Searching the Porphyromonas gingivalis genome with peptide fragmentation mass spectra

An approach is described for genomic database searching based on experimentally observed proteolytic fragments, e.g., isolated from 1D or 2D gels or analyzed directly, that can be applied to unfinished prokaryotic genomic data in the absence of annotations or previously assigned open reading frames (ORFs). This variation on the database search is in contrast to the more familiar use of peptide mass spectral fragmentation data to search fully annotated inferred protein databases, e.g., OWL or SWISS-PROT. We compared the SEQUEST search results from a six reading frame translation of the Porphyromonas gingivalis genome DNA sequence with those from computationally derived ORFs created using publicly available genomics software tools. The ORF approach eliminated many of the artifacts present in output from the six reading frame search. The method was applied to uninterpreted tandem mass spectrometric data derived from proteins secreted by the periodontal pathogen Porphyromonas gingivalis in response to the gingival epithelial cell environment, a model system for the study of host-pathogen interactions relevant to human periodontal disease.     

Molecular genetics of Porphyromonas gingivalis: gingipains and other virulence factors

Porphyromonas gingivalis is a black-pigmented anaerobic gram-negative bacterium that is a major pathogen of chronic adult periodontitis, an inflammatory disease of tooth-supporting tissues. P. gingivalis possesses a number of potential virulence factors. Among them, cell-surface-associated and secreted proteinases such as Arg-gingipain and Lys-gingipain have received much attention because they can degrade various host proteins and cause inflammation. Molecular genetic analysis is extremely powerful to evaluate the significance of each virulence factor in a pathogenic microorganism. This review will describe the introduction of molecular genetics to analysis of pathogenesis of P. gingivalis and the findings that have been obtained using knockout mutants of various potential virulence factors, especially proteinases.     

Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire

The gingipains are cell surface Arg- and Lys-specific proteinases of the bacterium Porphyromons gingivalis, which has been associated with periodontitis, a disease that results in the destruction of the teeth-s supporting tissues. The proteinases are encoded by three genes designated rgpA, rgpB and kgp. Arg-specific proteolytic activity is encoded by rgpA/B and the Lys-specific activity by kgp. RgpA and Kgp are polyproteins comprising proteinases with C-terminal adhesin domains that are proteolytically processed. After processing, the domains remain non-covalently associated as complexes on the cell surface. RgpB is also a cell surface proteinase but does not associate with adhesin domains. Using gene knockout P. gingivalis mutants, the proteolytic processing of the gingipain domains has been shown to involve the gingipains themselves as well as C-terminal processing by a carboxypeptidase. A motif in the C-terminal domain of each protein/polyprotein has been identified that is suggested to be involved in attachment to LPS on the cell surface. RgpB lacks a C-terminal adhesin binding motif found in the catalytic domains of RgpA and Kgp. This adhesin binding motif is proposed to be responsible for the non-covalent association of the RgpA and Kgp catalytic domains into the cell surface complexes with the processed adhesin domains. The RgpA-Kgp proteinase-adhesin complexes, through the adhesin domains A1 and A3, have been implicated in colonization of P. gingivalis by binding to other bacteria in subgingival plaque and also binding to crevicular epithelial cells. The RgpA-Kgp complexes also bind to fibrinogen, laminin, collagen type V, fibronectin and hemoglobin. Amino acid sequences likely to be involved in binding to these host proteins have been identified in adhesin domains A1 and A3. It is proposed that these adhesins target the proteolytic activity to host cell surface matrix proteins and receptors. The continual cycle of binding and degradation of the surface proteins/receptors on epithelial, fibroblast and endothelial cells by the RgpA-Kgp complexes in the gingival tissue leading to cell death would contribute to inflammation, tissue destruction and vascular disruption (bleeding). P. gingivalis has an obligate growth requirement for iron and protoporphyrin IX, which it preferentially utilizes in the form of hemoglobin. Kgp proteolytic activity is essential for rapid hydrolysis of hemoglobin and it is suggested therefore that a major role of the RgpA-Kgp complexes is in vascular disruption and the binding and rapid degradation of hemoglobin for heme assimilation by P. gingivalis. The RgpA-Kgp complexes also have a major role in the evasion and dysregulation of the host-s immune response. It is proposed that host pro-inflammatory cytokines and cellular receptors close to the infection site may be rapidly and efficiently degraded by the gingipains while the proteinases at lower concentrations distally could result in the promotion of an inflammatory response through activation of proteinase-activated receptors and cytokine release. The culmination of this dysregulation would be tissue destruction and bone resorption. In animal models of disease the RgpA-Kgp complex when used as a vaccine to produce a high titre antibody response protects against challenge with P. gingivalis. Using recombinant domains of RgpA and Kgp as vaccines, it has been demonstrated that the A1 and A3 domains confer protection.     

Mechanism of visible light phototoxicity on Porphyromonas gingivalis and Fusobacterium nucleatum

Phototoxicity of visible light laser on the porphyrin-producing bacteria, Porphyromonas gingivalis, in the absence of photosensitizers and under aerobic conditions was shown in previous studies. Recently, we found that the noncoherent visible light sources at wavelengths of 400-500 nm, commonly used in restorative dentistry, induced a phototoxic effect on P. gingivalis, as well as on Fusobacterium nucleatum, and to a lesser extent on the Streptococci sp. To elucidate the mechanism of this phototoxic effect, P. gingivalis and F. nucleatum were exposed to light (1) under aerobic and anaerobic environments and (2) in the presence of scavengers of reactive oxygen species (ROS). Phototoxic effect was not observed when the bacteria were exposed to light under anaerobic conditions. Dimethyl thiourea, a hydroxyl radical scavenger, was effective in reducing phototoxicity (P 相似文献   

Suppression of virulence of Porphyromonas gingivalis by potent inhibitors specific for gingipains

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that is implicated as a major etiologic agent of adult periodontal disease. This bacterium is asaccharolytic and possesses strong potency for proteolysis. It produces a novel class of cysteine proteinases, termed gingipains, in the cell-associated and secretory forms. Gingipains consist of arginine-X-specific cysteine proteinases (Arg-gingipains, Rgps) and lysine-X-specific cysteine proteinase (Lys-gingipain, Kgp). Previous studies using various P. gingivalis mutants deficient in Rgp- and/or Kgp-encoding genes have revealed that both enzymes are important for the bacterium both to exhibit its virulence and to survive in periodontal pockets. Mammalian internal proteinase inhibitors such as cystatins, a1-antichymotrypsin, and tissue inhibitor of metalloproteinases (TIMPs) have little or no effects on the proteolytic activities of these enzymes, suggesting the evasion of the bacterium from host defense mechanisms. Recent epidemiological reports have shown a significant relation between periodontal diseases and systemic diseases such as cardiovascular diseases and diabetes. Thus, the development of potent inhibitors specific for gingipains provides new therapeutic approaches to treat periodontal diseases and the related systemic diseases. More recently, we have developed novel synthetic inhibitors specific for Rgp and Kgp, based on the specificity and efficacy of cleavage of histatins by each enzyme. We have also isolated a novel and potent inhibitor of Rgp from the culture supernatant of Streptomyces species strain FA-70, now designated as FA-70C1. Here we summarized the usefulness of these new inhibitors in providing a broader application in studies of this important class of enzymes.     

Effect of Dosimetric and Physiological Factors on the Lethal Photosensitization of Porphyromonas gingivalis in vitro

The aims of this study were to (1) determine the effect of dosimetric and physiological factors on the lethal photosensitization of Porphyromonas gingivalis using tolui-dine blue O (TBO) and light from a helium/neon (HeNe) laser; (2) determine the influence of sensitizer concentration, preirradiation time, serum and growth phase on sensitizer uptake by P. gingivalis. The dosimetric factors studied were concentration of TBO, light dose and preirradiation time. The physiological factors were presence of serum, pH and bacterial growth phase. Sensitizer uptake by P. gingivalis under various conditions was determined using tritiated TBO (³H-TBO). In the presence of TBO, a light dose-dependent increase in kill was attained (100% kill at 4.4 J). There was no significant effect on the numbers killed when TBO was increased from 12.5 to 50 µg/mL. An increase in preirradiation time gave slightly increased kills. High kills were achieved at all three pH (6.8–8.0). Although kills were substantial in the presence of serum, they were significantly less than those obtained in the presence of saline. Cells in all three growth phases were susceptible to lethal photosensitization, although stationary phase cells were slightly less susceptible. Maximum uptake of TBO occurred within 60 s and uptake in serum was less than in saline. The uptake by the log phase cells was greater at lower concentrations of sensitizer (50 µg/mL), compared to the other two phases.     

Synthetic tetra-acylated derivatives of lipid A from Porphyromonas gingivalis are antagonists of human TLR4

Tetra-acylated lipid As derived from Porphyromonas gingivalis LPS have been synthesized using a key disaccharide intermediate functionalized with levulinate (Lev), allyloxycarbonate (Alloc) and anomeric dimethylthexylsilyl (TDS) as orthogonal protecting groups and 9-fluorenylmethoxycarbamate (Fmoc) and azido as amino protecting groups. Furthermore, an efficient cross-metathesis has been employed for the preparation of the unusual branched R-(3)-hydroxy-13-methyltetradecanic acid and (R)-3-hexadecanoyloxy-15-methylhexadecanoic acid of P. gingivalis lipid A. Biological studies have shown that the synthetic lipid As cannot activate human and mouse TLR2 and TLR4 to produce cytokines. However, it has been found that the compounds are potent antagonist of cytokine secretion by human monocytic cells induced by enteric LPS.     

Structural confirmation of the dihydrosphinganine and fatty acid constituents of the dental pathogen Porphyromonas gingivalis

Porphyromonas gingivalis, a recognized periodontal pathogen, is a source of sphinganine bases, fatty acids, free ceramides as well as complex lipids that potentiate interleukin-1b-mediated secretory responses in gingival fibroblasts. The purpose of this study is the structural verification of the sphinganine bases and fatty acids that had been proposed as major components of the complex lipids found in P. gingivalis. The putative C17, C18, and C19 sphinganine bases were prepared from Garner's aldehyde (1) or from a protected serine Weinreb's amide (2). We confirmed that isobranched sphinganine bases are the major structural feature of the ceramides observed from P. gingivalis. We also prepared a C17 unsaturated fatty acid, along with an isobranched C17 3-hydroxy fatty acid, and determined that the major component of the active lipids was the latter.     

Phototoxic effect of visible light on Porphyromonas gingivalis and Fusobacterium nucleatum: an in vitro study

The antibacterial effect of visible light irradiation combined with photosensitizers has been reported. The objective of this was to test the effect of visible light irradiation without photosensitizers on the viability of oral microorganisms. Strains of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Streptococcus faecalis in suspension or grown on agar were exposed to visible light at wavelengths of 400-500 nm. These wavelengths are used to photopolymerize composite resins widely used for dental restoration. Three photocuring light sources, quartz-tungsten-halogen lamp, light-emitting diode and plasma-arc, at power densities between 260 and 1300 mW/cm2 were used for up to 3 min. Bacterial samples were also exposed to a near-infrared diode laser (wavelength, 830 nm), using identical irradiation parameters for comparison. The results show that blue light sources exert a phototoxic effect on P. gingivalis and F. nucleatum. The minimal inhibitory dose for P. gingivalis and F. nucleatum was 16-62 J/cm2, a value significantly lower than that for S. mutans and S. faecalis (159-212 J/cm2). Near-infrared diode laser irradiation did not affect any of the bacteria tested. Our results suggest that visible light sources without exogenous photosensitizers have a phototoxic effect mainly on Gram-negative periodontal pathogens.     

Disinfect Porphyromonas gingivalis Biofilm on Titanium Surface with Combined Application of Chlorhexidine and Antimicrobial Photodynamic Therapy

Various antimicrobial modalities have been proposed to treat peri‐implantitis but resulted in limited outcomes. The aim of this in vitro study was to evaluate the disinfection efficacy of combined application of chlorhexidine digluconate (CHX) and antimicrobial photodynamic therapy (aPDT) of titanium surfaces previously contaminated with Porphyromonas gingivalis biofilm. P. gingivalis biofilms were grown on 32 polished and 32 sandblasted large‐grit acid‐etched (SLA) titanium surfaces. Titanium disks were allocated into four groups as follows: (1) immersed in phosphate‐buffered saline (PBS), (2) immersed in 0.2% CHX, (3) application of aPDT and (4) immersed in 0.2% CHX and subsequent aPDT. Residual bacteria were determined by microbial culture analysis and by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) imaging. Combination protocol (CHX+ aPDT) was the most effective in eradicating P. gingivalis (P < 0.05) on both polished and SLA surfaces. There was no significant difference in the number of remaining P. gingivalis between polished titanium disks and the SLA ones in four groups (P > 0.05). Under the limitation of this study, combined technique of preceding application of CHX and subsequent aPDT was shown to be an efficient method in reducing P. gingivalis numbers in both polished and SLA titanium surfaces.     

Glycosylation of the Arg-gingipains of Porphyromonas gingivalis and comparison with glycoconjugate structure and synthesis in other bacteria

Post-translational modification of proteins by covalent attachment of sugars to the protein backbone (protein glycosylation) is the most common post-translational modification in the eucaryotic cell. However, the addition of carbohydrates to proteins of Eubacteria and Archaea has been demonstrated and accepted only recently. There is now a rapidly expanding list of bacterial glycoproteins that have been characterised from a variety of different organisms including many important pathogens. The Arg-gingipains of Porphyromonas gingivalis are recent additions to this list. In this review we present a summary of our investigations on the structure of the glycan additions to these proteolytic enzymes, the genetics of the glycosylation process and some of the effects on enzyme function and recognition. These findings are placed in the context of the current status of understanding of glycoconjugate structure and synthesis in other bacteria. Given the importance of glycosylation of eucaryotic proteins to their stability, structure, resistance to proteolysis and recognition, the modifications to the proteases described in the present report are likely to have a functional role in the properties of these enzymes in periodontal disease.     

A Study of the Uptake of Toluidine Blue O by Porphyromonas gingivalis and the Mechanism of Lethal Photosensitization

The purpose of the study was to determine the distribution of the photosensitizer toluidine blue O (TBO) within Porphyromonas gingivalis and the possible mechanism(s) involved in the lethal photosensitization of this organism. The distribution of TBO was determined by incubating P. gingivalis with tritiated TBO (³H-TBO) and fractionating the cells into outer membrane (OM), plasma membrane (PM), cytoplasmic proteins, other cytoplasmic constituents and DNA. The percentage of TBO in each of the fractions was found to be, 86.7, 5.4, 1.9, 5.7 and 0.3%, respectively. The involvement of cytotoxic species in the lethal photosensitization induced by light from a helium-neon (HeNe) laser and TBO was investigated by using deuterium oxide (D₂O), which prolongs the lifetime of singlet oxygen, and the free radical and singlet oxygen scavenger L-tryptophan. There were 9.0 log₁₀ and 2 log₁₀ reductions in the presence of D₂O and H₂O (saline solutions), respectively, at a light dose of 0.44 J (energy density = 0.22 J/cm²), suggesting the involvement of singlet oxygen. Decreased kills were attained in the presence of increasing concentrations of L-tryptophan. The effect of lethal photosensitization on whole cell proteins was determined by measuring tryptophan fluorescence, which decreased by 30% using 4.3 J (energy density = 4.3 J/ cm²) of light. Effects on the OM and PM proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was evidence of change in the molecular masses of several PM proteins and OM proteins compared to controls. There was evidence of damage to the DNA obtained from irradiated cells. Scanning electron microscopic studies showed that there was coaggre-gation of P. gingivalis cells when sensitized and then exposed to laser light. These results suggest that lethal photosensitization of P. gingivalis may involve changes in OM and/or PM proteins and DNA damage mediated by singlet oxygen.     

Quantitative Detection for Porphyromonas gingivalis in Tooth Pocket and Saliva by Portable Electrochemical DNA Sensor Linked with PCR

Here, a quantitative electrochemical analysis of periodontal bacteria in gingival crevicular fluid (GCF) and saliva by direct polymerase chain reaction (PCR) is presented. The electrochemical measurement was performed by mixing with PCR products and electrochemical indicator (bisbenzimidazole trihydrochloride). The peak current of indicator is reduced due to slower diffusion when the dye intercalates into the amplified DNA, and the degree of reduction in the peak current is correlates with the quantity of amplified DNA. Therefore, a quantitative analysis is possible by using our electrochemical method at the end point of PCR. In the GCF testing, The number of Porphyromonas gingivalis (Pg) detected by our electrochemical method at the end point of PCR were almost same compared with that were calculated by the conventional method of quantitative real? time PCR. In the saliva testing, the relationship between number of Pg in saliva and average pocket depth, and age‐dependence were also clearly observed. Since the saliva sample is obtained in a non‐invasive manner, this method is useful for the primary screening of periodontal disease. Moreover, our detection method is simple and uses a hand‐held potentiostat making it suitable for development of an on‐site periodontal diagnosis system.     

Unique Properties of Heme Binding of the Porphyromonas gingivalis HmuY Hemophore-like Protein Result from the Evolutionary Adaptation of the Protein Structure

To acquire heme, Porphyromonas gingivalis uses a hemophore-like protein (HmuY). HmuY sequesters heme from host hemoproteins or heme-binding proteins produced by cohabiting bacteria, and delivers it to the TonB-dependent outer-membrane receptor (HmuR). Although three-dimensional protein structures of members of the novel HmuY family are overall similar, significant differences exist in their heme-binding pockets. Histidines (H134 and H166) coordinating the heme iron in P. gingivalis HmuY are unique and poorly conserved in the majority of its homologs, which utilize methionines. To examine whether changes observed in the evolution of these proteins in the Bacteroidetes phylum might result in improved heme binding ability of HmuY over its homologs, we substituted histidine residues with methionine residues. Compared to the native HmuY, site-directed mutagenesis variants bound Fe(III)heme with lower ability in a similar manner to Bacteroides vulgatus Bvu and Tannerella forsythia Tfo. However, a mixed histidine-methionine couple in the HmuY was sufficient to bind Fe(II)heme, similarly to T. forsythia Tfo, Prevotella intermedia PinO and PinA. Double substitution resulted in abolished heme binding. The structure of HmuY heme-binding pocket may have been subjected to evolution, allowing for P. gingivalis to gain an advantage in heme acquisition regardless of environmental redox conditions.     

Light-induced coumarin cyclopentannelation

[reaction: see text] Whereas cyclopentenylcarbenes resulting from photocycloaddition of 4-alk-1-ynylcoumarins to 2,3-dimethylbut-2-ene undergo tandem cyclization to hitherto unknown tetracyclic (4-hetera)cyclopent[b,c]acenaphthylenes, the corresponding cyclopentenylnitrenes stemming from 4-cyanocoumarins and the same alkene are converted into tricyclic imines via H-abstraction.     

Light-induced helix formation

This study shows that incorporation of [Rub2m-OH]2+ at the N-terminus of the Fs peptide enhances its stability by approximately 0.15 kcal/mol through the mechanism of dipole-dipole coupling at the excited state, suggesting that photoinduced charge generation at a well-controlled and specific location provides a convenient means to trigger helix-coil transition on nanosecond or even faster time scales.     

Light-induced carbocyclization of iodoalkenes

The direct irradiation of iodoalkenes leads to the formation of carbon-centered radical by homolysis of the C–I bond. The photoreaction is used in cyclizations with formation of six membered rings.     

Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis: structure, function and assembly of multidomain protein complexes

Gingipains, extracellular cysteine proteinases of Porphyromonas gingivalis, constitute the major virulence factor of this periodontopathogenic bacterium. They are the product of three genes, two coding for an Arg-specific (RgpA and RgpB) and one for a Lys-specific proteinase (Kgp). Proteinase domains of RgpA and RgpB are virtually identical; however, the gene encoding the former enzyme is missing a large segment coding for hemaglutinin / adhesin (HA) domains. The latter domains are present also in Kgp. The tertiary structure of RgpB revealed that the proteinase domain of gingipains has a protein fold referred to as the caspase-hemoglobinase fold. On this basis, they are also evolutionary related to other highly specific proteinases including clostripain, caspases, legumains and separase (clan CD of cysteine peptidases). Gingipains are produced as large preproproteins and are subject to elaborate, not yet fully understood, secretion, glycosylation, activation, and maturation processes. How they traverse the outer membrane is unknown, although it can be hypothesized that they use an autotransporter pathway. Apparently during transport through the periplasm the LPS-like glycan moiety is added at the conserved C-terminal portion of progingipains. At the cell surface pro-gingipains fold into partially active, single-chain zymogens and undergo autocatalytic, intermolecular processing. Two sequential cleavages within the profragment domain enhance zymogen activity and in the case of RgpA and Kgp are followed by excision of the individual HA domains. These domains are further truncated at the C-terminus by concerted action of Kgp and carboxypeptidase and form a non-covalent multidomain, multifunctional complex anchored into the outer membrane by the glycated, C-terminal HA domain. This hypothetical scenario is a reasonable explanation for the occurrence of many forms of gingipains.     

Light-induced Dehydrodimerization of 3-Hydroxypyrroles

Irradiation (λ > 280 nm) of 3-hydroxy-1H-pyrrole-2-carboxylates 1 in CH₃CN gives the [2.2′-bi(3-oxo-2,3-dihydro-1H-pyrrole)]-2-,2′-dicarboxylates 2 in reasonable to good yields. The corresponding N-methylpyrroles 3 only undergo slow photodecomposition under similar conditions. Several 2-methyl-3-oxo-2,3-dihydro-1H-pyrrole-2-carboxylates 4 and 5 were synthesized to compare their spectral data with those of the dehydrodimers 2 . A X-ray structure analysis was performed for diethyl [2,2′-bi(4,5-dimethyl-3-oxo-2,3-dihydro-1 H-pyrrole)]-2,2′-dicarboxylate ( 2b ). The originally proposed [3,3′-bi(3H-pyroole)] structure for compounds 2a - e proves incorrect.