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Pili or fimbriae, which are filamentous structures present on the surface of bacteria, were purified from a periodontal pathogen, Porphyromonas gingivalis, in 1980s. The protein component of pili (stalk pilin), which is its major component, was named FimA; it has a molecular weight of approximately 41 kDa. Because the molecular weight of the pilin from P. gingivalis is twice that of pilins from other bacterial pili, the P. gingivalis Fim pili were suggested to be formed via a novel mechanism. In earlier studies, we reported that the FimA pilin is secreted on the cell surface as a lipoprotein precursor, and the subsequent N‐terminal processing of the FimA precursor by arginine‐specific proteases is necessary for Fim pili formation. The crystal structures of FimA and its related proteins were determined recently, which show that Fim pili are formed by a protease‐mediated strand‐exchange mechanism. The most recent study conducted by us, wherein we performed cryoelectron microscopy of the pilus structure, provided evidence in support of this mechanism. As the P. gingivalis Fim pili are formed through novel transport and assembly mechanisms, such pili are now designated as Type V pili. Surface lipoproteins, including the anchor pilin FimB of Fim pili that are present on the outer membrane, have been detected in certain Gram‐negative bacteria. Here, we describe the assembly mechanisms of pili, including those of Type V and other pili, as well as the lipoprotein transport mechanisms. [ABSTRACT FROM AUTHOR]

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement‐producing protein architectures. Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility. [ABSTRACT FROM AUTHOR]

The type IX secretion system (T9SS) was originally discovered in Porphyromonas gingivalis, one of the pathogenic bacteria associated with periodontal disease and is now known to be present in many members of the phylum Bacteroidetes. The T9SS secretes a number of potent virulence factors, including the highly hydrolytic proteases called gingipains, across the outer membrane in P. gingivalis. To understand the entire machinery of T9SS, an exhaustive search for T9SS‐related genes in P. gingivalis using the mariner family transposon (Tn) and Tn‐seq analysis was performed. Seven hundred and two Tn insertion sites in Tn mutants with no colony pigmentation that is associated with Lys‐gingipain (Kgp) defectiveness were determined, and it was found that the Tn was inserted in the kgp gene and 54 T9SS‐related candidate genes. Thirty‐three out of the 54 genes were already known as T9SS‐related genes. Furthermore, deletion mutant analysis of the remaining 21 genes revealed that they were not related to the T9SS. The 33 T9SS‐related genes include a gene for PGN_0297, which was found to be associated with the T9SS components PorK and PorN. A PGN_0297 gene deletion mutant was constructed, and it was found that the mutant showed no colony pigmentation, hemagglutination or gingipain activities, indicating that PGN_0297 was an essential component of the T9SS. The 33 genes did not include the six genes (gppX, omp17, porY, rfa, sigP and wzx) that were also reported as T9SS‐related genes. gppX deletion and insertion mutants were constructed, and it was found that they did not show deficiency in the T9SS. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis is a Gram‐negative anaerobic bacterium that has been considered to be one of the bacteria associated with progression of human periodontitis. Subgingival biofilms formed by bacteria, including P. gingivalis, induce chronic inflammation, and activation of inflammasome in the gingival tissue. However, the mechanisms of P. gingivalis‐triggering inflammasome activation and the role of bacteria–host interactions are controversial. In this study, we investigated the potential of P. gingivalis for triggering inflammasome activation in human cells and mouse models. We demonstrated that secreted or released factors from bacteria are involved in triggering NLR family, pyrin‐domain containing 3 protein (NLRP3) inflammasome in a gingipain‐independent manner. Our data indicated that released active caspase‐1 and mature IL‐1β are eliminated by proteolytic activity of secreted gingipains. These results elucidate the molecular bases for the mechanisms underlying P. gingivalis‐triggered inflammasome activation. Gram‐negative anaerobic bacterium Porphyromonas gingivalis triggers the activation of NLRP3 inflammasome mediated by secreted/released factors from bacteria. The inflammasome induced caspase‐1 activation following processing of IL‐1β. On the other hand, secreted bacterial gingipains proteolytically degrade the cytokines including IL‐1β, as one of bacterial evasion systems against host immune responses. [ABSTRACT FROM AUTHOR]

Summary: The periodontal pathogen Porphyromonas gingivalis secretes many potent virulence factors using the type IX secretion system (T9SS). T9SS cargo proteins that have been structurally determined by X‐ray crystallography are composed of a signal peptide, functional domain(s), an immunoglobulin (Ig)‐like domain and a C‐terminal domain. Role of the Ig‐like domains of cargo proteins in the T9SS has not been elucidated. Gingipain proteases, which are cargo proteins of the T9SS, were degraded when their Ig‐like domains were lacking or truncated. The degradation was dependent on the activity of a quality control factor, HtrA protease. Another T9SS cargo protein, HBP35, which has a thioredoxin domain as a functional domain, was analyzed by X‐ray crystallography, revealing that HBP35 has an Ig‐like domain after the thioredoxin domain and that the hydrophobic regions of the thioredoxin domain and the Ig‐like domain face each other. HBP35 with substitution of hydrophobic amino acids in the Ig‐like domain was degraded depending on HtrA. These results suggest that the Ig‐like domain mediates stability of the cargo proteins in the T9SS. Cargo proteins via the type IX secretion system (T9SS) have an immunoglobulin (Ig)‐like domain after a functional domain. T9SS cargo proteins lacking their Ig‐like domains were degraded by a quality control protease, HtrA, suggesting that the Ig‐like domain mediates stability of the cargo proteins in the T9SS. Hydrophobic regions of a ‐sheet structure of the Ig‐like domain facing to the functional domain were suggested to be particularly important for the stabilization by the Ig‐like domain. [ABSTRACT FROM AUTHOR]

ABSTRACT: Prevotella melaninogenica is a gram‐negative anaerobic commensal bacterium that resides in the human oral cavity and is isolated as a pathogen of suppurative diseases both inside and outside the mouth. However, little is known about the pathogenic factors of P. melaninogenica. The periodontal pathogens Porphyromonas gingivalis and Tanerella forsythia secrete virulence factors such as protease and bacterial cell surface proteins via a type IX secretion system (T9SS) that are involved in pathogenicity. P. melaninogenica also possesses all known orthologs of T9SS. In this study, a P. melaninogenica GAI 07411 mutant deficient in the orthologue of the T9SS‐encoding gene, porK, was constructed. Hemagglutination and biofilm formation were decreased in the porK mutant. Furthermore, following growth on skim milk‐containing medium, the diameters of the halos surrounding the porK mutant were smaller than those of the wild‐type strain, suggesting a decrease in secretion of proteases outside the bacterium. To investigate this in detail, culture supernatants of wild‐type and porK mutant strains were purified and compared by two‐dimensional electrophoresis. In the mutant strain, fewer spots were detected, indicating fewer secreted proteins. In infection experiments, the mortality rate of mice inoculated with the porK mutant strain was significantly lower than in the wild‐type strain. These results suggest that P. melaninogenica secretes potent virulence factors via the T9SS that contribute to its pathogenic ability. [ABSTRACT FROM AUTHOR]

ABSTRACT: Many members of the phylum Bacteroidetes, such as Flavobacterium johnsoniae, can glide over a solid surface: an ability called gliding motility. It can be usually observed on agar plates as thin, flat, spreading colonies with irregular, feathery edges; this phenomenon is called colony spreading. Colony spreading of F. johnsoniae on 1.5% agar plates containing poor nutrients is dose‐dependently inhibited by addition of D‐glucose, as previously reported. Accordingly, here, we created mutants (by transposon mutagenesis) that partially suppressed glucose‐mediated inhibition of colony spreading. Among the isolates, we found that one had a transposon insertion in Fjoh_4565, tentatively named mfsA, which encodes a major facilitator superfamily (MFS) transporter previously shown to be required for growth on glucose, N‐acetyl‐glucosamine, and chitin. We constructed an mfsA deletion mutant and found that the mutant showed no glucose‐mediated acceleration of growth or glucose uptake. The mfsA gene complemented the phenotype of a glucose‐negative Escherichia coli. These results suggest that the mfsA gene encodes the sole MFS transporter of glucose in F. johnsoniae and that glucose uptake is partially required for the glucose‐mediated inhibition of F. johnsoniae colony spreading. [ABSTRACT FROM AUTHOR]

This work is focused on montmorillonite (MMT)-based 'support-activators' (S-As) for the metallocene-catalyzed propylene polymerization. This catalyst was previously industrialized; however, for further technological advances, the activation mechanism is investigated. The chemical and morphological requirements of the S-A are surveyed using both commercially available raw clay minerals (non-acid-treated) and acid-treated clay minerals. The S-A possessing strong-acid sites (p Ka < −8.2) gives a highly active catalyst. Acid treatment of MMT induces morphological changes as well as the formation of strong acid sites. Based on pore size distribution analysis and atomic force microscopy observations, it is concluded that the strong acid sites are located in the small pores around the edge of the clay mineral (not in the interlayer), where the structure is disordered by the acid treatment. [ABSTRACT FROM AUTHOR]

Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium. [ABSTRACT FROM AUTHOR]

The photoinduced conversion via the aci-nitro into the nitroso form was studied for 4,5-dimethoxy-2-nitrobenzyl alcohols attached to various leaving groups: amino acids histidine (NHis) and aspartate (NAsp) as well as their fluorenylmethoxycarbonyl derivatives (FHis) and (FAsp). In addition, two peptides containing either of the two amino acids were studied, carrying the photoreactive group attached to a histidine (PHis), or to an aspartate (PAsp). The aci-nitro forms with maximum at λaci = 420 nm were observed for FHis and FAsp after the decay of a triplet-triplet absorption, analogous to those of other 2-nitrobenzyl type compounds. For both FHis and FAsp the quantum yield of photoconversion Φp is 0.03 and for the peptides PHis and PAsp ca 0.01 and 0.005, respectively. [ABSTRACT FROM AUTHOR]

The oral anaerobic bacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, requires heme for its growth. Non-iron metalloporphyrins, In-PPIX and Ga-PPIX, were examined for antibacterial effects on P. gingivalis. Both In-PPIX and Ga-PPIX caused retardation of P. gingivalis growth in a dose-dependent fashion. Microarray and qPCR analyses revealed that In-PPIX treatment upregulated the expression of several genes encoding proteins including ClpB and ClpC, which are members of the Clp (caseinolytic protease, Hsp100) family, and aRNR, aRNR-activating protein and thioredoxin reductase, whereas In-PPIX treatment had no effect on the expression of genes encoding proteins involved in heme uptake pathways, Hmu-mediated, Iht-mediated and Tlr-mediated pathways. P. gingivalis ihtA and ihtB mutants were more resistant to In-PPIX than was the wild-type parent, whereas hmuR and tlr mutants did not show such resistance to In-PPIX. The results suggest that In-PPIX is incorporated by the Iht-mediated heme uptake pathway and that it influences protein quality control and nucleotide metabolism and retards growth of P. gingivalis. [ABSTRACT FROM AUTHOR]

The article focuses on the hemagglutination and platelet aggregation stimulated by Porphyromonas gingivalis cells, one of the most important etiological agents of chronic periodontitis. It says that the study of M.A. Curtis and colleagues reveals that proteinase preparation from P. gingivalis is capable of inducing platelet aggregation with purified platelets. Moreover, P. gingivalis that enters the human circulatory system ensuing transient bacteremia may induce infective endocarditis.

Evidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet-rich plasma (PRP). Mechanism of P. gingivalis-induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg-gingipain (Rgp) and Lys-gingipain (Kgp) did not suppress P. gingivalis-induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis-induced platelet aggregation using washed platelets. Mutant analysis revealed that P. gingivalis-induced platelet aggregation in PRP depended on Rgp-, Kgp- and haemagglutinin A (HagA)-encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis-induced platelet aggregation in PRP. P. gingivalis cell-reactive IgG in plasma, and FcγRIIa receptor and to a lesser extent GPIbα receptor on platelets were found to be a prerequisite for P. gingivalis-induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis. [ABSTRACT FROM AUTHOR]

Background: It is well known that the moderately roughened surfaces of dental implants enhance direct bone-implant contact. However, rough implant surfaces, as compared to smooth surfaces, are thought to pose a higher risk of bacterial infection when exposed to the oral cavity. Purpose: This study was focused on evaluating the photocatalytic bactericidal effects of anatase titanium dioxide (TiO2) on gram-negative anaerobic bacteria known to be associated with periimplantitis. Materials and Methods: A film of photocatalytic anatase TiO2 was added onto the surface of commercially pure titanium disks by plasma source ion implantation (PSII) followed by annealing. The photocatalytic properties of the film were confirmed by the degradation of methylene blue. Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum cells were incubated anaerobically and seeded on the disk. The disks were then exposed to ultraviolet A (UVA) illumination from black light in an anaerobic environment. After illumination, the number of viable cells was counted in terms of colony-forming units. Results: The anatase TiO2 film added by the PSII method and annealing exhibited a strong photocatalytic reaction under UVA illumination. The viability of both types of bacteria on the photocatalytic TiO2 film was suppressed to less than 1% under UVA illumination within 120 minutes. Conclusion: The bactericidal effect of the TiO2 photocatalyst is of great use for sterilizing the contaminated surface of dental implants. [ABSTRACT FROM AUTHOR]

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

Bacterial cell surface filaments play significant roles in adherence to and invasion of host cells. They are generated by the chaperone/usher pathway system (class I fimbriae), the type II secretion system (type IV pili) and the nucleation-dependent polymerization system (Curli filaments) that are categorized by their modes of expression and assembly. In this study, we found that the periodontal pathogen Porphyromonas gingivalis expressed the major structural components of two cell surface filaments (fimbrilin and the 75 kDa protein) that had extremely long prosequences in their primary gene products. N-terminal amino acid sequencing of the prosequences, treatment of P. gingivalis cells with globomycin, an inhibitor for lipoprotein-specific signal peptidase, amino acid substitution of the cysteine residue of the prosequence of fimbrilin and [3H]-palmitic acid labelling implied that fimbrilin and the 75 kDa protein were matured through their lipoprotein precursor forms. Accumulation of precursor forms of fimbrilin and the 75 kDa protein on the cell surface of the gingipain-null mutant revealed that Arg-gingipain processed these precursors on the surface to yield their mature forms, which subsequently assembled into the filamentous structures, suggesting that the transport and assembly of the major component proteins appear to be novel. [ABSTRACT FROM AUTHOR]

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

The obligately anaerobic bacterium Porphyromonas gingivalis produces characteristic black-pigmented colonies on blood agar. It is thought that the black pigmentation is caused by haem accumulation and is related to virulence of the microorganism. P. gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of its N-terminal amino acid sequence indicated that the 19 kDa protein was encoded by an internal region (HGP15 domain) of an arginine-specific cysteine proteinase (Arg-gingipain, RGP)-encoding gene (rgp1) and was also present in genes for lysine-specific cysteine proteinases (prtP and kgp) and a haemagglutinin (hagA) of P. gingivalis. The HGP15 domain protein was purified from an HGP15-overproducing Escherichia coli and was found to have the ability to bind to haemoglobin in a pH-dependent manner. The anti-HGP15 antiserum reacted with the 19 kDa haemoglobin-binding protein in the envelope of P. gingivalis. P. gingivalis wild-type strain showed pH-dependent haemoglobin adsorption, whereas its non-pigmented mutants that produced no HGP15-related proteins showed deficiency in haemoglobin adsorption. These results strongly indicate a close relationship among HGP15 production, haemoglobin adsorption and haem accumulation of P. gingivalis. [ABSTRACT FROM AUTHOR]

The ferritin-encoding gene (cft) of Campylobacter jejuni was cloned and sequenced. The nucleotide sequence of cft had a 501 bp open reading frame for a protein with 167 amino acids and a predicted molecular mass of 19180 Da, and showed a high similarity to that of Helicobacter pylori and Escherichia coli ferritin genes. To determine the biological function of ferritin in C. jejuni, a ferritin-deficient mutant was constructed. The growth of ferritin-deficient strain SNA1 was clearly inhibited under iron deprivation. The ferritin-deficient mutant was more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These findings demonstrate that ferritin in C. jejuni makes a significant contribution to both iron storage and protection from intracellular iron overload, and resulting iron-mediated oxidative stress. [ABSTRACT FROM AUTHOR]

The superoxide dismutase (SOD) of Porphyromonas gingivalis, an obligate anaerobe, was purified from Escherichia coli (sodA sodB mutant) harboring the P. gingivalis SOD-encoding gene. The purified protein contained both iron and a small amount of manganese. Iron- and manganese-reconstituted SOD, which contained one of these metals exclusively, showed specific activities of 1000 and 1200 U/mg/mol of metals/subunit, respectively. These values were similar to the specific activity of the native enzyme purified from the recombinant E. coli strain. The Fe-reconstituted enzyme was inactivated by 10 mM hydrogen peroxide to about 5 % of its original activity after a 15 min incubation at 25 +C at pH 7.8, whereas the Mn-reconstituted enzyme showed no inactivation after 80 min. A concomitant increase in absorbance at 320 nm was observed with inactivation of the Fe-reconstituted enzyme. Amino acid analysis of the inactivated Fe-reconstituted enzyme showed a decrease of about 0.7 residues of tryptophan/subunit, a value similar to the iron content of the iron-reconstituted enzyme. Three major peptides of the digests of the purified SOD with lysylendopeptidase were separated by a reverse-phase HPLC monitoring at 280 nm. One of the peptides, corresponding to the residues from Gly149 to Lys176, decreased in the HPLC eluent of the H2O2-inactivated SOD to 20 % of the amount measured for native SOD. Since this peptide contains only one tryptophan residue, it was concluded that the decomposed tryptophan residue is Trp159, which is located midway between the third and fourth metal ligands, Asp157 and His161, and is conserved in aligned amino acid sequences of all known Fe-SODs and Mn-SODs. Based on these results, we propose that the differences in hydrogen peroxide sensitivities observed for the Fe-SODs and Mn-SODs may be caused by the difference in the identity of the active site metal in the Fe-SODs and Mn-SODs and a tuning of the properies of the iron center... [ABSTRACT FROM AUTHOR]

The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharides (LPSs) designated O-LPS and A-LPS, which are a conventional O-antigen polysaccharide and an anionic polysaccharide that are both linked to lipid A-cores, respectively. However, the precise mechanisms of LPS biosynthesis remain to be determined. In this study, we isolated a pigment-less mutant by transposon mutagenesis and identified that the transposon was inserted into the coding sequence PGN&#95;2005, which encodes a hypothetical protein of P. gingivalis ATCC 33277. We found that (i) LPSs purified from the PGN&#95;2005 mutant were shorter than those of the wild type; (ii) the PGN&#95;2005 protein was located in the inner membrane fraction; and (iii) the PGN&#95;2005 gene conferred Wzz activity upon an Escherichia coli wzz mutant. These results indicate that the PGN&#95;2005 protein, which was designated WzzP, is a functional homolog of the Wzz protein in P. gingivalis. Comparison of amino acid sequences among WzzP and conventional Wzz proteins indicated that WzzP had an additional fragment at the C-terminal region. In addition, we determined that the PGN&#95;1896 and PGN&#95;1233 proteins and the PGN&#95;1033 protein appear to be WbaP homolog proteins and a Wzx homolog protein involved in LPS biosynthesis, respectively., (© 2013 The Authors. Microbiology Open published by John Wiley & Sons Ltd.)

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.

Although amphotericin B (AmB) is a major polyene antibiotic against invasive fungal infection, administration to patients sometimes causes inflammatory side effects, which limits the usage of the antibiotic. We studied the intracellular signaling that was induced by AmB. p65 (RelA) of nuclear factor-kappaB (NF-kappaB), a well-known signaling molecule as an inducer of proinflammatory cytokines, was phosphorylated by AmB in RAW264.7 cells, a monocyte-like cell line. Among chemical inhibitors of signaling molecules, U-73122 (phospholipase C (PLC) inhibitor), Gö6976 (protein kinase C (PKC) inhibitor), BAPTA-AM (calcium chelator), LFM-A13 (Bruton's tyrosine kinase (Btk)-specific inhibitor), and PP2 (c-Src kinase inhibitor) suppressed AmB-induced phosphorylation of p65 and translocation of p65 into the nucleus. U-73122 and Gö6976 reduced AmB-mediated induction of proinflammatory cytokines (tumor necrosis factor (TNF)-alpha and interleukin (IL)-6) in RAW264.7 cells. Furthermore, AmB-induced activation of NF-kappaB was observed in toll-like receptor (TLR) 2-expressed cells, and the activation of NF-kappaB was inhibited by U-73122, whereas peptidoglycan-induced NF-kappaB activation, which was also dependent on TLR2, was not inhibited by U-73122. Finally, U-73122 partially suppressed in vivo production of TNF-alpha and IL-6 induced by AmB administration in BALB/c mice. These results suggested that the signaling from AmB stimulation to proinflammatory cytokine production is mediated by TLR2, Btk, PLC, PKC, c-Src and NF-kappaB. These signaling molecules may become a target for chemotherapy suppressing AmB-induced proinflammatory cytokine production.

Porphyromonas gingivalis autolyzes in the culture media. To examine in more detail the molecular components of the autolysate, two-dimensional gel electrophoresis was performed. Many protein spots varied both in number and volume. One of these spots included Arg-gingipain (Rgp) as determined by N-terminal amino acid sequencing. Corresponding to the increase in spot volume, Rgp activity also increased during autolysis. The results of this study suggested that Rgp and other proteins in the P. gingivalis autolysate may be involved with the prolongation of periodontal disease, even after the death of P. gingivalis cells.