Participation of DPPIV produced by P.gingivalis in periodontal disease.

牙龈卟啉单胞菌是成人牙周炎的主要致病菌，也被认为是老年人吸入性肺炎等全身疾病的病原体。该细菌产生二肽基氨基肽酶IV(DPPIV)，它是一种丝氨酸蛋白酶，能从多肽链的N端裂解X-Pro或X-ALA二肽。本研究的目的是阐明牙龈假单胞菌DPPIV作为致病因子的病理功能。我们从牙龈假单胞菌中克隆了DPPIV的编码基因(DPP)，构建了DPP缺失的等基因突变体，并进行了动物实验。野生型表现出比突变体更高的毒力，表现为更高的死亡率，更慢的疾病恢复，以及更大程度的病变发展。组织病理学观察，野生型对结缔组织的破坏比突变型更严重。然后我们研究了这种酶参与组织破坏的分子机制。该酶具有明胶酶活性，可通过宿主来源的明胶酶、基质金属蛋白酶(MMP2和MMP9)加速明胶的降解。被宿主来源的胶原酶-1或8裂解的I型胶原被DPPIV进一步降解，这种酶与纤维连接蛋白结合，并介导牙龈假单胞菌与纤维连接蛋白的黏附。在DPP缺失突变体中未检测到粘附性。通过引入野生型DPP基因和突变基因恢复了活性，突变基因中负责DPPIV外肽酶活性的氨基酸Ser593通过定点突变发生了改变。DPPIV以剂量依赖的方式抑制人牙龈成纤维细胞和NIH3T3细胞与纤维连接蛋白的黏附。这些结果显示了DPPIV的新的生物学活性，并表明了其在牙周炎和其他系统性疾病进展中的病理作用。

Porphyromonas gingivalis is a major pathogen associated with adult periodontitis and is also suggested to be an etiologic agent of systemic diseases such as aspiration pneumonia in the elder. This bacterium produces dipeptidyl aminopeptidase IV (DPPIV), which is a serine protease that cleaves X-Pro or X-Ala dipeptide from the N-terminal end of the polypeptide chain. Our purpose of this investigation was to clarify pathological functions of P.gingivalis DPPIV as a virulence factor. We cloned the gene (dpp) coding for DPPIV from P.gingivalis, constructed an isogenic dpp null mutant, and subjected to animal experiments. The wild type showed higher virulence than the mutant, as revealed by a higher mortality, a slower recovery from illness, and a greater extent of lesion development. Destruction of the connective tissue caused by the wild type was severer than that by the mutant as observed histopathologically. We then investigated the molecular mechanisms by which this enzyme participated in the tissue destruction. This enzyme possessed a gelatinase activity and accelerated gelatin degradation by host-derived gelatinase, matrix metalloproteinases (MMP-2 and MMP-9). Type I collagen cleaved with host-derived collagenase, MMP-1 or MMP-8, was further degraded by DPPIV.This enzyme bound to fibronectin and mediated adhesion of P.gingivalis to fibronectin. Adhesion was not detected in the dpp-null mutant. The activity was restored by introducing not only the wild type dpp gene but the mutant gene, in which the amino acid responsible for DPPIV exopeptidase activity, Ser 593, was altered by site-directed mutagenesis. Adhesion of human gingival fibroblast and NIH3T3 cells to fibronectin was inhibited by DPPIV in a dose dependent manner. These results exhibit novel biological activities of DPPIV and indicate the pathological roles for progression of periodontitis and other systemic diseases.