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.     

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.     

Photosensitized oxidation and inactivation of pyocyanin, a virulence factor of Pseudomonas aeruginosa

Pyocyanin (PyO-) (1-hydroxy-5-methylphenazine) is a cytotoxic compound secreted by Pseudomonas aeruginosa, an omnipresent bacterium and a human pathogen. We report that visible light illumination in the presence of rose bengal, or riboflavin, in aerated solutions (pH 7.0-7.2) induces irreversible loss of the pigment's characteristic absorption band at 690 nm, indicating its oxidation. This photobleaching was paralleled by generation of a multiline Electron Paramagnetic Resonance (EPR) spectrum attributed to a PyO(-)-derived radical. The reaction was dependent on the presence of air, sensitizers and light, was inhibited by sodium azide and was unaffected by ethanol. This suggests that PyO- was oxidized largely via singlet oxygen and that hydroxyl radicals were not involved. The photochemically modified pigment was less efficient in oxidizing NAD(P)H and generated less superoxide (by approximately 50%) than the intact PyO-, indicating its partial inactivation. 1-Methoxy-5-methylphenazine, a PyO- analog in which the -O- moiety was replaced by the methoxy group (-OMe), was resistant to oxidation, suggesting that oxidation of PyO- involves its phenolate moiety. These results also suggest that photosensitization could be a potentially useful method for inactivation of PyO- and, possibly, detoxification of superficial wounds (skin, eye) infected with P. aeruginosa.     

Enzyme activation and inactivation induced by low doses of irradiation

Activation phenomenon has been observed with two sets of enzymes under the conditions of low dosage irradiation. Activation was registered for angiotensin-converting enzyme under in vitro γ-irradiation (0.662 MeV, pulse duration approx 10s) at dose levels of 1–3 Gy and under X-ray irradiation (approx 9 keV, pulseduration approx 10^?9s) at dose levels of 2×10^?5 Gy. An activation effect has also occurred for native and recombinant horseradish peroxidase and tobacco peroxidase under γ-irradiation. The phenomenon observed is rationalized in terms of a kinetic model suggesting the existence of at least one activated enzyme conformation induced by radiolysis. The activity oscillations registered in dense plasma focus experiments were rationalized using the same model with the corresponding kinetic equation converted into the form describing the decaying oscillations caused by exciting force. The model analysis is presented.     

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.     

Design of host defence peptides for antimicrobial and immunity enhancing activities

Host defense peptides are a vital component of the innate immune systems of humans, other mammals, amphibians, and arthropods. The related cationic antimicrobial peptides are also produced by many species of bacteria and function as part of the antimicrobial arsenal to help the producing organism reduce competition for resources from sensitive species. The antimicrobial activities of many of these peptides have been extensively characterized and the structural requirements for these activities are also becoming increasingly clear. In addition to their known antimicrobial role, many host defense peptides are also involved in a plethora of immune functions in the host. In this review, we examine the role of structure in determining antimicrobial activity of certain prototypical cationic peptides and ways that bacteria have evolved to usurp these activities. We also review recent literature on what structural components are related to these immunomodulatory effects. It must be stressed however that these studies, and the area of peptide research, are still in their infancy.     

Antibiotic activities of host defense peptides: more to it than lipid bilayer perturbation

Defensins are small basic amphiphilic peptides (up to 5 kDa) that have been shown to be important effector molecules of the innate immune system of animals, plants and fungi. In addition to immune modulatory functions, they have potent direct antimicrobial activity against a broad spectrum of bacteria, fungi and/or viruses, which makes them promising lead compounds for the development of next-generation antiinfectives. The mode of antibiotic action of defensins was long thought to result from electrostatic interaction between the positively charged defensins and negatively charged microbial membranes, followed by unspecific membrane permeabilization or pore-formation. Microbial membranes are more negatively charged than human membranes, which may explain to some extent the specificity of defensin action against microbes and associated low toxicity for the host. However, research during the past decade has demonstrated that defensin activities can be much more targeted and that microbe-specific lipid receptors are involved in the killing activity of various defensins. In this respect, human, fungal and invertebrate defensins have been shown to bind to and sequester the bacterial cell wall building block lipid II, thereby specifically inhibiting cell wall biosynthesis. Moreover, plant and insect defensins were found to interact with fungal sphingolipid receptors, resulting in fungal cell death. This review summarizes the current knowledge on the mode of action and structure of defensins from different kingdoms, with specific emphasis on their interaction with microbial lipid receptors.     

Interaction between non-heme iron of lipoxygenases and cumene hydroperoxide: basis for enzyme activation, inactivation, and inhibition

Lipoxygenase catalysis depends in a critical fashion on the redox properties of a unique mononuclear non-heme iron cofactor. The isolated enzyme contains predominantly, if not exclusively, iron(II), but the catalytically active form of the enzyme has iron(III). The activating oxidation of the iron takes place in a reaction with the hydroperoxide product of the catalyzed reaction. In a second peroxide-dependent process, lipoxygenases are also inactivated. To examine the redox activation/inactivation dichotomy in lipoxygenase chemistry, the interaction between lipoxygenase-1 (and -3) and cumene hydroperoxide was investigated. Cumene hydroperoxide was a reversible inhibitor of the reaction catalyzed by lipoxygenase-1 under standard assay conditions at high substrate concentrations. Reconciliation of the data with the currently held kinetic mechanism requires simultaneous binding of substrate and peroxide. The enzyme also was both oxidized and largely inactivated in a reaction with the peroxide in the absence of substrate. The consequences of this reaction for the enzyme included the hydroxylation at C beta of two amino acid side chains in the vicinity of the cofactor, Trp and Leu. The modifications were identified by mass spectrometry and X-ray crystallography. The peroxide-induced oxidation of iron was also accompanied by a subtle rearrangement in the coordination sphere of the non-heme iron atom. Since the enzyme retains catalytic activity, albeit diminished, after treatment with cumene hydroperoxide, the structure of the iron site may reflect the catalytically relevant form of the cofactor.     

Selective chemical inactivation of AAA proteins reveals distinct functions of proteasomal ATPases

BACKGROUND: The 26S proteasome contains six highly related ATPases of the AAA family. We have developed a strategy that allows selective inhibition of individual proteasomal ATPases in the intact proteasome. Mutation of a threonine in the active site of Sug1/Rpt6 or Sug2/Rpt4 to a cysteine sensitizes these proteins to inactivation through alkylation by the sulfhydryl modifying agent NEM. Using this technique the individual contributions of Sug1 and Sug2 to proteasome function can be assessed. RESULTS: We show that both Sug1 and Sug2 can be selectively alkylated by NEM in the context of the intact 26S complex and as predicted by structural modeling, this inactivates the ATPase function. Using this technique we demonstrate that both Sug 1 and 2 are required for full peptidase activity of the proteasome and that their functions are not redundant. Kinetic analysis suggests that Sug2 may have an important role in maintaining the interaction between the 19S regulatory complex and the 20S proteasome. In contrast, inhibition of Sug1 apparently decreases peptidase activity of the 26S proteasome by another mechanism. CONCLUSIONS: These results describe a useful technique for the selective inactivation of AAA proteins. In addition, they also demonstrate that the functions of two related proteasomal AAA proteins are not redundant, suggesting differential roles of proteasomal AAA proteins in protein degradation.     

Design, folding, and activities of metal-assembled coiled coil proteins

Metal ions serve many purposes in natural proteins, from the stabilization of tertiary structure to the direction of protein folding to crucial roles in electron transfer and catalysis. There is considerable interest in creating metal binding sites in designed proteins to understand the structural role of metal ions and to design new metalloproteins with useful functions. The de novo design of metalloproteins and the role of metals in the folding of designed proteins are reviewed here, with particular focus on the design, folding, and activities of the [M(bpy-peptide)(3)](2+) structure. This maquette is constructed by the covalent attachment of 2,2'-bipyridine to the N-termini of amphiphilic peptides, and it is assembled into a folded trimeric coiled coil by the addition of a six-coordinate transition metal ion and the resulting hydrophobic collapse of the peptides. The [M(bpy-peptide)(3)](2+) structure has been employed in diverse applications, ranging from electron transfer pathway studies to the study of optimal hydrophobic packing in a virtual library to the construction of receptors and biosensors.     

Influence of redox activation of NAMI-A on affinity to serum proteins: transferrin and albumin

Imidazolium trans-tetrachloridodimethylsulfoxideimidazoleruthenate(III), NAMI-A, is a ruthenium drug exhibiting unique properties under clinical studies such as ability to inhibit the process of metastases without exerting tumor toxicity. Its place of action is concentrated at the extracellular level, therefore, the transformation and fate of this drug upon injection is of great importance. This study focuses on evaluation of the reducing potency of blood stream on interaction with two serum proteins: albumin and transferrin. It was investigated by applying various simplified serum models preserving physiological concentration of proteins and the amount of Ru complex as found in patients. It was shown that ruthenation of albumin is slightly increased while transferrin decreased upon addition of reductant in blood stream (ascorbate, glutathione, and cysteine) at physiological concentration. Interestingly, in serum models comprising low-molecular-mass components the amount of the reductant in the solution had a pronounced effect on the Ru content, in particular in transferrin. Supplementation of serum models with glutathione at concentration enough for complete reduction of NAMI-A selectively enhanced the binding of Ru complex to transferrin while ruthenation of albumin was almost unchanged. Spectrofluorimetric studies revealed that reduction has a marginal effect on binding affinity, therefore, both Ru(III) and (II) derivatives equally can compete for binding to transferrin.     

Fluorous biphasic catalysis with dirhodium(II) perfluorocarboxylates: selective silylation of alcohols under fluorous biphasic conditions

Dirhodium(II) perfluorocarboxylates bearing C7–C13 perfluoroalkyl chains have been prepared and used as catalysts under fluorous biphasic conditions. They were found to be active and recyclable catalysts for the silylation of alcohols with triethylsilane. Hydrophobic, primary alcohols are preferentially silylated by the fluorous biphasic catalytic system in comparison with hydrophilic or secondary ones. This opens the way to the development of selective silylation protocols.     

牙龈卟啉菌表面蛋白的生物活性和免疫原性研究

为了更好地研究牙龈卟啉菌(Prophyromonas gingivalis，Pg)表面蛋白(Surface-associated proteins，SAP)的生物活性，实验用SAP体外刺激小鼠颅骨和人外周血单个核细胞，分别用自动生化分析仪和放免方法检测培养液中Ca2 和IL-6的含量．同时，采用ELISA和旧法分别测定SAP免疫小鼠的抗体效价和诱导的脾细胞转化程度．结果显示：一方面SAP是体外诱导骨吸收和前炎性因子合成的介质；另一方面SAP免疫的小鼠产生了抗SAP特异性抗体和脾细胞特异增生反应．这说明具有生物活性的SAP诱导了实验小鼠产生体液免疫应答和细胞免疫应答．     

Colorimetric detection of proteins based on target-induced activation of aptazyme

The detection of protein is vital to fundamental research as well as practical applications. However, most detection methods depend on antibody-based assays which are faced with many shortcomings. Herein, we propose a colorimetric method for protein assays based on target-triggered activation of aptazyme, which may offer simple, rapid and cost-effective detection of the target protein. In this method, the conformation change of aptazyme induced by target protein is designed to be associated with aptazyme activation. Consequently, in the presence of the target protein, the designed DNA linkers will be cleaved into two fragments that fail to cross-link gold nanoparticles (GNPs), thus the color of GNP solution remains red, while the color will be changed in the absence of the target. Because of the advantages of aptazyme such as economic synthesis, stable, easy modification and its ability to accomplish signal recognition and signal amplification simultaneously, the method is thermostable, simple and cost-efficient. In this work, we have taken the detection of vascular endothelial growth factor (VEGF) as an example, which can present an analytical performance with as low as 0.1 nM detection limit, spanning a detection range of 3 orders of magnitude. What is more, the principle of this proposed new method can be extended as a universal assay method not only for the detection of analytes which have an aptamer but also for those analytes that have ligands.     

The role of retroviral dUTPases in replication and virulence

Several retroviruses, including equine infectious anemia virus (EIAV), visna virus, caprine arthritis-encephalitis virus (CAEV) and feline immunodeficiency virus (FIV) encode dUTPase. The role of this enzyme in the replication of these viruses has been scrutinized, with particular emphasis on potential roles for dUTPase in virulence and viral mutation rate. Overall, the results of these studies have indicated a central role for dUTPase in facilitating productive viral replication in non-dividing cells. The requirement for dUTPase in EIAV, which replicates exclusively in macrophages, may be the most stringent. Studies of dUTPase mutants of virulent EIAV clones suggest that the enzyme is a major determinant of virulence. In contrast, FIV readily replicates in dividing cell populations such as CD4+ and CD8+ T cells, and B cells as well as in non-dividing macrophages. Thus, the virus burden and disease sequelae are lowered in cats infected with a dUTPase-minus FIV relative to cats infected with wild type FIV, but not totally abrogated. Growth in macrophages is attenuated with the DU-minus FIV with evidence of a 5 to 8-fold increase in G-->A transition mutations in viral integrants present in macrophages. These findings are consistent with an increase in uracil misincorporation in the absence of dUTPase, resulting in transition mutations that cripple the virus. Effects on virus replication and disease production have also been noted for dUTPase-deleted CEAV and visna virus. While HIV and SIV do not encode dUTPase some reports suggest that other viral and host cell factors may substitute for its activity. Betaretroviruses also encode dUTPase and while several of these cause significant disease, the role of dUTPase in their replication and pathogenesis is currently unknown.     

A versatile ruthenium precursor for biphasic catalysis and its application in ionic liquid biphasic transfer hydrogenation: conventional vs task-specific catalysts

The synthesis of a novel imidazolium-tagged ruthenium complex, which represents a versatile precursor for aqueous and ionic liquid biphasic catalysis, is reported. Its utility is demonstrated in the highly enantioselective ionic liquid biphasic transfer hydrogenation of acetophenone and is compared to conventional (untagged) complexes.     

Design of a bacterial host for site-specific incorporation of p-bromophenylalanine into recombinant proteins

Introduction of a yeast suppressor tRNA (ytRNA(Phe)(CUA)) and a mutant yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) into an Escherichia coli expression host allows in vivo incorporation of phenylalanine analogues into recombinant proteins in response to amber stop codons. However, high-fidelity incorporation of non-natural amino acids is precluded in this system by mischarging of ytRNA(Phe)(CUA) with tryptophan (Trp) and lysine (Lys). Here we show that ytRNA(Phe)(CUA) and yPheRS can be redesigned to achieve high-fidelity amber codon suppression through delivery of p-bromophenylalanine (pBrF). Two strategies were used to reduce misincorporation of Trp and Lys. First, Lys misincorporation was eliminated by disruption of a Watson-Crick base pair between nucleotides 30 and 40 in ytRNA(Phe)(CUA). Loss of this base pair reduces mischarging by the E. coli lysyl-tRNA synthetase. Second, the binding site of yPheRS was redesigned to enhance specificity for pBrF. Specifically, we used the T415A variant, which exhibits 5-fold higher activity toward pBrF as compared to Trp in ATP-PP(i) exchange assays. Combining mutant ytRNA(Phe)(CUA) and yPheRS (T415A) allowed incorporation of pBrF into murine dihydrofolate reductase in response to an amber codon with at least 98% fidelity.     

Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis

Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.