Comparative study of two outer membrane protein genes from Porphyromonas gingivalis

A periodontal pathogen, Porphyromonas gingivalis possesses either a 53 kD (Ag53) or a 67 kD (Ag67) outer membrane protein (OMP). Almost all sera from patients with periodontal diseases reacted strongly with either Ag53 or Ag67. In previous work the cloning and sequencing of the 53 kD outer membrane protein gene designated pga53 from P. gingivalis FDC381, was reported and the presence of a gene homologous to pga53 in P. gingivalis ATCC 33277 demonstrated. In the present work this pga53-homologous gene from P. gingivalis ATCC 33277 was isolated and characterized. Nucleotide sequence analysis revealed that this gene encoded Ag67, and the gene was designated pga67. The deduced amino acid sequence and composition of pga67 was similar to the amino acid composition and N-terminal partial sequence of Ag67. An open reading frame of pga67 consisted of 1,692 nucleotides encoded as 564 amino acids, including a 49 amino acid signal sequence. The comparative analysis between pga67 and pga53 revealed that (1) the deduced amino acid sequence showed a 30.1% homology; (2) signal sequence and proline-rich regions at the C-terminus were the most conserved regions; (3) considerable differences were found mainly in the middle part of the OMPs; and (4) obvious differences in the two-dimensional models were evoked. These differences between pga67 and pga53 may explain the antigenic diversity between Ag67 and Ag53 OMPs.     

Involvement of CD14 on human gingival fibroblasts in Porphyromonas gingivalis lipopolysaccharide-mediated interleukin-6 secretion

The lipopolysaccharides (LPS) of Porphyromonas gingivalis are implicated in the initiation and development of periodontal diseases. However, the mechanisms underlying P. gingivalis LPS-mediated periodontal destruction are still unknown. Here, it was found that P. gingivalis LPS activates human gingival fibroblasts (HGF) to release interleukin 6 (IL-6) via CD14. Flow-cytometric analysis showed that HGFs bind to fluorescein-isothiocyanate (FITC)-labelled LPS, and express CD14 on their surfaces. The binding of FITC LPS was competitively suppressed by unlabelled synthetic lipid A as well as by LPS. LPS-induced IL-6 production was inhibited by anti-CD14 monoclonal antibody in a dose-dependent manner. The binding of FITC LPS to HGF was abrogated by anti-CD14 monoclonal antibody. Engagement of LPS initiated the protein tyrosine phosphorylation of several intracellular proteins including extracellular signal-regulated kinase (ERK) 1 and 2, and these events were suppressed by the anti-CD14 monoclonal. These results suggest that CD14 is a cell surface binding site for LPS and is involved in the LPS-mediated activation of HGF.     

Binding of hemoglobin by Porphyromonas gingivalis

Nitric oxide which was released in aqueous solutions (> or = 10 microM) of direct NO-donors such as 3-morpholinesydnonimine (SIN-1) and S-nitroso-N-acetyl-penicillamine (SNAP) consumed avidly sulfhydryl groups of N-acetylcysteine > cysteine > glutathione. In case of SIN-1 generation of nitrites run in parallel to disappearance of sulfhydryl groups of N-acetylcysteine and glutathione, however, for a pair of SIN-1 and cysteine the rate of formation of nitrites was much slower than the rate of consumption of sulfhydryl groups. We infer that kinetics of formation and breakdown of S-nitrosothiols varies depending on the type of a thiol which reacts with a NO-donor. Indirect NO-donors such as glyceryl trinitrate (GTN), molsidomine (MSD) or sodium nitroprusside (NaNP) at concentrations < 100 microM did not consume sulfhydryl groups of cysteine unless pretreated with the xanthine/xanthine oxidase system. We suppose that in this last case superoxide anions react with nitric oxide to form peroxynitrites with a higher potency than nitric oxide itself to destroy sulfhydryl groups. We conclude that out of three studied thiols N-acetylcysteine is the best substrate for the formation of S-nitrosothiols, while S-nitrosocysteine is the slowest releaser of nitric oxide. Moreover, unlike SIN-1 and SNAP, NaNP is not a direct NO-donor but behaves rather like GTN. Minute amounts of nitric oxide released either from NaNP or GTN gain from superoxide anions an amplification as SH-scavengers.     

Distribution of Porphyromonas gingivalis in adult periodontitis patients

Porphyromonas gingivalis (P. gingivalis) is considered to be a pathogenic factor in adult or rapidly progressive periodontitis. The purpose of this study was to evaluate the distribution of P. gingivalis in the dentition of adult periodontitis patients using a nonradioactive DNA probe, and to compare the presence of P. gingivalis with clinical parameters. Twelve adult periodontitis patients were examined. Subgingival plaque samples were taken from 4 sites of all the remaining teeth using a paper point. At the same time, probing depth and bleeding on probing (BOP) were also recorded. Plaque samples were investigated using a whole genomic DNA probe from P. gingivalis (ATCC 33277) modified with bisulfite. The detection, percentage and amounts of P. gingivalis present were statistically compared with probing depth and BOP in each patient. P. gingivalis was detected in all patients examined. The detection percentage was 35% of all sample sites. When the probing depth was over 4 mm or BOP was positive, the detection percentage of P. gingivalis significantly increased (P < 0.01). As more P. gingivalis was identified, the percentage of sites with deep probing depth or that were BOP positive increased significantly (P < 0.01). However, P. gingivalis was also detected in clinically healthy sites, and P. gingivalis negative sites with deep probing depth or that were BOP positive existed in the same patient. These results indicate that P. gingivalis play an important role, but is not the only microorganism responsible for adult periodontitis.     

Porphyromonas gingivalis as putative pathogen in ovine periodontitis

Ovine periodontitis has clinical features similar to early onset periodontitis in man. Porphyromonas gingivalis has been implicated as a putative pathogen in this disease and its role in periodontitis in 107 sheep was investigated. The organism was recovered from most diseased sites at a significant percentage level of the total bacterial count. Antibody assays of 107 animals using an ELISA did not distinguish between diseased (46) and control adult (33) sheep for either P. gingivalis or any of the other putative periodontopathogens tested, but did differentiate adult sheep from healthy lambs (28) for all bacteria except one. It is suggested that sheep rather than human bacterial strains should be used in antibody studies of ovine periodontitis.     

Porphyromonas gingivalis invades human pocket epithelium in vitro

The present study examined the adhesive and invasive potential of Porphyromonas gingivalis interacting with human pocket epithelium in vitro. Pocket epithelial tissue, obtained during periodontal surgery of patients with advanced periodontal disease, generated a stratified epithelium in culture. P. gingivalis strains W50 and FDC 381 (laboratory strains), OMGS 712, 1439, 1738, 1739 and 1743 (clinical isolates) as well as Escherichia coli strain HB101 (non-adhering control) were tested with respect to epithelial adhesion and invasion. Adhesion was quantitated by scintillation spectrometry after incubation of radiolabeled bacteria with epithelial cells. The invasive ability of P. gingivalis was measured by means of an antibiotic protection assay. The epithelial multilayers were infected with the test and control strains and subsequently incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). The number of internalized bacteria surviving the antibiotic treatment was assessed after plating lyzed epithelial cells on culture media. All tested P. gingivalis strains adhered to and entered pocket epithelial cells. However, considerable variation in their adhesive and invasive potential was observed. E. coli strain HB101 did not adhere or invade. Transmission electron microscopy revealed that internalization of P. gingivalis was preceded by formation of microvilli and coated pits on the epithelial cell surfaces. Intracellular bacteria were most frequently surrounded by endosomal membranes; however, bacteria devoid of such membranes were also seen. Release of outer membrane vesicles (blebs) by internalized P. gingivalis was observed. These results support and extend previous work from this laboratory which demonstrated invasion of a human oral epithelial cell-line (KB) by P. gingivalis.     

Porphyromonas gingivalis proteinases in periodontitis, a review

OBJECTIVE: Only the simultaneous analysis of periodic and nonlinear properties of heart rate fluctuations (HRF) can describe completely this complex physiological process. Up to now there is, apart from a study of our own, no systematic and correlative investigation using both parameter groups, also not in early development. Thus, we tried to describe in this manner these properties of HRF, the corresponding mean arterial pressure fluctuations (MAPF) and respiratory movements (RM) and their mutual relations in neonatal pig. METHODS: In 6 term newborn piglets, periodic properties of HRF, RM, and MAPF were analyzed by spectral and coherence analysis, and deterministic-chaotic properties by calculation of correlation dimension (CD), Lyapunov exponent (LE), and construction of phase space plots. The assumption of deterministic chaotic components was supported by Theiler's test for nonlinearity, by always positive leading LEs, and by the results of a nonlinear deterministic model. These analyses were done in sleep states, general anaesthesia, hypoxic hypoxia, in ventilated state, and during cholinergic and additional beta-adrenergic blockade. RESULTS: In all experimental states, HRF and MAPF have periodic and nonlinear, very probably deterministic-chaotic properties, but in different relations. In anaesthetized piglets, periodic properties of HRF and MAPF dominate. In hypoxia the decreasing LE and CD of HRF and CD of MAPF were connected with increasing MAPF power density. Cholinergic blockade caused a decreased overall HRF and MAPF power and a decreasing LE and CD, but beta-adrenergic blockade decreased a small part of power density of both in 0.02-0.08 Hz only. The results of CD, LE, Theiler's test and the low dimensional deterministic model data suggested mainly deterministic-chaotic properties in the nonlinear part of HRF and MAPF. CONCLUSIONS: Already in neonatal piglets, both periodic and nonlinear, very probably deterministic chaotic properties of HRF and MAPF exist which change both during hypoxia and cholinergic blockade. They are partly cholinergically and--to a small extent--also beta-adrenergically mediated. The decrease of nonlinear complexity of HRF and MAPF during hypoxia suggests characteristic pathological change even in early development.     

Role of Arg-gingipain A in virulence of Porphyromonas gingivalis

In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37 degrees C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism.     

High-performance liquid chromatographic separation of Porphyromonas gingivalis fimbriae

A right lung cancer case is presented, aged 65 years, obese, submitted to a right lung resection. Stress is laid on the difficult evolution concerning the haemodynamics and particularly the breathing owing to the association of risk factors.     

Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis

Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.     

Prevalence of Porphyromonas gingivalis and periodontal health status

Periodontitis is a common, progressive disease that eventually affects the majority of the population. The local destruction of periodontitis is believed to result from a bacterial infection of the gingival sulcus, and several clinical studies have provided evidence to implicate Porphyromonas gingivalis. If P. gingivalis is a periodontal pathogen, it would be expected to be present in most subjects with disease and rarely detected in subjects with good periodontal health. However, in most previous studies, P. gingivalis has not been detected in the majority of subjects with disease, and age-matched, periodontally healthy controls were not included for comparison. The purpose of the study reported here was to compare the prevalence of P. gingivalis in a group with periodontitis to that of a group that is periodontally healthy. A comprehensive sampling strategy and a sensitive PCR assay were used to maximize the likelihood of detection. The target sequence for P. gingivalis-specific amplification was the transcribed spacer region within the ribosomal operon. P. gingivalis was detected in only 25% (46 of 181) of the healthy subjects but was detected in 79% (103 of 130) of the periodontitis group (P < 0.0001). The odds ratio for being infected with P. gingivalis was 11.2 times greater in the periodontitis group than in the healthy group (95% confidence interval, 6.5 to 19.2). These data implicate P. gingivalis in the pathogenesis of periodontitis and suggest that P. gingivalis may not be a normal inhabitant of a periodontally healthy dentition.     

Evidence for the absence of hyaluronidase activity in Porphyromonas gingivalis

The aim of the present study was to evaluate the ability of Porphyromonas gingivalis to degrade hyaluronic acid. No hyaluronidase activity was detected using a turbidimetric method, whereas a standard plate assay showed a positive reaction for P. gingivalis. We postulated that the high proteolytic activity of P. gingivalis may account for this observation. A modified plate assay was designed to avoid false-positive reactions caused by proteolytic bacteria. The new assay, based on the formation of a water-insoluble salt between hyaluronic acid and the polyanion cetylpyridinium chloride, indicated that P. gingivalis does not have hyaluronidase activity. By this modified plate method, it was found that among 24 different oral bacterial species tested, Propionibacterium acnes and Prevotella oris were the only species that possess hyaluronidase activity.     

Secretion of Porphyromonas gingivalis fimbrillin polypeptides by recombinant Streptococcus gordonii

The fimbriae of Porphyromonas gingivalis plays an important role in the pathogenesis of periodontal disease. A structural subunit of the P. gingivalis fimbriae, fimbrillin, has been shown to promote adherence of the bacteria to host surfaces and also induce an immune response. Biologically active domains of fimbrillin responsible for adherence or eliciting immune responses have been determined. In a previous study, we engineered the human oral commensal organism Streptococcus gordonii to express such biologically active domains on the surface of the bacteria as a vaccine delivery system. In this study we report an alternative approach of secreting fimbrillin polypeptide domains into the medium by modification of the surface-expression system described earlier. Such recombinant S. gordonii, in addition to being a source for antigen presentation to trigger a protective immune response, may have the added advantage of directly blocking the fimbriae-mediated adherence of P. gingivalis to the oral cavity following implantation. This approach can also be utilized for secreting other biologically important therapeutic molecules on mucosal surfaces for modulating local microenvironments.     

Vascular permeability enhancing activity of Porphyromonas gingivalis protease in guinea pigs

The effects of hemofiltration (HF) on serum levels of intact parathyroid hormone (PTH), as well as its mid-C regional and C-terminal fragments and bone Gla-protein (bone gamma-carboxyglutamic acid, osteocalcin), were investigated in 17 patients during one session of HF. During HF there was a significant decrease in the serum concentrations of both intact PTH (p < 0.01), mid-C regional PTH (p < 0.01) and C-terminal PTH (p < 0.01) as well as in the osteocalcin concentrations (p < 0.01). There was a significant increase in serum calcium (p < 0.05) during the procedure and this increase correlated with the reduction in PTH (r = 0.56, p < 0.05). Mid-C regional PTH and osteocalcin were found in the ultrafiltrate of all patients, while intact PTH was found in the ultrafiltrate of 8 of 17 patients and C-terminal PTH was detected only in the ultrafiltrate of one patient. The data demonstrate that the permeability of HF membranes is high enough to cause filtration of both intact PTH, PTH fragments and osteocalcin. An inhibited secretion of PTH probably played a major part in reducing intact PTH in serum during HF.     

Proteases of Porphyromonas gingivalis: what don't they do?

The gram-negative anaerobic bacterium Porphyromonas gingivalis has been strongly associated with the causation of human periodontal diseases. One distinguishing property of these organisms that has been implicated in periodontal destruction is the expression of potent protease activity. Recent biochemical and genetic approaches have clearly demonstrated that at least five distinct proteases are elaborated by these organisms. The utilization of monospecific mutants defective in individual proteases has demonstrated that protease activity is important in virulence but also has suggested the complexity of the functions of the enzymes in the physiology of these microorganisms. This review summarizes current progress in assessing the role of these enzymes in periodontal inflammation and discusses some unresolved issues relevant to the significance of P. gingivalis proteases in virulence.     

Structures involved in the interaction of Porphyromonas gingivalis fimbriae and human lactoferrin

We report a 21-year-old woman presenting with a slowly progressive tetraparesis, optic nerve atrophy on both sides, and autonomic disturbances since early childhood. The patient has been carefully followed up for 5 years with clinical and ancillary investigations. The results and the time course strongly suggest an underlying degenerative syndrome affecting parts of three major systems: autonomic, motor and visual. Some symptoms resemble familial dysautonomia (FD, Riley-Day syndrome), however, hallmarks of FD, such as absence of fungiform papillae of the tongue, abnormal reaction on intradermal histamine injection, absent tendon reflexes, are missing, and central motor disturbances have not been described in FD. We consider this syndrome a slowly progressive multisystemic degeneration with two unusual hitherto unreported features: the combination of affected systems (autonomic and motor systems, optic nerves), and the early onset.     

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis

Porphyromonas gingivalis, a periodontal pathogen can invade primary cultures of gingival epithelial cells. This invasion was significantly inhibited (74-81%) by thapsigargin and 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid, acetoxymethyl ester (BAPTA/AM), but not by EDTA or amiloride. Release of Ca2+ from an intracellular store and the subsequent increase in cytosolic [Ca2+] may, therefore, be involved in the invasion process, while Ca2+ influx is not. Moreover, cytosolic [Ca2+] was found to increase transiently in about 30% of gingival epithelial cells acutely exposed to P. gingivalis, but not in unexposed cells, or in cells exposed to noninvasive Escherichia coli. These findings indicate that P. gingivalis invasion of epithelial cells is correlated with activation of [Ca2+]-dependent host cell signaling systems.