Induction of matrix metalloproteinases and a collagen-degrading phenotype in fibroblasts and epithelial cells by secreted Porphyromonas gingivalis proteinase

Periodontitis is characterized by advancement of a narrow band of epithelium (1–10 cells wide) through the collagenous periodontal ligament in response to bacterial accumulation and infection. A modulating role by epithelial cells in the progression of periodontitis was hypothesized due to the close proximity of the advancing epithelium to both the etiological bacteria and to the collagen fibers of the ligament. We demonstrate that rat mucosal epithelial cells and human fibroblasts are similarly stimulated to degrade a collagen type I cellular substrate by thiol-dependent activity released by the major periodontal pathogen Porphyromonas gingivalis. A purified, extracellular bacterial thiolproteinase from P. gingivalis ATCC 33277 stimulated mucosal epithelial cells to upregulate expression of collagenase and stromelysin, and to degrade a collagen type I fibril matrix. Stimulation of the epithelial cells with this purified proteinase was associated with morphological changes in the cells and with accumulation of secreted latent procollagenase throughout the culture medium. Release of active collagenase was minimal and collagen degradation by the epithelial cells was discreet and localized subcellularly suggesting the possibility that activation of the secreted procollagenase was cell-associated. We conclude that a collagen-degrading phenotype can be stimulated in relatively quiescent mucosal epithelial cells and fibroblasts by the presence of bacterial proteinase. These experiments suggest roles for the P. gingivalis thiol-proteinase and the epithelial cell in the pathogenesis of periodontal disease and demonstrate the potential for dysregulation of extracellular matrix remodeling events during healing of other bacterially infected wounds.     

Porphyromonas gingivalis mediates the shedding and proteolysis of complement regulatory protein CD46 expressed by oral epithelial cells

Introduction:  Human cells express membrane-bound complement regulatory proteins to prevent complement-mediated autologous tissue damage. In this study, we hypothesized that Porphyromonas gingivalis , the major etiological agent of chronic periodontitis, causes the shedding or proteolysis of the complement regulatory protein CD46 expressed by oral epithelial cells.
Methods:  Oral epithelial cells were treated with a culture of P. gingivalis before measurement of membrane-bound and shed CD46 by enzyme-linked immunosorbent assay (ELISA). The effect of soluble recombinant CD46 on secretion of interleukin-8 (IL-8) by epithelial cells was evaluated by ELISA. The susceptibility of soluble recombinant CD46 to proteolytic degradation by cells and purified Lys-gingipain of P. gingivalis was investigated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/western immunoblotting analysis.
Results:  Oral epithelial cells treated with a culture of P. gingivalis showed a lower reactivity with antibodies directed to CD46. ELISA revealed that such a treatment resulted in increased amounts of CD46 in the conditioned media suggesting that P. gingivalis caused the shedding of membrane-anchored CD46. Stimulation of epithelial cells with soluble recombinant CD46 induced IL-8 secretion in a dose-dependent manner. Whole cells and purified Lys-gingipain of P. gingivalis degraded recombinant CD46 in a dose-dependent manner.
Conclusion:  This study showed the ability of P. gingivalis to induce the shedding/ proteolysis of CD46 from the surface of oral epithelial cells. This may render host cells susceptible to the complement system and contribute to tissue damage and the inflammatory process in periodontitis.     

Sequence analysis of kgp in Porphyromonas gingivalis isolates from periodontitis patients

The aim of the present study was to determine sequence variation in the Lys-x-specific protease (Kgp) encoding gene kgp of Porphyromonas gingivalis and to analyze its association with periodontal disease severity. Pooled subgingival plaque samples were obtained from the six most severely affected sites of 102 patients with periodontitis. Sequence analysis of the kgp gene in 23 clinical P. gingivalis isolates resulted in the identification of two distinct kgp types (kgp-I and kgp-II) according to sequence differences in the region encoding the catalytic domain. Restriction analysis revealed that 59 of the 102 patients were colonized by kgp-I and 43 by kgp-II. Patients harboring kgp-I or kgp-II showed no significant difference in the severity of periodontal disease as assessed by pocket probing depth and bleeding on probing following adjustment for smoking habit and age. Moreover, no differences in proteolytic activity of Kgp-I and Kgp-II were detected. The results indicated that two kgp types are maintained in natural populations of P. gingivalis.     

牙龈蛋白的生物学特性及临床意义

牙龈蛋白是一类结构和功能十分相似的蛋白质,具有多种生物学活性和免疫原性。牙龈蛋白分为牙龈蛋白酶R（Rgp）和牙龈蛋白酶K（Kgp）,具有结合、吸收、聚集血红蛋白的能力,可以降解和灭活免疫球蛋白,有效抑制中性粒细胞产生氧,抑制其杀菌作用,使牙龈卟啉单胞菌细胞避免被噬菌细胞攻击。Kgp能分解纤维蛋白原和纤维蛋白,从而干扰血凝块的形成使凝血酶时间延长,引起病变组织出血。Rgp能促进牙龈成纤维细胞产生干细胞生长因子,参与牙周病的炎症和修复过程。牙龈蛋白能破坏血管内皮细胞中细胞因子的反应系统,降低其黏附性和细胞活性,在引起牙周病的同时参与心血管疾病发生。牙龈蛋白能直接作用于结合上皮,降解细胞之间的连接,破坏结合上皮结构和功能的完整性,为牙龈卟啉单胞菌侵入牙周组织进一步发挥致病作用创造条件。在免疫功能上,抗牙龈蛋白抗体可避免上牙槽骨的吸收。四环素族药物等抑制剂可抑制牙龈蛋白活性,提高宿主对牙龈卟啉单胞菌的抵抗力。     

Sequence variations in rgpA and rgpB of Porphyromonas gingivalis in periodontitis

Objective:  The aim of the present study was to determine sequence variations in the active centre of the Arg-X-specific protease encoding genes rgpA and rgpB of clinical Porphyromonas gingivalis isolates and to analyse their prevalence in periodontitis patients before and 3 months after mechanical periodontal therapy.
Background:  Genetic diversity at nucleotides 281, 283, 286 and 331 has been shown to result in amino acid substitutions in the catalytic domain of RgpA and RgpB that affect the substrate specificity and thus may influence the efficacy of Arg-X-protease specific inhibitors.
Methods:  Sequence analysis of rgpA and rgpB genes in clinical P. gingivalis strains isolated from subgingival plaque samples of 82 periodontitis patients before and 3 months after mechanical supra- and subgingival debridement was performed.
Results:  No specific variation within the rgpA sequence was observed. However, the rgpB sequence in the region of the active centre showed five different rgpB genotypes, which were named NYPN, NSSN, NSSK, NYPK and DYPN according to the derived amino acid substitution. Porphyromonas gingivalis genotype NYPN was detected in 27 patients (32.9%) before and in 8 patients (9.8%) after therapy, NSSN in 26 (31.7%) and 10 (12.2%), NSSK in 22 (26.8%) and 2 (2.4%), NYPK in 5 (6.2%) and 1 (1.2%), and DYPN in 1 patient (1.2%) and 0 patients (0%), respectively. Only one patient (1.2%) harboured two P. gingivalis rgpB genotypes (NSSK/NYPN) before treatment; these were no longer detected after therapy.
Conclusion:  The results indicate that five rgpB genotypes are maintained in natural populations of P. gingivalis. These data may be of importance with regard to the development of specific rgpB inhibitors.     

牙龈卟啉单胞菌PG1055基因在临床菌株中的表达

目的应用基因芯片技术检测PG1055基因在不同人群的牙龈卟啉单胞菌(P.gingivalis)中分布,探讨这些基因与牙周临床指数之间的关系。方法取龈下菌斑进行细菌分离培养,以临床采集样本提取的DNA为探针,以抑制消减杂交技术获得P.gingivalisW83的特异基因片段PG1055为目标序列,采用Cy5荧光标记目标序列。应用基因芯片技术检测PG1055基因在牙周病患者及健康人群的牙龈卟啉单胞菌中的分布。结果PG1055基因在牙周病患者及健康人群中的检出率有统计学差异,并且与牙周临床指数相关。结论PG1055基因与P.gingivalis的致病性有关。     

Selective inhibition of Porphyromonas gingivalis growth by a factor Xa inhibitor, DX-9065a

BACKGROUND: Porphyromonas gingivalis is a causative bacterium of adult periodontitis. However, there is no drug specific for P. gingivalis and for its virulence factor. OBJECTIVES: The objective of this study was to examine the effects of a new selective inhibitor of activated factor X, DX-9065a, on growth of Porphyromonas gingivalis and other periodontopathic bacteria. METHODS: We incubated P. gingivalis and other periodontopathic bacteria in the presence or absence of DX-9065a and examined the effect of DX-9065a on bacterial growth and trypsin-like activity in its cultures. We also examined the effects of DX9065a on amidolytic activity of purified trypsin-like proteinases (gingipains RgpA and RgpB), from P. gingivalis and on trypsin-like activity in gingival crevicular fluids from patients with adult periodontitis. RESULTS: DX-9065a selectively inhibited the growth of P. gingivalis and Prevotella intermedia, and its effect on P. gingivalis was bactericidal. Trypsin-like proteinase activity was detected in P. gingivalis, and the activity was strongly inhibited by DX-9065a. DX-9065a even inhibited amidolytic activity of RgpA and RgpB from P. gingivalis. Furthermore, trypsin-like proteinase activity in gingival crevicular fluids was strongly inhibited by DX-9065a. CONCLUSIONS: DX-9065a inhibits P. gingivalis growth in part through to its ability to inhibit the trypsin-like proteinase activity in P. gingivalis and may be useful for a new drug for treatment of adult periodontitis.     

Analysis of Porphyromonas gingivalis PG27 by deletion and intragenic suppressor mutation analyses

PG27 is required for secretion of virulence factor gingipains, and has recently been proposed as LptO, which is involved in O-deacylation of lipopolysaccharide. In the present study, a predicted 14 anti-parallel β-strand structure of PG27 was ascertained. Deletion study showed that the region from Asp382 to the C-terminal His391 of PG27 is dispensable for the function of PG27. Analysis of C-terminal deletion mutants revealed that the region in strand S14 (Asn369-Gly385) is important for activity. Of the gingipain-defective mutants, ΔThr378-His391 and ΔPhe377-His391 produced amounts of PG27 comparable to those produced by wild-type cells, suggesting that Thr378-Phe381 contains essential residues for the function of PG27. In contrast, ΔPhe381-His391, ΔAla380-His391, ΔLeu379-His391 and ΔArg376-His391 produced no detectable PG27. The defects of the ΔAla380-His391 mutant were suppressed by changing either Ala346 or Ala359 of PG27 to valine. Importantly, Ala346 and Ala359 are located close to Leu379 in the structural model of PG27. A359V compensated for the instability of PG27, but not the gingipain-defective phenotypes, of other deletion mutants tested, suggesting that Ala380 and Phe381 of PG27 are important for the stability of PG27. Lastly, we found that the C-terminal region of PG27 may be located in the periplasm. Taken together, these findings fit well with a predicted β-barrel structure model for PG27, and show that strand S14 is important for its function.