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   

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.     

Effect of High Intensity Blue Light on Fusobacterium nucleatum Membrane Integrity

Oral malodour is considered to be caused mainly by the production of volatile sulfide compounds (VSC) by anaerobic gram-negative oral bacteria. Previous studies showed that these bacteria were susceptible to blue light phototoxicity mediated by the production of reactive oxygen species (ROS). In the present study, we tested the effect of blue light on the integrity Fusobacterium nucleatum's membrane, cellular proteins and DNA. Bacterial samples were exposed to high intensity blue light for 0, 70, 140 and 280 s (i.e. fluences of 0, 96, 192 and 384 J cm⁻², respectively). Following light exposure, bacterial samples were examined for membrane damage using fluorescence microscopy, intra-cellular protein analysis using electrophoresis (SDS-PAGE) and DNA fragmentation using ultra–filtration. Results showed that the increasing exposure of bacterial samples to blue light caused increased membrane permeability concomitant with a reduction in intra-cellular proteins and DNA fragments content. These results suggest that membrane damage is the main effect of high intensity blue light exposure on malodour producing bacteria.     

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.     

具核梭杆菌具核亚种蛋白质组及其在癌症pH环境下的差异蛋白质组分析

利用LC-MS/MS对F. nucleatum的蛋白质表达谱进行了深入研究, 共鉴定到1198个蛋白质的表达, 占基因组58%的编码基因. 通过在pH 6.5(癌症组织pH环境)和pH 7.5(正常组织pH环境)两种条件下F. nucleatum蛋白质表达的定量蛋白质组学分析, 发现处于癌症微环境pH条件下蛋白质表达的变化明显集中于金属离子转运, 硫胺素代谢, 糖代谢等过程和功能. 产丁酸发酵的能量代谢通路中的七个代谢酶在蛋白质组数据中找到, 其中有五个在癌症pH条件下显著下调, 这五个酶包含了丁酸盐代谢的整个过程, 这为结直肠癌患者肠道丁酸盐含量降低提供了新的证明. 此外, 癌症pH条件下F. nucleatum的丁酸代谢受到抑制, 造成产丁酸能力下降, 可能是其促进结直肠癌发展在代谢水平上的原因之一.     

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.     

Structural and Functional Analysis of the Pyridoxal Phosphate Homeostasis Protein YggS from Fusobacterium nucleatum

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family.     

The effect of visible light on systems containing polyvinyl alcohol and cupric ions

Conclusions The action of light in the Cu(II) d-d transition region on systems containing polyvinyl alcohol and cupric ions leads to a decrease in the cupric ion concentration and the appearance of a new band. The system returns to the initial state in the dark.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 3, pp. 676–678, March, 1986.     

The different behavior of rutile and anatase nanoparticles in forming oxy radicals upon illumination with visible light: an EPR study

Photoexcited TiO(2) has been found to generate reactive oxygen species, yet the precise mechanism and chemical nature of the generated oxy species especially regarding the different crystal phases remain to be elucidated. Visible light-induced reactions of a suspension of titanium dioxide (TiO(2)) in water were investigated using electron paramagnetic resonance (EPR) coupled with the spin-trapping technique. Increased levels of both hydroxyl (˙OH) and superoxide anion (˙O(2)(-)) radicals were detected in TiO(2) rutile and anatase nanoparticles (50 nm). The intensity of signals assigned to the ˙OH and ˙O(2)(-) radicals was larger for the anatase phase than that originating from rutile. Moreover, illumination with visible (nonUV) light enhanced ˙O(2)(-) formation in the rutile phase. Singlet oxygen was not detected in water suspension of TiO(2) neither in rutile nor in anatase nanoparticles, but irradiation of the rutile phase with visible light revealed a signal, which could be attributed to singlet oxygen formation. The blue part of visible spectrum (400-500 nm) was found to be responsible for the light-induced ROS in TiO(2) nanoparticles. The characterization of the mechanism of visible light-induced oxy radicals formation by TiO(2) nanoparticles could contribute to its use as a sterilization agent.     

Effects of visible light on the skin

Electromagnetic radiation has vast and diverse effects on human skin. Although photobiologic studies of sunlight date back to Sir Isaac Newton in 1671, most available studies focus on the UV radiation part of the spectrum. The effects of visible light and infrared radiation have not been, until recently, clearly elucidated. The goal of this review is to highlight the effects of visible light on the skin. As a result of advances in the understanding of skin optics, and comprehensive studies regarding the absorption spectrum of endogenous and exogenous skin chromophores, various biologic effects have been shown to be exerted by visible light radiation including erythema, pigmentation, thermal damage and free radical production. It has also been shown that visible light can induce indirect DNA damage through the generation of reactive oxygen species. Furthermore, a number of photodermatoses have an action spectrum in the visible light range, even though most of the currently available sunscreens offer, if any, weak protection against visible light. Conversely, because of its cutaneous biologic effects, visible light is used for the treatment of a variety of skin diseases and esthetic conditions in the form of lasers, intense pulsed light and photodynamic therapy.     

Bactericidal performance of photocatalytic titanium dioxide particle mixture under ultraviolet and fluorescent light: an in vitro study

When ultraviolet (UV) light comes into contact with titanium dioxide (TiO₂), a variety of free radicals are released to provide a potent oxidizing power. Few reports are available, however, evaluating the bactericidal effects of TiO₂ particle under UV light and fluorescent light (FL) in the same line of research for clinical applications. In the present study, we set out to evaluate the in vitro photocatalytic bactericidal effects on Staphylococcus aureus, which is one of the most common pathogens of infectious disease, in an aqueous system of TiO₂ particles irradiated by UV and FL. A TiO₂ particle mixture containing 0.019 mg/ml of TiO₂ was prepared. A bacterial solution was added dropwise to the mixture, and the resulting product was irradiated by UV or FL light. The colony‐forming units were counted and the bacterial survival rate was calculated. Control samples maintained a relatively high bacterial survival rate. In the TiO₂ mixture group, however, the bacterial survival rate decreased steadily, reaching 9.4% after 60 min of exposure to UV light and 10.9% after 60 min of FL irradiation. Distributing the TiO₂ particles in a water mixture produces highly efficient light absorption and enables greater and more frequent adhesion with bacteria, allowing a high degree of photocatalytic antibacterial action. Although the quantity was inferior to UV, our TiO₂ particles were able to show effective bactericidal activity even under FL. The TiO₂ particle mixture is expected to prove effective in preventing postoperative infection. Copyright © 2009 John Wiley & Sons, Ltd.     

Systematic study of parameters influencing the action of Rose Bengal with visible light on bacterial cells: comparison between the biological effect and singlet-oxygen production

As part of a project to study different methods for the disinfection of effluent water, the inactivation of different microorganisms (Escherichia coli, Deinococcus radiodurans and spores of Bacillus subtilis) using a combination of a photosensitizer (Rose Bengal) with simulated sunlight and oxygen was determined under various environmental conditions (temperature, pH index). In parallel, the singlet-oxygen (1O2) production was also measured under the same conditions. Whereas the vegetative cells could be inactivated much more efficiently at increased temperature and altered index of pH, the production of 1O2 remained essentially the same under these alterations. Additionally, the relations among the sensitivities of different cell types to be killed by our photodynamic treatments (PDT) were opposite to those found after exposure to ionizing radiation. The results of photodynamic experiments do not reflect the cells' capacity to repair DNA strand breaks. Spores of B. subtilis, as a nonvegetative system, could not be inactivated by illuminations up to 100 J cm-2. Together, these findings indicate that DNA is not the primary target, the inactivation of which leads to the killing of our test organisms. Instead, the cellular envelope appears to be the component being assaulted by our PDT.     

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.     

In vitro and in vivo fluorescent imaging of a monofunctional chelated platinum complex excitable using visible light

The biological fluorescent distribution of a model antitumor monofunctional platinum(II) complex bearing a 7-nitro-2,1,3-benzoxadiazole fluorophore can be visualized in breast carcinoma MCF-7 cells, pulmonary carcinoma A549 cells, kidney epithelial 293T cells, and zebrafish larva.     

Cu2O cubic and polyhedral structures versus commercial powder: Shape effect on photocatalytic activity under visible light

A simple precipitation-reduction method was used to prepare cubic and polyhedral Cu₂O structures. Their morphological, structural, optical and electronic properties were analyzed and compared with those of commercial Cu₂O by means of SEM, TEM, XRD, UV–vis DRS, Photoluminescence Spectroscopy and Time Resolved Microwave Conductivity. Methyl orange (MO) photodegradation with visible light (blue-light LEDs, λ = 450–470 nm) was taken as a model reaction to study the photocatalytic activity. According to the results, Cu₂O edge-and corner-truncated polyhedral particles significantly decreased the MO initial concentration by adsorption and photocatalysis, whereas, Cu₂O cubic particles did not show MO adsorption but slightly higher photocatalytic activity than the polyhedral particles. Commercial Cu₂O showed MO adsorption and high electron mobility but it was completely inactive. These results were explained in terms of the crystalline defects that influence both, the adsorption capacity and the photocatalytic activity.     

A generic method of visible light sensitization for perovskite-related layered oxides: Substitution effect of lead

Nearly complete substitution of lead into layered perovskites is a generic method of visible light sensitization for these ultraviolet (UV)-active materials. Thus, CaBi₄Ti₄O₁₅ (Aurivillius phase), K_0.5La_0.5Ca_1.5Nb₃O₁₀ (Dion-Jacobson phase), and Sr₃Ti₂O₇ (Ruddlesden-Popper phase) are photocatalysts, all working under UV light, but their analogs containing lead (PbBi₄Ti₄O₁₅, K_0.5La_0.25Bi_0.25Ca_0.75Pb_0.75Nb₃O₁₀, and PbTiO₃) absorb visible light and exhibit photocatalytic activity of decomposition of water under visible light.     

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.     

Boron-doped TiO2: Characteristics and photoactivity under visible light

Boron-doped TiO₂ was prepared by the sol-gel method and by grinding TiO₂ powder with a boron compounds (boric acid and boric acid triethyl ester followed by calcinations at temperature range 200 to 600°C. Three types of pristine TiO₂: ST-01 (Ishihara Sangyo Ltd., Japan; 300 m²/g), P25 (Degussa, Germany, 50 m²/g), A11 (Police S.A., Poland 12 m²/g) were used in grinding procedure. The photocatalytic activity of obtained powders in visible light was estimated by measuring the decomposition rate of phenol (0.21 mmol/dm³) in an aqueous solution. The photocatalysts were characterized by X-ray diffraction (XRD), X-ray photoelectron emission spectroscopy (XPS), UV-VIS absorption and BET surface area measurements. The best photoactivity under visible light was observed for B-TiO₂ modified with 2 wt% of boron prepared by grinding ST-01 with dopant followed by calcinations at 400°C. This photocatalyst contains 16.9 at.% of carbon and 6.6 at.% of boron in surface layer and its surface area is 192 m²/g.     

A visible light photocatalyst: effects of vanadium substitution on ETS-10

Hybrid density functional theory/molecular mechanics (DFT/MM) methods have been used to investigate the effects of vanadium substitution in ETS-10. Models have been developed to contain varying concentrations of V(IV) and V(V) within the O-M-O (M = Ti, V) chain. Most of the V-substituted models have a localized mid-gap state. The occupation of this localized state depends upon the dopant oxidation state, leading to the addition of multiple low energy transitions. A linear correlation has been identified between band gap energies estimated using ground state orbital energies and those calculated using the more accurate and computationally demanding time-dependent DFT (TDDFT) method for a variety of transition metal substituted models of ETS-10. Consistent with experimental data for V substitution, our models predict a decrease in the optical band gap with increasing [V], due to a lowering of the delocalized d-orbital states at the bottom of the conduction band with increasing V d-orbital character. This effect is more pronounced in the case of V(V) substitution than V(IV). Excitation energies for the V-doped models, calculated with TDDFT methods correlate well with experimental data, allowing for the assignment of specific optical transitions to experimental UV-Vis spectra. The electronic structure of V-substituted ETS-10 at high V concentration demonstrates band gap energies within the visible range of the spectrum. Additionally, at high [V] the band gap energy and presence of low energy electron traps can be controlled by the relative concentration of V(IV) and V(V) along the O-M-O chain, establishing V-substituted ETS-10 as a promising visible light photocatalyst.     

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.