黏附于唾液受体蛋白和形成菌斑的细菌。这些结果表明，这种利用非牙周病原菌的重组方法可能适用于治疗性预防牙龈卟啉单胞菌在口腔中的黏附。
