Development of inhibitory peptide in adherence of periodontopathic bacteria to dental plaqu

我们先前发现牙龈卟啉菌毛特异性结合唾液酸性脯氨酸富蛋白（PRP）， PRP的活性结构域为PQGPPQ。在本研究中，我们检测了PRP中c端区对应的合成肽（PQGPPPQGGRPQGPPQGQSPQ; pepPRP-C）对菌毛与唾液蛋白相互作用的影响。pepPRP-C显著抑制菌毛与碱性富脯氨酸糖蛋白（PRG）包被羟基磷灰石（HAP）微球以及羟基磷灰石微球上PRP的结合。在覆盖实验中，pepPRP-C明显减少了毛与各种唾液成分之间的相互作用，包括PRPs， PRGs和较小分子大小的成分。此外，pepPRP-C可以抑制牙龈假单胞菌与多种链球菌的共聚集，表明该肽可能是一种有效的抑制牙龈假单胞菌粘附牙菌斑的抑制剂。然后生成分泌pepPRP-C的重组戈登氏链球菌，作为防止牙龈卟啉卟啉口腔定植的可能方法模型。通过对合成的寡核苷酸进行自互补退火，获得了编码pepPRP-C的重复DNA片段（pepPRP-C）。在框架中，PepPRP-C下游连接到一个启动子和一个编码下行链球菌葡萄糖基转移酶1信号肽的基因。将连接的片段插入pVA838衍生的质粒pMNK-4中。将构建的质粒转化到S.gordonii G9B中，成功分泌重组pepPRP-C （r-pepPRP-C）。含有r-pepPRP-C的细菌培养上清液对牙龈假单胞菌细胞和菌毛与PRP结合的抑制作用分别达到72%和77%。r-pepPRP-C浓缩物还能抑制牙龈假单胞菌与多种链球菌的共聚集。因此，pepPR？c分泌系统可能可用于预防牙龈假单胞菌引起的牙周炎。

We previously showed that P.gingivalis fimbriae specifically bind to salivary acidic proline-rich proteins (PRP) and that the active domain in PRP is PQGPPQ.In this study, we examined the effect of synthetic peptide (PQGPPPQGGRPQGPPQGQSPQ ; pepPRP-C) corresponding to C-terminal region in PRP on the interaction between fimbriae and salivary proteins. The pepPRP-C significantly inhibited the binding of fimbriae to basic proline-rich glycoproteins (PRG)-coated hydroxyapatite (HAP) beads as well as to PRP on HAP beads. In the overlay assay, the pepPRP-C clearly diminished the interactions between the fimbriae and the various salivary components, including PRPs, PRGs, and the components with smaller molecular sizes. Moreover, pepPRP-C inhibited the coaggregation of P.gingivalis with various streptococcal strains, suggesting that this peptide may act as an effective inhibitor against P.gingivalis adherence to dental plaque. Then the recombinant Streptococcus gordonii secreting pepPRP-C was generated as a model of a possible approach to prevent the oral colonization of P.gingivalis. A duplicate DNA fragment (pepPRP-C) encoding pepPRP-C was obtained by self-complementary annealing of synthetic oligonucleotides. PepPRP-C was connected downstream to a promoter and a gene encoding a signal peptide of Streptococcus downei glucosyltransferase 1 in frame. The linked fragments were inserted into the plasmid pMNK-4 derived from pVA838. The constructed plasmid was transformed to S.gordonii G9B, which successfully secreted the recombinant pepPRP-C (r-pepPRP-C). The concentrated bacterial culture supernatant containing r-pepPRP-C inhibited the binding of P.gingivalis cells and fimbriae to PRP up to 72% and 77%, respectively. The r-pepPRP-C concentrate also inhibited the coaggregation of P.gingivaLis with various streptococcal strains. Therefore, the pepPR? -C secretion system might be available for prevention against P.gingivalis-induced periodontitis.