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

我们先前的研究表明，牙龈假单胞菌菌毛与唾液酸性富含脯氨酸的蛋白(PRP)特异结合，并且PRP中的活性结构域是PQGPPQ。在本研究中，我们研究了与PRP中C-末端对应的合成肽(PQGPPPQGGRPQGPPQGQSPQ；PepPRP-C)对菌毛与唾液蛋白相互作用的影响。多肽PRP-C显著抑制菌毛与碱性富含脯氨酸糖蛋白(PRG)包被的羟基磷灰石(HAP)小球和HAP小球上的PRP的结合。在重叠分析中，PepPRP-C明显减少了菌毛与各种唾液成分之间的相互作用，包括PRPS、PRGS和分子尺寸较小的成分。此外，PepPRP-C还能抑制牙龈假单胞菌与各种链球菌的共聚集，提示该多肽可能是一种有效的抑制牙龈假单胞菌黏附牙菌斑的药物。然后，重组戈登链球菌分泌多肽PRP-C，作为一种可能的预防牙龈假单胞菌口腔定植的方法的模型。通过人工合成的寡核苷酸自互补退火法获得了编码PepPRP-C的重复DNA片段(PepPRP-C)。PepPRP-C在下游连接到一个启动子和一个编码唐氏链球菌糖基转移酶1信号肽的基因。将连接片段插入pVA838表达载体pMNK-4中。将构建的重组表达载体转化戈登氏链霉菌G9B，成功地分泌重组多肽PRP-C(r-PepPRP-C)。含r-PepPRP-C的浓缩细菌培养上清液对牙龈假单胞菌和菌毛与PRP的结合抑制率分别高达72%和77%。R-PepPRP-C浓缩物还能抑制牙龈假单胞菌与各种链球菌的共聚集。因此，Peppr？-C分泌系统有可能用于预防牙周炎。

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.