Identification of key determinants in Porphyromonas gingivalis host‐cell invasion assays

The periodontal pathogen Porphyromonas gingivalis can invade host cells, a virulence trait which may contribute to the persistence of infection at subgingival sites. Whilst the antibiotic protection assay has been commonly employed to investigate and quantify P. gingivalis invasion, data obtained have varied widely and a thorough investigation of the factors influencing this is lacking. We investigated the role of a number of bacterial and host‐cell factors and report that the growth phase of P. gingivalis, source (laboratory strain vs. clinical strain), host‐cell identity (cell line vs. primary), host‐cell lysis method, and host‐cell passage number had no significant effect on bacterial invasion. However, incubation time, host‐cell seeding density, method of quantification (viable count vs. DNA), and whether host cells were plated or in suspension, were shown to influence invasion. Also, cells isolated by rapid adhesion to fibronectin exhibited higher levels of P. gingivalis invasion, possibly as a result of increased levels of active α5β1 integrin. Interestingly, this may represent a population of cells with stem cell‐like properties. This study provides important new information by identifying the most important factors that influence P. gingivalis invasion assays and may help to explain variations in the levels previously reported.     

Subgingival colonization by Porphyromonas gingivalis

Porphyromonas gingivalis, a gram-negative anaerobe, is a major causative agent in the initiation and progression of severe forms of periodontal disease. In order to cause periodontal disease, P. gingivalis must colonize the subgingival region, a process that involves several distinct steps and multiple gene products. The organism must first navigate within the oral fluids in order to reach the hard or soft tissues of the mouth. Retention and growth of bacteria on these surfaces is facilitated by a repertoire of adhesins including fimbriae, hemagglutinins and proteinases. Once established subgingivally, P. gingivalis cells participate in intercellular communication networks with other oral prokaryotic cells and with eukaryotic cells. The establishment of these multiple interactive interfaces can lead to biofilm formation, invasion of root dentin and internalization within gingival epithelial cells. The resulting bacterial and host cellular locations, products and fate contribute to the success of P. gingivalis in colonizing the periodontal region.     

Invasive differences among Porphyromonas gingivalis strains from healthy and diseased periodontal sites

Background and Objective:  The purpose of this study was to determine any difference between Porphyromonas gingivalis isolates from periodontally healthy sites as compared to those from diseased sites with respect to the ability to invade host cells.
Material and Methods:  Subgingival plaque samples were obtained from periodontally healthy and diseased sites using paper points. P .  gingivalis colonies were isolated and tested, using an antibiotic protection assay, for their ability to invade KB cells. P. gingivalis 381 and Escherichia coli MC1061 were used as controls.
Results:  Mean values of 16.79 ± 0.86 × 10³ colony-forming units/mL and 26.14 ± 2.11 × 10³ colony-forming units/mL were observed in invasion assays for isolates from periodontally healthy and diseased sites, respectively. P .  gingivalis present in diseased sites had significantly greater invasive abilities than strains isolated from healthy sites. No statistical difference was noted between male or female subjects concerning the degree of invasion; isolates from diseased sites from both genders had significantly greater invasion abilities than those from healthy sites. A significant correlation was found between the increased invasive capabilities of P .  gingivalis isolates vs. an increased probing depth.
Conclusion:  The increased invasion noted with P .  gingivalis isolates from diseased sites vs. healthy sites, and the increased invasive capabilities with increasing probing depth, indicate that P .  gingivalis isolates have a varying ability to invade host cells in the periodontal pocket.     

Immunologic environment influences macrophage response to Porphyromonas gingivalis

Macrophages adapt both phenotypically and functionally to the cytokine balance in host tissue microenvironments. Recent studies established that macrophages contribute an important yet poorly understood role in the development of infection‐elicited oral bone loss. We hypothesized that macrophage adaptation to inflammatory signals encountered before pathogen interaction would significantly influence the subsequent immune response of these cells to the keystone oral pathobiont Porphyromonas gingivalis. Employing classically activated (M1) and alternatively activated (M2) murine bone‐marrow‐derived macrophage (BMDMø), we observed that immunologic activation of macrophages before P. gingivalis challenge dictated phenotype‐specific changes in the expression of inflammation‐associated molecules important to sensing and tuning host response to bacterial infection including Toll‐like receptors 2 and 4, CD14, CD18 and CD11b (together comprising CR3), major histocompatibility complex class II, CD80, and CD86. M2 cells responded to P. gingivalis with higher expression of tumor necrosis factor‐α, interleukin‐6, monocyte chemoattractant protein‐1, macrophage inflammatory protein‐1α, regulated on activation normal T cell expressed and secreted, and KC than M1 cells. M1 BMDMø expressed higher levels of interleukin‐10 to P. gingivalis than M2 BMDMø. Functionally, we observed that M2 BMDMø bound P. gingivalis more robustly than M1 BMDMø. These data describe an important contribution of macrophage skewing in the subsequent development of the cellular immune response to P. gingivalis.     

Effects of metronidazole on Porphyromonas gingivalis biofilms

Subgingival bacteria exist within a biofilm consisting of cells and extracellular matrix which may afford organisms protection from both antibiotics and components of the host immune system. MIC values for planktonic Porphyromonas gingivalis treated with metronidazole were compared with those obtained for the same strain in biofilms associated with hydroxyapatite (HA) surfaces. The treated biofilms were examined for growth and studied by scanning electron microscopy. A broth assay resulted in an MIC of 0.125 μg/ml for metronidazole against P. gingivalis . P. gingivalis biofilms exhibited growth after treatment with 20μg/ml metronidazole, which was 160 times the MIC for planktonic organisms. The results of this study indicate that biofilm-associated P. gingivalis may be resistant to metronidazole at concentrations which are usually attained by systemic administration.     

Identification and characterization of novel glycoproteins involved in growth and biofilm formation by Porphyromonas gingivalis

Porphyromonas gingivalis has been implicated as a major pathogen associated with chronic periodontitis. To extend our knowledge of post‐translational protein glycosylation in P. gingivalis, a proteomic analysis involving two‐dimensional polyacrylamide gel electrophoresis combined with carbohydrate staining and mass spectrometry was performed. Four novel glycoproteins, PGN0743, PGN0876, PGN1513 and PGN0729, in P. gingivalis ATCC 33277 were identified. These four identified glycoproteins possess a range of biochemical activities and cellular localization. PGN0743 contains a sequence motif identifying it as a FKBP‐type cis‐trans isomerase, which has activity usually associated with chaperone functions. PGN0876 and PGN1513 contain tetratricopeptide repeat domains that mediate protein–protein interactions. PGN0729 encodes the outer membrane protein 41 precursor, which was previously identified as Pgm6, and is homologous to the OmpA protein in Escherichia coli. Several different types of glycoprotein were identified, suggesting that P. gingivalis possesses a general mechanism for protein glycosylation. PGN0743‐deficient and PGN0876‐deficient mutants were constructed to examine the role(s) of the two identified glycoproteins. Both mutants showed a decreased growth rate under nutrient‐limited conditions and reduced biofilm formation activity. These results suggest that the novel glycoproteins PGN0743 and PGN0876 play an important role in the growth and colonization of P. gingivalis.     

Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

The ability for DNA mismatch repair, after oxidative stress‐induced DNA damage, is critical for the persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket. Our previous report demonstrated that, in contrast to other organisms, the repair of oxidative stress‐induced DNA damage involving 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) may occur by a yet‐to‐be described mechanism in P. gingivalis. 8‐oxoG does not block DNA replication; rather, it mispairs with adenine, which can be repaired by the MutY glycosylase. To determine the function of the P. gingivalis MutY homologue in DNA repair, it was insertionally inactivated using the ermF‐ermAM antibiotic cassette and used to create a mutY‐deficient mutant (FLL147) by allelic exchange mutagenesis. FLL147 had an increased rate of spontaneous mutation and was more sensitive to hydrogen peroxide compared with the wild‐type W83 strain. DNA oligomers containing a site‐specific 8‐oxoG:A mispair was repaired similarly in both the P. gingivalis mutY‐defective mutant and wild‐type strains. The P. gingivalis mutY homologue was shown to complement the mutY mutation in Escherichia coli. In a gel mobility shift assay, the purified recombinant MutY is able to bind an oligo containing an 8‐oxoG:A mispair. Taken together, MutY may play the expected role in oxidative stress resistance in P. gingivalis. However, there may exist other redundant mechanism(s) for the removal of 8‐oxoG:A mismatch in this organism.     

Antigenic cross-reactivity among Porphyromonas gingivalis serotypes

The goal of our research program is to develop a Porphyromonas gingivalis vaccine. Vaccine development requires identification of antigenic components shared by the many clonal types of P. gingivalis. The purpose of the present study was to evaluate the extent and nature of antigenic cross-reactivity among serotypes of P. gingivalis and to identify shared antigenic components. Strains selected to represent serotypes A-D were 33277, A7A1-28 W50 and 381, respectively. Using intact cells, antibodies were raised in rabbits. Titers were assessed by enzyme-linked immunosorbent assay (ELISA) using intact cells as antigen, Western blots were prepared and biologic activity was measured as opsonization (chemiluminescence expressed as mV) and enhancement of phagocytosis and killing by polymorphonuclear leukocytes. Extensive cross-reactivity that varied greatly among serotypes was observed by ELISA. The Western blots showed an even greater extent of cross-reactivity, with shared protein components at approximately 140, 130, 37, 32 and 28 kDa and a shared variable molecular mass smear considered to be lipopolysaccharide and other carbohydrate. Additional protein components at 110, 85, 35 and 20 kDa appeared to be shared by some but not all serotypes. In the functional assays, strains 33277 and 381 were equally well opsonized by anti-33277 and anti-381 (500-650 mV) but opsonized to a much lesser extent by anti-A7A1-28 and anti-W50 (roughly 125 mV and 350 mV respectively). A7A1-28 and W50 were opsonized by all four immune sera almost equally but to a much lower extent (roughly 400 mV and 250 mV respectively). Enhancement of phagocytosis and killing in the presence of active complement mirrored opsonization with the exception that 381 was reasonably well opsonized by anti-A7A1-28 (400 mV) and anti-W50 (350 mV), but poorly killed. The protein components at 140, 130, 37 and 28 kDa shared by all of the four serotypes appear to have potential as vaccine candidate antigens.     

Virulence of six capsular serotypes of Porphyromonas gingivalis in a mouse model

Capsular structures of Porphyromonas gingivalis have been correlated to the pathogenicity in animal models. Six polysaccharide capsular serotypes have recently been described in P. gingivalis. In the present study, virulence of the P. gingivalis strains of the six capsular serotypes was compared with strains of the non-capsular serotype of P. gingivalis in a mouse model. All 18 encapsulated strains caused a spreading type of infection with exudate, often accompanied by ulceration and necrosis of the skin and a significant loss of body weight. All three non-encapsulated strains tested induced localized abscesses, although a spreading type of infection was occasionally observed. Illness of the mice was almost always associated with recovery of encapsulated P. gingivalis from blood, kidneys, lungs and spleens. A statistically significant contrast was found in the average weight change during the experiment between the mice infected with the K^- serotype strains and the mice infected with the encapsulated P. gingivalis strains. Differences in severity of illness and the recovery of P. gingivalis from mouse tissues were recorded between strains of the same serotype. The differences in virulence within a capsular serotype suggest that the capsule is an important but not the only determining virulence factor for P. gingivalis.     

牙龈卟啉单胞菌对人类免疫缺陷病毒-1致病性的影响

人类免疫缺陷病毒-1(HIV-1)是以CD4+细胞缺损和功能障碍为主要表现的获得性免疫缺陷综合征(AIDS)的主要病原。AIDS与牙周病有密切的关系,HIV-1的感染被广泛认为是重型牙周病的危险因素,而近来研究发现牙周病可能是HIV-1感染宿主细胞、诱导潜伏的HIV-1复制的促进因素。牙龈卟啉单胞菌(Pg)是慢性牙周炎的主要致病菌之一,该文综述Pg对HIV-1致病性的影响。     

牙龈卟啉单胞菌脂多糖对泡沫细胞凋亡基因的影响

目的 了解牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)脂多糖(lipopolysaccharide,LPS)在巨噬细胞吞噬氧化低密度脂蛋白(oxidized low density lipopmtein,oxLDL)形成泡沫细胞过程中和形成后对凋亡基因的影响,以期了解Pg影响动脉粥样硬化的可能机制.方法 以oxLDL、oxLDL+Pg-LPS刺激人急性单核细胞白血病单核细胞株THP-1源性巨噬细胞,以及oxLDL诱导巨噬细胞形成的泡沫细胞.采用吖啶橙-溴化乙锭双染色观察细胞凋亡,聚合酶链反应(PCR)芯片检测11种动脉粥样硬化相关凋亡基因的变化,实时PCR检测p53、c-Myc和半胱氨酸天冬氨酸蛋白酶(caspase)-3基因的变化.结果 Pg-LPS提高了巨噬细胞吞噬oxLDL形成泡沫细胞过程中和形成后的细胞凋亡率,分别为(5.47±0.93)%、(7.50 4-0.54)%;PCR芯片检测显示泡沫细胞形成过程中Pg-LPS上调了B细胞淋巴瘤-白血病-2相关蛋白Al的转录(>2倍),泡沫细胞形成后上调了B细胞淋巴瘤-白血病-2和B细胞淋巴瘤-白血病-2相关蛋白A1的转录(>2倍);在泡沫细胞形成过程中提高了p53和caspase-3的转录水平[分别为(4.50×10-3±4.02×10-4)和(5.30×10-2±4.58×10-3)],抑制了c-Myc的转录水平(1.53×10-2±5.77×10-4);在泡沫细胞形成后降低了p53转录水平(4.23×10-3±5.85×10-4),促进了caspase-3的转录水平(6.00×10-2±6.08×10-3),P<0.05.结论 Pg-LPS影响了泡沫细胞形成过程中和形成后细胞中多种凋亡基因的转录,促进了凋亡的发生.     

Genetic and antigenic analyses of Porphyromonas gingivalis FimA fimbriae

The periodontal pathogen Porphyromonas gingivalis generally expresses two distinct fimbriae, FimA and Mfa1, which play a role in biofilm formation. The fimA gene that encodes FimA fimbrilin is polymorphic, and polymerase chain reaction analysis has identified six genotypes called types I–V and Ib. We found recently that fimbriae exhibit antigenic heterogeneity among the genotypes. In the present study, we analysed the fimA DNA sequences of 84 strains of P. gingivalis and characterized the antigenicity of FimA fimbriae. Strains analysed here comprised 10, 16, 29, 13, 10 and 6 strains of types I, Ib, II, III, IV and V, respectively. DNA sequencing revealed that type Ib does not represent a single cluster and that type II sequences are remarkably diverse. In contrast, the fimA sequences of the other types were relatively homogeneous. Antigenicity was investigated using antisera elicited by pure FimA fimbriae of types I–V. Antigenicity correlated generally with the respective genotype. Type Ib strains were recognized by type I antisera. However, some strains showed cross‐reactivity, especially, many type II strains reacted with type III antisera. The levels of fimbrial expression were highly variable, and expression was positively correlated with ability of biofilm formation on a saliva‐coated plate. Further, two strains without FimA and Mfa1 fimbriae expressed fimbrial structures, suggesting that the strains produce other types of fimbriae.     

Identification of the genes associated with a virulent strain of Porphyromonas gingivalis using the subtractive hybridization technique

Background/aims:  Porphyromonas gingivalis , a major etiological organism implicated in periodontal disease, can be classified into virulent and avirulent strains. Our aim was to identify a gene for the virulence of P .  gingivalis .
Methods:  The subtractive hybridization technique was employed to identify the genes specific to P .  gingivalis W83, a virulent strain. In this study, P. gingivalis W83 was used as the tester strain, and P .  gingivalis ATCC 33277 was the driver strain. The prevalence of W83-specific genes was determined by Southern blot analysis of several P. gingivalis strains.
Results:  We obtained 575 colonies using the subtractive hybridization technique. From among these, 26 DNA fragments were subjected to a homology search using the BLAST program. Compared with strain ATCC 33277, strain W83 contained 12 unique clones. The specificities of the isolated DNA fragments were analyzed among four P. gingivalis strains by Southern blot analysis. Five genes showed specificity for strain W83 compared with strain ATCC 33277. All five genes were also identified in strain W50.
Conclusions:  The subtractive hybridization technique was effective in screening the two strains for specific DNA sequences, some of which might be responsible for determining virulence. The results suggested that several genes specific to strain W83 were associated with its virulence. Further analysis of these DNA fragments will provide important information on the pathogenesis of virulent P .  gingivalis strains.     

Antigenic cross‐reactivity among Porphyromonas gingivalis serotypes

The goal of our research program is to develop a Porphyromonas gingivalis vaccine. Vaccine development requires identification of antigenic components shared by the many clonal types of P. gingivalis. The purpose of the present study was to evaluate the extent and nature of antigenic cross‐reactivity among serotypes of P. gingivalis and to identify shared antigenic components. Strains selected to represent serotypes A–D were 33277, A7A1‐28 W50 and 381, respectively. Using intact cells, antibodies were raised in rabbits. Titers were assessed by enzyme‐linked immunosorbent assay (ELISA) using intact cells as antigen, Western blots were prepared and biologic activity was measured as opsonization (chemiluminescence expressed as mV) and enhancement of phagocytosis and killing by polymorphonuclear leukocytes. Extensive cross‐reactivity that varied greatly among serotypes was observed by ELISA. The Western blots showed an even greater extent of cross‐reactivity, with shared protein components at approximately 140, 130, 37, 32 and 28 kDa and a shared variable molecular mass smear considered to be lipopolysaccharide and other carbohydrate. Additional protein components at 110, 85, 35 and 20 kDa appeared to be shared by some but not all serotypes. In the functional assays, strains 33277 and 381 were equally well opsonized by anti‐33277 and anti‐381 (500–650 mV) but opsonized to a much lesser extent by anti‐A7A1‐28 and anti‐W50 (roughly 125 mV and 350 mV respectively). A7A1‐28 and W50 were opsonized by all four immune sera almost equally but to a much lower extent (roughly 400 mV and 250 mV respectively). Enhancement of phagocytosis and killing in the presence of active complement mirrored opsonization with the exception that 381 was reasonably well opsonized by anti‐A7A1‐28 (400 mV) and anti‐W50 (350 mV), but poorly killed. The protein components at 140, 130, 37 and 28 kDa shared by all of the four serotypes appear to have potential as vaccine candidate antigens.     

牙龈卟啉单胞菌脂多糖对人牙周膜成纤维细胞胶原吞噬作用的影响

目的研究牙周病致病菌牙龈卟啉单胞菌脂多糖（LPS）对体外培养的人牙周膜成纤维细胞（hPDLF）胶原吞噬作用的影响。方法将不同质量浓度的LPS加入体外培养的hPDLF 48 h后,采用荧光定位术和流式细胞技术检测hPDLF胶原吞噬率的变化。结果LPS导致hPDLF胶原吞噬率显著增加（P<0.05）。结论牙龈卟啉单胞菌脂多糖具有促进hPDLF吞噬胶原的作用,可能是牙周组织破坏机制之一。     

Porphyromonas gingivalis proteinases as virulence factors in the development of periodontitis

Porphyromonas gingivalis contains exceedingly high concentrations of cysteine proteinases with trypsin-like activity which have been implicated as virulence factors in adult-onset periodontitis. These enzymes, referred to as gingipains, cleave protein and peptide substrates after arginine (gingipain R) and lysine residues (gingipain K), and it has been found that neither is easily inhibited by host proteinase inhibitors. Examination of the properties of each proteinase clearly indicates a role(s) for both in the dysregulation of a number of normally tightly controlled pathways. The effects of such uncontrolled proteolysis are the development of edema (kallikrein/kinin pathway activation by gingipain R), neutrophil infiltration (complement pathway activation by gingipain R), and bleeding (degradation of fibrinogen by gingipain K). Since three of the major hallmarks of periodontitis involve increased crevicular flow, neutrophil accumulation at infected sites and bleeding on probing, it seems likely that both P. gingivalis -derived proteinases are important virulence factors in the development of periodontal disease.     

Studies on the aminopeptidase activities of Porphyromonas gingivalis

Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources. The aims of this study were to investigate the aminopeptidase activities of P. gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth. Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected. Experimental evidence was obtained suggesting that the Arg-gingipains of P. gingivalis can function as both an endopeptidase and an aminopeptidase. Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity. Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase. Lastly, a mutant of P. gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity. To investigate the key role of aminopeptidase activities in growth of P. gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation. Bestatin and actinonin were the only ones to inhibit growth of P. gingivalis. Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV).