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.     

大黄素-8-O-β-D-吡喃葡萄糖苷对牙龈卟啉单胞菌唾液酸酶活性及其毒力基因表达影响研究

目的    探讨大黄素-8-O-β-D-吡喃葡萄糖苷对牙龈卟啉单胞菌（P. gingivalis）唾液酸酶活性及其毒力基因表达的影响。方法    使用不同质量浓度的大黄素-8-O-β-D-吡喃葡萄糖苷（0.2、0.5、2、5、10 mg/mL）处理P. gingivalis W83（实验组）,用未加药物的P. gingivalis W83作对照（对照组）,采用荧光法检测大黄素-8-O-β-D-吡喃葡萄糖苷对P. gingivalis唾液酸酶活性的作用。5 mg/mL大黄素-8-O-β-D-吡喃葡萄糖苷作用于P. gingivalis W83,Real-time PCR法检测毒力基因fimA、fimR、fimS、kgp、rgpA和rgpB的表达情况。结果    大黄素-8-O-β-D-吡喃葡萄糖苷对P. gingivalis唾液酸酶活性产生了抑制作用,当其质量浓度为0.2、0.5、2、5、10 mg/mL时,对唾液酸酶活性的抑制率分别为11.4%、32.23%、40.21%、73.54%、84.31%。与对照组比较,实验组（5 mg/mL大黄素-8-O-β-D-吡喃葡萄糖苷处理）的fimA、fimR、fimS、kgp、rgpA和rgpB基因表达均下降,差异均有统计学意义（均P < 0.05）。结论    大黄素-8-O-β-D-吡喃葡萄糖苷可有效抑制P. gingivalis唾液酸酶活性,其抑制作用会降低细菌毒力基因表达,有望成为预防及治疗牙周炎的新型药物。     

Proteome analysis of Porphyromonas gingivalis cells placed in a subcutaneous chamber of mice

Introduction:  Porphyromonas gingivalis , an oral anaerobic bacterium, is considered a major pathogen for chronic periodontitis. Pathogenic bacteria usually upregulate or downregulate gene expression to combat the protective responses of their hosts.
Methods:  To determine what protein is regulated when P. gingivalis cells invade host tissues, we analyzed the proteome of P. gingivalis cells that were placed in a mouse subcutaneous chamber by two-dimensional gel electrophoresis and mass spectrometry.
Results:  Fourteen proteins were upregulated, while four proteins were downregulated. We focused on three upregulated proteins, PG1089 (DNA-binding response regulator RprY), PG1385 (TPR domain protein), and PG2102 (immunoreactive 61-kDa antigen), and constructed mutant strains that were defective in these proteins. Mouse abscess model experiments revealed that the mutant strain defective in PG1385 was clearly less virulent than the wild-type parent strain.
Conclusion:  These results indicate that the PG1385 protein is involved in P. gingivalis virulence and that the method used here is useful when investigating the P. gingivalis proteins responsible for virulence.     

Oxidative stress induces phosphorylation of the ABC transporter, ATP-binding protein, in Porphyromonas gingivalis

The Gram-negative anaerobic bacterium Porphyromonas gingivalis is a major causative agent of periodontal disease. Although P. gingivalis is an anaerobic bacterium, it exhibits aerotolerance and can survive in periodontal pockets, indicating that it must possess a mechanism for protection against oxidative stress, although the precise details are still unclear. Recently, phosphorylation signaling has been implicated in the regulation of bacterial virulence. In the present study, to examine the effect of oxidative stress on phosphorylation of proteins in P. gingivalis, we analyzed oxidative stress-induced alterations of phosphorylated proteins using two-dimensional electrophoresis with phosphoprotein staining coupled with MALDI-TOF mass spectrometry analysis. Among the phosphorylated proteins analyzed, we identified an increase in phosphorylation of the ABC transporter, ATP-binding protein (PG0258). Since the ABC transporter family is known to be involved in lipopolysaccharide (LPS) biosynthesis, we examined the level of LPS using an endotoxin assay and found that LPS production was increased in P. gingivalis. Our present findings suggest that the early response of P. gingivalis to oxidative stress could trigger the development and progression of periodontal disease through enhancement of LPS production by phosphorylation of the ABC transporter, ATP-binding protein.     

Surface components of Porphyromonas gingivalis

Background and Objective:  Research on Porphyromonas gingivalis , a periodontopathogen, has provided a tremendous amount of information over the last 20 years, which may exceed in part than that on other closely related members in terms of phylogenetic as well as proteomic criteria, including Bacteroides fragilis and B. thetaiotaomicron as major anaerobic, opportunistic pathogens in the medical field. In this minireview, we focused on recent research findings concerning surface components such as outer membrane proteins and fimbriae, of P. gingivalis .
Material and Methods:  Elucidation of the surface components in P. gingivalis was especially difficult because outer membrane proteins are tightly bound to lipopolysaccharide and they are resistant to dissociation and separation from each other, even during sodium dodecyl sulfate–polyacrylamide gel electrophoresis, unless samples are appropriately heated. In addition, P. gingivalis is asaccharolytic and therefore a potent proteolytic bacterium, another factor causing difficulty in research. The study of the surface components was carefully carried out considering these unique features in P. gingivalis when compared with other gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa .
Results:  Separation of outer membrane proteins, and characterization of OmpA-like proteins and RagAB as major proteins, is described herein. Our recent findings on FimA and Mfa1 fimbriae, two unique appendages in this organism, and on their regulation of expression are also described briefly.
Conclusion:  Surface components of P. gingivalis somehow have contact with host tissues and cells because of the outermost cell elements. Therefore, such bacterial components are potentially important in the occurrence of periodontal diseases.     

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.     

Scavenger receptor A is expressed by macrophages in response to Porphyromonas gingivalis, and participates in TNF-α expression

Introduction:  Porphyromonas gingivalis is a periodontopathic bacterium closely associated with generalized aggressive periodontal disease. Pattern recognition receptors (PRRs) participate in host response to this organism. It is likely that PRRs not previously recognized as part of the host response to P. gingivalis also participate in host response to this organism.
Methods and Results:  Employing qRT-PCR, we observed increased msr1 gene expression at 2, 6, and 24 h of culture with P. gingivalis strain 381. Flow cytometry revealed increased surface expression of SR-A protein by the 24 h time point. Macrophages cultured with an attachment impaired P. gingivalis fimA - mutant (DPG3) expressed intermediate levels of SR-A expression. Heat-killed P. gingivalis stimulated SR-A expression similar to live bacteria, and purified P. gingivalis capsular polysaccharide stimulated macrophage SR-A expression, indicating that live whole organisms are not necessary for SR-A protein expression in macrophage response. As SR-A is known to play a role in lipid uptake by macrophages, we tested the ability of low-density lipoprotein (LDL) to influence the SR-A response of macrophages to P. gingivalis , and observed no effect of LDL on P. gingivalis -elicited SR-A expression. Lastly, we observed that SR-A knockout (SR-A^−/−) mouse macrophages produced significantly more tumor necrosis factor (TNF)-α than wild type mouse macrophages cultured with P. gingivalis .
Conclusion:  These data identify that SR-A is expressed by macrophages in response to P. gingivalis , and support that this molecule plays a role in TNF-α production by macrophages to this organism.     

Immunization by Arg-gingipain A DNA vaccine protects mice against an invasive Porphyromonas gingivalis infection through regulation of interferon-gamma production

We previously demonstrated that a Porphyromonas gingivalis rgpA DNA vaccine induced protective immune responses against P. gingivalis infection in mice. In the present study, reduction in lethality against infection by lethal doses of P. gingivalis was observed in the rgpA DNA vaccine-immunized mice. Cytokine levels in the mouse model with nonlethal doses of infection by P. gingivalis were evaluated to analyze the mechanism of protection by immunization with the rgpA DNA vaccine. After nonlethal challenge with invasive P. gingivalis W50, production of interleukin (IL)-2, IL-4, IL-5 and IL-12 was elevated; however, interferon (IFN)-gamma was lower in the serum of the DNA vaccine-immunized mice than in the serum of nonimmunized mice. The regulation of IFN-gamma production elicited by immunization with the rgpA DNA vaccine may play a significant role in protection against P. gingivalis infection in mice.     

Single gavage with Porphyromonas gingivalis reduces acute systemic nitric oxide response in mice

Introduction:  Porphyromonas gingivalis , an important periodontal pathogen, can also induce host responses in distant tissues. P. gingivalis induces nitric oxide (NO) production in immune system cells and non-immune system cells, therefore NO might be involved in an acute systemic host response.
Methods:  Eighteen female BALB/c mice were perorally inoculated with 10⁸ colony-forming units live P. gingivalis ATCC 33277. Plasma nitrite and nitrate (NOx) and NO production in lungs, aorta, heart, liver, spleen, kidneys, and brain were measured at intervals after inoculation and compared with levels in 11 control animals.
Results:  NOx levels were significantly ( P  = 0.017) lower at 7, 13, and 25 h after P. gingivalis inoculation. A similar trend in NO production occurred in most tested organs, but never reached statistical significance. The correlation between NOx in plasma and NO in liver was positive (Spearman correlation coefficient = 0.81, P  = 0.0025) and marginal for kidney (0.58, P  = 0.059).
Conclusion:  Single peroral inoculation of mice with P. gingivalis reduces the acute systemic NO response. As NO is important for host defense, the reduction of NO levels after exposure is likely to delay the host response, increasing the chances that infection with P. gingivalis will become established.     

牙龈卟啉菌蛋白酶基因rgpA与rgpB蛋白酶区的克隆和表达

目的克隆牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)rgpA、rgpB蛋白酶区,构建rgpA、rgpB蛋白酶区原核表达系统。方法采用聚合酶链式反应(PCR)从PgATCC33277菌株中扩增rgpA、rgpB蛋白酶区,T-A克隆后测定核苷酸序列,构建pET42a的rgpA/rgpB蛋白酶区表达载体,在E.coli BL21DE3宿主菌中用不同浓度的IPTG诱导其表达。结果所克隆的PgATCC33277菌株rgpA、rgpB基因蛋白酶区的核苷酸序列与报道的相应核苷酸序列同源性分别为98.9%、99.0%,氨基酸序列同源性分别高达99.2%、99.1%。pET42a-rgpA/rgpB-E.coli BL21DE3系统的蛋白表达量为细菌总蛋白的60%左右。结论本研究成功地构建PgrgpA、rgpB蛋白酶区高效表达系统,为测定RgpA、RgpB免疫原性及作用提供前提。     

Binding of the periodontitis associated bacterium Porphyromonas gingivalis to glycoproteins from human epithelial cells

Introduction:  In the present study we examined the ability of the periodontal pathogen Porphyromonas gingivalis to adhere to glycoconjugates on intact cells and to protein preparations of epithelial cells (KB cells).
Methods:  The KB cell protein preparation was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by Western blotting. The membranes were used in overlay assays with labeled P. gingivalis . Flow cytometry was used to analyze attachment of bacteria to intact KB cells.
Results:  Glycoconjugate expression on the KB cells and in the protein preparation was confirmed. Binding was detected to several bands on the Western blots. Flow cytometry showed a distinct increase in fluorescence for strain FDC 381. Preincubation of the bacteria with mannose, fucose, N -acetylglucosamine and N -acetylgalactosamine inhibited the binding to KB cells by approximately 30% whereas preincubation with N -acetylneuraminic acid reduced the binding by 60%.
Conclusion:  These results indicate that carbohydrate structures are involved in the binding process of P. gingivalis to oral epithelial cells and that neuraminic acid plays a significant role in the adhesion process.     

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.     

Prevalence of fimA genotypes of Porphyromonas gingivalis and periodontal health status in Chinese adults

Background and Objective:  Porphyromonas gingivalis fimbriae play a key role in colonization of the oral cavity. The fimA gene, which encodes fimbrillin ( FimA ), can be classified into six types (I–V and Ib) according to nucleotide sequence. In the present study, we investigated the relationship between the prevalence of P. gingivalis -specific fimA genotypes and periodontal health status in Chinese adults.
Material and Methods:  One-hundred and fifteen patients with chronic periodontitis and 136 periodontally healthy adults were selected. P. gingivalis detection, determination of fimA genotypes, and the co-existence of Actinobacillus actinomycetemcomitans and Tannerella forsythia with various fimA types, were assessed by the polymerase chain reaction. Odds ratios and 95% confidence intervals were calculated for associating the fimA -specific genes with periodontitis.
Results:  P. gingivalis was detected in 22.1% of healthy subjects and in 81.7% of the patients. A single fimA genotype was detected in most samples. In healthy adults, the most prevalent fimA genotype was type I (66.7%). However, type II was detected most frequently (43.6%) in the patient group, followed by type IV (30.9%). The frequency of co-existing A. actinomycetemcomitans and T. forsythia was highest in type II fimA -positive sites. Statistical analysis revealed that periodontitis was associated with occurrences of type I (odds ratio 0.97), Ib (odds ratio 13.26), II (odds ratio 36.62), III (odds ratio 4.57), IV (odds ratio 22.86) and V (odds ratio 1.19).
Conclusion:  P. gingivalis type II followed by type IV were considered as disease-associated strains that account for the pathogenesis of chronic periodontitis in Chinese adults.     

Effect of enamel matrix derivative on periodontal ligament cells in vitro is diminished by Porphyromonas gingivalis

BACKGROUND: Enamel matrix derivative (EMD) has been shown to possess a mitogenic effect to induce effective periodontal regeneration, however, it is unclear whether periodontal pathogens can modulate the effect of EMD. The present study examined the influence of Porphyromonas gingivalis on EMD-stimulated periodontal ligament (PDL) cells. METHODS: P. gingivalis ATCC33277 and its mutants deficient in fimbriae (delta fimA) or gingipains (delta rgpA delta rgpB, delta kgp, and delta rgpA delta rgpB delta kgp) were employed. PDL cells were grown on EMD-coated dishes and infected with P. gingivalis wild strain or a mutant. Cell migration and proliferation were then evaluated with an in vitro wound healing assay. The expression of transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor I (IGF-I) mRNA by PDL cells was examined. Further, the degradation and phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) as well as paxillin in infected PDL cells were estimated using Western blot analysis. RESULTS: P. gingivalis ATCC33277 inhibited the migration and proliferation of PDL cells on EMD-coated dishes, and the mutants delta fimA, delta rgpA delta rgpB, and delta kgp showed the same effects. Further, each of these organisms diminished the expression of TGF-beta1 and IGF-I mRNA, as well as the phosphorylation of ERK1/2 from EMD-stimulated PDL cells. In addition, total paxillin protein was markedly degraded by both the wild-type strain and each of the mutants except for delta rgpA delta rgpB delta kgp, which showed a negligible effect in all of the assays with EMD-stimulated PDL cells. CONCLUSION: These results suggest that P. gingivalis diminishes the effect of EMD on PDL cells in vitro through a cooperative action of gingipains.     

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.     

Participation of the secreted dipeptidyl and tripeptidyl aminopeptidases in asaccharolytic growth of Porphyromonas gingivalis

Background and Objective:  Porphyromonas gingivalis secretes gingipains, endopeptidases essential for the asaccharolytic growth of this bacterium. P. gingivalis also secretes dipeptidyl aminopeptidases (DPPIV and DPP-7) and a tripeptidyl aminopeptidase (PTP-A), although their role in asaccharolytic growth is unclear. The present study was carried out to elucidate the role of these dipeptidyl/tripeptidyl aminopeptidases on the asaccharolytic growth of P. gingivalis .
Material and Methods:  Knockout mutants for the DPPIV ( dpp ), dpp7 and/or PTP-A genes were constructed. Brain–heart infusion medium supplemented with sterile hemin and menadione (BHIHM) was used as a complex medium, and the minimal medium used was GA, in which the sole energy source was a mixture of immunoglobulin G and bovine serum albumin. Growth of P. gingivalis was monitored by measuring the optical density of the culture.
Results:  All knockout mutants for DPPIV , dpp7 and PTP-A grew as well as strain W83 in BHIHM. In GA, growth of single-knockout and double-knockout mutants was similar to that of W83, whereas growth of a triple-knockout mutant (83-47A) was reduced. We purified recombinant DPPIV and recombinant PTP-A from recombinant Escherichia coli overproducers, and purified DPP-7 from the triple-knockout mutant 83-4A. GA supplemented with the three purified dipeptidyl/tripeptidyl aminopeptidases supported the growth of 83-47A.
Conclusion:  DPPIV, DPP-7 and PTP-A contribute to the normal growth of P. gingivalis by cleaving substrate peptides into short-chain polypeptides that are efficient energy sources for P. gingivalis.     

Mixed infection of Porphyromonas gingivalis and Bacteroides forsythus in a murine abscess model: involvement of gingipains in a synergistic effect

Several microorganisms including Porphyromonas gingivalis and Bacteroides forsythus have been implicated to be etiologically important agents of periodontal disease. In this study, we determined the ability of combinations of periodontopathogenic microorganisms to cause tissue destruction in a murine abscess model. Although all bacterial combinations used in this study produced larger abscesses than did monoinfection of each bacterium, the combination of P. gingivalis and B.forsythus showed a synergistic effect on abscess formation. Since these two bacteria have been frequently found together in lesions of periodontitis, these results suggest the significance of their co-infection in the progression of periodontitis. P. gingivalis produces extracellular and cell-associated cysteine proteinases (gingipains) which appear to be involved in its virulence. The rgpA rgpB double and kgp mutants induced significantly smaller abscesses than the wild type. Moreover, the rgpA rgpB kgp triple (gingipain-null) mutant hardly showed lesion formation at all with the experimental conditions used in this study, indicating that these genes encoding gingipains are important for virulence of P. gingivalis. Mixed infection of these P. gingivalis mutants with B. forsythus showed an additive effect on abscess formation, indicating that the gingipains of P. gingivalis may play an important role in the pathological synergism between P. gingivalis and B. forsythus.