Inhibition of tissue-reparing signaling by P.gingivalis infection

牙龈假单胞菌是一种引起牙周病的革兰氏阴性菌，已知可侵入多种宿主细胞，如上皮细胞和成纤维细胞。感染牙周组织的慢性炎症导致组织破坏。在这项研究中，我们调查了该病原体的两个主要毒力因素的参与；菌毛和一种叫做牙龈蛋白酶的蛋白酶对受感染宿主组织的破坏和再生的抑制。首先，我们构建了牙龈卟啉单胞菌缺乏菌毛和缺乏牙龈蛋白酶的突变体。利用这两个突变体，我们发现：1)牙龈卟啉卟啉突变体和牙龈蛋白酶缺乏突变体对人牙周韧带细胞（pdl）的侵袭均减少。2)野生型牙龈卟啉单胞菌在无血清培养基中感染导致pdl脱离培养皿。然而，缺乏牙龈蛋白酶的突变体并没有引起pdl的分离。3)野生型牙龈假单胞菌感染可诱导FAK和paxillin等整合素相关信号分子降解。然而，在感染牙龈素缺乏突变体时，没有观察到FAK和paxillin的降解。4)通过抓痕实验，我们发现牙龈卟啉单胞菌的感染强烈抑制了受损pdl单层的再生。细菌感染抑制了细胞的运动，也抑制了细胞的生长。5)Gingipain-deficient突变体对感染的细胞单层再生无抑制作用。另一方面，纤原缺陷突变体确实抑制了再生过程。这些结果强烈提示牙龈痛在抑制感染牙周组织再生中起重要作用。

P.gingivalis, a periodontopathic Gram-negative bacteria, is known to invade into a variety of host cells such as epithelial cells and fibroblasts. A chronic inflammation of infected periodontal tissue results in tissue destruction. In this study, we investigated the involvement of the two major virulent factors of this pathogen ; fimbriae and a protease called gingipain on the destruction and inhibition of regeneration of infected host tissues.At first, we constructed fimbriae-deficient and gingipain-deficient mutants of P.gingivalis. Using these two mutants, we found that :1)Both fimbriae- and gingipain-deficient mutants of P.gingivalis showed decreased invasion in human periodontal ligament cells (PDLs).2)Infection of wild type of P.gingivalis in serum-free medium resulted in a detachment of PDLs from the culture dishes. However, gingipain-deficient mutant did not induce such detachment of PDLs.3)Infection of wild type of P.gingivalis induced a degradation of integrin-associated signaling molecules such as FAK and paxillin. However, the degradation of FAK and paxillin was not observed during infection with gingipain-deficient mutant.4)By using a scratch assay, we found that the infection of P.gingivalis strongly inhibited the regeneration of damaged monolayers of PDLs. The bacterial infection inhibited the cellular movement as well as the cellular growth.5)Gingipain-deficient mutant did not show the inhibitory effect on the regeneration of the infected cellular monolayers. On the other hand, fimbriae-deficient mutant did inhibit the regeneration process.These results strongly suggested that gingipain of P.gingivalis plays an important role in the inhibition of regeneration of the infected periodontal tissues.